RISK ASSESSMENT OF METHYLMERCURY FROM FISH
CONSUMPTION IN OAHU, HAWAII USING HAIR AS A BIOMARKER
OF EXPOSURE
_______________
A Thesis
Presented to the
Faculty of
San Diego State University
_______________
In Partial Fulfillment
of the Requirements for the Degree
Master of Science in Public Health
with a Specialization in
Toxicology
_______________
by
Alethea L. Ramos
Summer 2012
iii
Copyright © 2012
by
Alethea L. Ramos
All Rights Reserved
iv
Ua Mau Ke Ea O Ka ‘Aina I Ka Pono –
The Life of the Land is Perpetuated in Righteousness
--Hawai’i State Motto
v
ABSTRACT OF THE THESIS
Risk Assessment of Methylmercury from Fish Consumption in
Oahu, Hawaii Using Hair as a Biomarker of Exposure
by
Alethea L. Ramos
Master of Science in Public Health with a Specialization in
Toxicology
San Diego State University, 2012
The main purpose of this study was to assess the risk of methylmercury exposure
from fish consumption in adults living in Oahu, Hawaii and to determine if demographic
variables, fish consumption frequency, and fish parts eaten were associated with
methylmercury body burden assessed through hair. The ultimate goal was to inform any
guidelines on fish consumption, with consideration of cultural and lifestyle factors for
populations that are susceptible to exposure. Persons were recruited at public areas from 5
areas of the island of Hawaii which included 110 adults (57 men and 53 women) during
December, 2010 and January, 2011. Hair samples were obtained in order to measure
methylmercury body burden concentrations, and a questionnaire assessed their fish
consumption, demographic information, and general awareness about methylmercury and
fish consumption. Hair samples were analyzed for total mercury through the Milestone
Direct Mercury Analyzer (Milestone, Shelton, CT). Older men had the highest hair mercury
levels, as compared to younger men and women of all age groups. Men > 45 years had a
median hair mercury level of 2.00 ug/g as compared to younger men with a median 0.97 ug/g
(p<0.05). Hair concentrations from older women had a median of 1.22 ug/g of mercury, as
compared to 0.57 ug/g for younger women. Risk indices were calculated, and the average HI
value among male residents was 1.61, which is above the safety criteria of 1.0. Among
women, the average HI value was 0.92, however, among women of childbearing age, 38%
had a HI > 1.0, indicating that both men and women were potentially at risk. Fish
consumption was a significant contributing factor to increased hair mercury concentrations;
significant variables included frequency of fish consumption, portion size of fish meal,
frequency of fish consumption in conjunction with portion size, amount of fish parts
consumed, and whether or not target organs (brain, head, heart) were consumed. This
present study addressed methylmercury exposure among a population of healthy adults,
which is uncommon for methylmercury studies since usually only susceptible populations,
such as women of childbearing age and children, are examined. Although we did
demonstrate that women were a susceptible population, as evidenced by values over 1.0 for a
third of the women of childbearing age, men also appeared as a susceptible population, with
the highest methylmercury values. Recent studies have established that cardiotoxicity is an
adverse health effect of methylmercury exposure for the men at body burden levels found in
this study. Since fish is an important staple for Oahu residents, proper guidelines for safe
fish consumption should include consuming less than ¼ pound of fish per meal at a
vi
frequency of less than one day per week, along with primarily consuming fish meat and
discarding all other organs due to high methylmercury content. The benefits of fish
consumption should also be highlighted, and if safe fish consumption practices are followed,
residents can reap the health benefits without excessive toxicant exposure.
vii
TABLE OF CONTENTS
PAGE
ABSTRACT ...............................................................................................................................v
LIST OF TABLES ................................................................................................................... ix
ACKNOWLEDGEMENTS .......................................................................................................x
CHAPTER
1
INTRODUCTION .........................................................................................................1 2
REVIEW OF THE LITERATURE - BACKGROUND ................................................7 Physical and Chemical Properties............................................................................7 Exposure Assessment...............................................................................................8 Toxicokinetics ........................................................................................................10 Transport of Methylmercury to Target Organs ......................................................14 Blood-Brain Barrier ...............................................................................................15 Neurochemical Effects of Mercurials ....................................................................16 Neurophysiological Effects of Mercurials .............................................................18 Cardiotoxicity ........................................................................................................19 Other Health Effects ..............................................................................................22 Oral Reference Dose (RfD) ...................................................................................28 Hair as a Biomarker of Exposure ...........................................................................29 Analytical Methods for Analyzing Methylmercury ...............................................32 3
MATERIALS AND METHODS .................................................................................34 Subjects ..................................................................................................................34 viii
Individual Dietary Surveys ....................................................................................35 Subjects and Biomarker Assessment .....................................................................36 Analytical Procedure ..............................................................................................37 Calibration and Standardization .............................................................................38 Calculation of Risk Indices ....................................................................................41 Statistical Analysis .................................................................................................42 4
RESULTS ....................................................................................................................43 Demographic Factors in Relation to Hair Mercury Levels ....................................43 Fish Consumption Factors in Relation to Hair Mercury Levels ............................47 Hazard Index and Cardiotoxicity ...........................................................................50 5
DISCUSSION ..............................................................................................................54 Relationship Between Demographic Factors and Hair Mercury Levels ...............54 Relationship Between Fish Consumption Factors and Hair Mercury Levels ........55 Men as a Susceptible Population to Cardiotoxicity ...............................................57 Benefits of Fish Consumption ...............................................................................59 Study Confounders and Limitations ......................................................................60 How To Protect the Public from Methylmercury Exposure ..................................62 6
CONCLUSION ............................................................................................................70 BIBLIOGRAPHY ....................................................................................................................72
APPENDIX
SURVEY ADMINISTERED TO OAHU RESIDENTS .............................................79 ix
LIST OF TABLES
PAGE
Table 1. Physical and Chemical Properties of Mercuric Compounds .......................................8 Table 2. Working Hg Standard Concentrations Used for 5-Point Calibration Curve .............39 Table 3. Median, 75th and 95th Percentiles, Minimum and Maximum Hair Mercury
Levels (ug/g) of Hair Sampled on December 2010 to January 2011 from
Residents Living Throughout Oahu, Hawaii. Hair Mercury Levels are
Categorized According to Gender, and Subcategorized by Age (Younger than,
and Including 45 Years of Age; Older than, and Including 46 Years of Age) ............44 Table 4. Demographic Variables as Reported by Residents in Relation to Median,
Minimum, and Maximum Hair Mercury Levels..........................................................45 Table 5. Fish Consumption Factors as Reported by Residents in Relation to Median,
Minimum, and Maximum Hair Mercury Levels..........................................................48 Table 6. Consumption of Different Fish Parts in Relation to Median, Minimum, and
Maximum Hair Levels as Reported by Residents........................................................49 Table 7. Various Fish Cooking Methods as Reported by Residents in Relation to
Median, Minimum, and Maximum Hair Mercury Levels ...........................................50 Table 8. Percent of At-Risk (HI > 1) and No-Risk (HI < 1) Populations Based on
Age, Gender, Region of Residency, Length of Residency, Presence of
Amalgam Fillings, Frequency of Fish Consumption, Portion Size per Meal,
and Consumption of Target Organs .............................................................................52 Table 9. Percentage of Total Population Reporting Sentiments on Public Health
Information About Methylmercury Exposure Through Fish Consumption ................63 Table 10. Consumption of Individual Fish Species from Oahu, Hawaii in Relation to
Median, Minimum, and Maximum Hair Levels as Reported by Residents.................64 x
ACKNOWLEDGEMENTS
I would like to acknowledge the divine inspiration and appreciation for being born
and raised in Hawaii, around its culture, and among the people that gave me insight to pursue
this study. Much gratitude is extended to the people of Oahu, Hawaii for their Aloha Spirit,
their openness for volunteering in this study, and their generous hospitality for letting me into
their lives; not only for donating their hair and time to complete the survey, but for also
making the environment enjoyable while conducting this study.
I would also like to extend my appreciation to INALAB, Inc., for the opportunity to
work within the realm of environmental and occupational science in order to hone in on my
analytical skills needed to conduct the chemical analysis portion of this study. I appreciate
Dr. Hagadone for his support and encouragement, and lending me his network within the
industry to direct me to people and resources to help in this study; additionally, for allowing
me to use his laboratory to conduct a pilot investigation. Thank you also to the employees
for volunteering as subjects for the study.
I greatly appreciate the Toxicology/Environmental Health Department at San Diego
State University for feeding my knowledge regarding Public Health and Environmental
Toxicology. Thank you to Christina Meyer for trusting and giving me free reign to conduct
mercury analysis on the hair samples using the DMA and for helping me with prep work.
Thank you to my parents for assisting me financially to pursue my master’s degree,
and for their unconditional support, strength, and patience despite the stress they have
encountered while pursuing my master’s degree and during the completion of my thesis.
xi
Last, but not least, thank you to Dr. DePeyster, Dr. Quintana, Dr. Hoh, and Dr.
Chatfield for their expertise, and time out of their busy schedules to lead me through this
investigation. Thank you to Dr. DePeyster for her assistance in guiding me through the IRB
process, and as an additional brain to scrutinize the feasibility and direction of the study.
Thank you to Dr. Quintana for her guidance in the statistical analysis portion and supervision
during the completion of the study.
Without everyone’s efforts, this study would not have been possible.
1
CHAPTER 1
INTRODUCTION
In the 1950’s, one of the most severe incidents of industrial pollution and mercury
poisoning occurred on the southwestern coast of Kyushu Island, Japan around Minamata
Bay. Chisso Corporation, a local petrochemical and plastics company, dumped an estimated
27 tons of methylmercury into Minamata Bay for a period of 37 years (Griesbauer, 2007).
Methylmercury chloride was used as a catalyst in producing acetaldehyde, a chemical used to
produce plastics. In order to meet the growing demand for plastics, the manufacturing plant
expanded, and the drainage site of the factory’s runoff was moved to the mouth of the
Minamata River, which disseminated contaminated wastewater into surrounding waters of
Shiranui Sea. Methylmercury was found in the industrial waste as a byproduct of this
process, and was dumped into the bay for more than a decade. The bay became a highly
toxic environment which harbored local fish that residents relied on for sustenance. After
consuming methylmercury contaminated fish and shellfish, residents exhibited acute
methylmercury poisoning which entailed severe neurological impairments, and resulted in
900 deaths. An estimated 2 million people suffered from health problems or were left
permanently disabled. This form of acute methylmercury poisoning is now termed
Minamata Disease. Symptoms include sensory disorders of the four extremities, loss of
feeling, cerebellar ataxia, tunnel vision or blindness, smell and hearing impediments, and
disequilibrium syndrome. The factory continued releasing methylmercury into the affected
bodies of water until 1968. Fishing around Shiranui Sea was never prohibited, and residents
2
living around that area continued to be exposed to polluted waters for 20 years, resulting in
chronic methylmercury poisoning (Ekino, Susa, Ninomiya, Imamura, & Kitamura, 2007).
Certain populations are susceptible to methylmercury exposure, due to lifestyle and
cultural factors. Asian and Pacific Islanders (API) have origins from the Far East, Southeast
Asia, Indian subcontinent, and Pacific Islands; they comprise a majority of the United States
growing immigrant population. Many of their lifestyle and cultural practices have followed
them to the United States. Among those practices, eating seafood reflects practices of their
homeland, and fish due to economic and cultural reasons. Therefore, API’s consume more
seafood of different varieties, and eat different parts of fish that other cultures may avert
(Sechena et al., 1999). Many of the recipes used by API immigrants reflect cooking practices
from their home countries. For instance, some API’s may employ fish organs that are
targeted for methylmercury accumulation, such as fish brain, into their diet. Economic
factors may also affect where people collect or purchase fish. In King county, API’s fish
regularly in urban areas that are Superfund sites, which is an uncontrolled or abandoned
place where hazardous waste is located, and is adjacent to several API communities. Due to
language barriers, many immigrants may not be able to read posted advisory sites, or have
access to cleaner fishing areas due to lack of transportation. Methods of preparation can also
affect exposure and heighten health risks to methylmercury toxicity. For instance, API’s
who reside in King County, WA, consume the entire crab, which includes the hepatopancreas
(digestive organ of the crab), an organ that contains high concentrations of PCBs (Judd,
Griffith, & Faustman, 2004). API’s also imbibe the water that the crab has been cooked in,
which harbors higher concentrations of contaminants with low boiling points. Cultural and
lifestyle factors are not accounted for in most assessments for average US consumers and
3
should be taken into consideration so public health solutions are met within cultural contexts
(Judd et al., 2004).
In addition to cultural susceptibility, recreational anglers and subsistence fishermen
that frequently consume locally caught fish from contaminated waters, or those who consume
predatory oceanic species, such as shark and swordfish, are exposed to mercury in
comparison to those who consume similar or lesser amount of commercially marketed fish
from various sources. Methylmercury exposure is also increased in individuals who
regularly eat fish and other seafood compared to those who occasionally or never eat fish or
other seafood (World Health Organization [WHO], 2008).
Since Hawaii is a coastal region, fish is a popular staple among the locals. In 1997,
Pacific Business News reported that fish consumption in Hawaii, which was 90 pounds per
person per year, doubled the national average of 40 pounds per person a year. Due to a high
consumption frequency of fish, residents of Hawaii are a susceptible population for adverse
effects from high methylmercury exposure. The types of fish, the amount consumed, and
how it’s prepared are often driven by cultural and lifestyle factors. Hawaii is comprised of a
large demographic of Asian-Pacific Islanders (API).
The main source of environmental methylmercury is from methylation of inorganic
mercury. Much of the source of background mercury levels is volcanic in origin from
magmatic degassing and rock weathering, as well as evaporation from bodies of water.
Mount Kilauea is the active volcano found on the Big Island that has emitted mercury
outputs, as much as 100-fold, during eruptions. Increase of air mercury levels have been
detected during full-scale eruptions, locally, as well as considerable distances from the
volcanic site (Raine, Siegel, & McMurtry, 1995).
4
Other sources of exposure include anthropogenic sources, such as agricultural
pesticides, antifouling paints, mercury fulminate in percussion caps, mercurous chloride in
fireworks, and both the legal and illegal dumping of laboratory equipment as well as
batteries. Mining of mercury yields 10,000 tons of mercury per year. Man-made emissions
from the combustion of fossil fuels account for 25% of emissions released into the
atmosphere. Mercury is used as a cathode in the electrolysis of a sodium chloride solution to
produce chlorine gas and caustic soda. It is utilized in the electrical industry to create lamps,
arc rectifiers, and mercury battery cells. In addition, it is implemented in the home industry
in creating switches, thermostats, and barometers, and in the manufacturing of laboratory and
medical equipment. Mercury is also widely used in creating dental amalgam fillings (WHO,
1990).
Another source of exposure is generated as chemical byproducts from Asian coalfired power plants that travel long distances through warm ocean currents, and raise mercury
levels in the North Pacific Ocean. These findings may explain why mercury levels are
increasing in the eastern North Pacific when no local source is apparent, and can cause an
increase of mercury levels in fish. A study conducted by Sunderland and colleagues from the
University of Washington in Seattle measured mercury concentrations at 16 ocean sites
between Hawaii and Alaska. Atmospheric and ocean circulation models were created from
data acquired through monitoring. Models pinpointed the source of mercury to coal fired
power plant emissions from a heavily populated coast of Asia. According to the model,
mercury was deposited into the western Pacific Ocean then ocean currents carried it to the
eastern North Pacific study sites within two years (Potera, 2009).
5
Through the biogeochemical cycling of mercury, methylmercury introduces into the
food chain. In the atmosphere, mercury takes the form of elemental mercury vapor. The
global cycling of mercury is primarily composed of inorganic forms. Inorganic species of
mercury do not readily accumulate within the human food chain. Therefore, in order for
bioaccumulation and biomagnification to occur, a speciation change from inorganic to
methylated forms of mercury is essential. Through photochemical oxidation from sunlight,
elemental mercury converts to inorganic mercury, and returns to the earth’s surface through
rainfall, and accumulates into soil and surface waters. A fraction of the mercury load
evaporates back to mercury vapor into the atmosphere. Another fraction accumulates as the
highly insoluble form, mercuric sulfide, into the bottom sediment of oceans. Through
methylation, sulfate-reducing bacteria that are found in the ocean sediments convert
inorganic mercury to organic mercury as a protective mechanism for the microbial
community. Methylmercury in coastal marine sediments is a significant source for
methylmercury found in marine fishes. Other sources of inorganic mercury methylation
include bacteria within fish intestines, as well as outer slime of fishes (Griesbauer, 2007).
Methylmercury then enters the food chain through the absorption of methylmercury
into the cell walls of phytoplankton through rapid diffusion and protein binding.
Phytoplankton are eaten by phytoplankton consumers, which are consumed by higher trophic
level predatory fish. Through bioaccumulation, methylmercury accumulates and has an
affinity for certain tissues within fish. As predatory fishes consume lower trophic level
animals, methylmercury accumulates with increased consumption of contaminated
organisms. Through biomagnification, methylmercury concentrations increase as trophic
levels increase. Thus, smaller fish that are lower in the food chain have lower concentrations
6
of methylmercury found within their tissues while larger fishes higher in the food chain have
higher concentrations of methylmercury. Larger predatory fishes, such as tuna, shark, and
swordfish contain a substantial amount of mercury compared to lower predatory fishes.
Factors, such as size and age of fish, microbial activity in sediment, mercury content in
sediment, water chemistry characteristics (dissolved organic content, salinity, pH, and redox
potential), dictate mercury levels found in fish (Griesbauer, 2007).
The goal of this study is to assess the risk of methylmercury exposure from fish
consumption among sampled residents of Oahu, Hawaii using hair as a biomarker of
exposure. Risk indices were calculated in order to propose guidelines on proper fish
consumption that will not pose a risk to human health, with consideration of cultural and
lifestyle factors for populations that may be susceptible to disease.
Hypothesis: body burdens of methylmercury, as assessed by concentrations in hair,
will be found above the Risk Index of 1, and high levels will be associated with high fish
consumption. This study will aid in considering whether consuming fish is harmful to the
residents of Oahu, and will potentially inform residents to lessen their exposure to
methylmercury.
7
CHAPTER 2
REVIEW OF THE LITERATURE - BACKGROUND
PHYSICAL AND CHEMICAL PROPERTIES
Elemental mercury is found as a silver liquid at room temperature in its zero
oxidation state, Hg0. It volatilizes readily to mercury vapor and remains stable in ambient
atmospheric conditions for years, which aids in the global cycling of mercury. Through
oxidation, two oxidation states are formed. Mercurous chloride is the first oxidation state
where one electron is removed from the mercury atom and two mercury atoms are present.
Mercuric ion is the second oxidation state where two electrons are removed. This form of
mercury is responsible for inorganic and organic mercury compounds, thus playing a key role
in the toxicity property of mercury (Clarkson & Magos, 2006). Methylmercury can be
commonly found as methylmercuric chloride- a stable, white, crystallized salt. It is a free ion
only in small quantities. Table 1 (National Research Council Committee on the
Toxicological Effects of Methylmercury, 2000) shows the molecular formula, molecular
structure, molecular weight, solubility, density, and oxidation state of various compounds of
mercury. Mercury compounds, such as mercuric chloride, are highly water soluble. Organic
methylmercury compounds, such as methylmercury chloride, are less water soluble and
highly lipophilic. Dimethylmercury, a toxic byproduct from chemical synthesis of
methylmercury, is also highly lipophilic. The solubility of mercuric compounds plays a
significant role in their differential toxicity (National Research Council Committee on the
Toxicological Effects of Methylmercury, 2000).
8
Table 1. Physical and Chemical Properties of Mercuric Compounds
Chemical name
Molecular
formula
Molecular
structure
Molecular
weight
Solubility
Density
Elemental
Mercury
Mercuric
Chloride
Mercurous
Chloride
Methylmercuric
Chloride
Dimethylmercur
y
Hg0
HgCl2
Hg2Cl2
CH3HgCl
C2H6Hg
Cl-Hg-Cl
Cl-Hg-Hg-Cl
CH3-Hg-Cl
CH3-Hg-CH3
271.52
472.09
251.1
230.66
0.100 g/L at
21oC
4.06 g/cm3 at
20oC
1 g/L at 21oC
200.59
-5
5.6 x 10 g/L
at 25oC
13.534 g/cm3 at
25oC
69 g/L at 20oC
5.4 g/cm3 at
25oC
-3
2.0 x 10 g/L
at 25oC
7.15 g/cm3 at
19oC
3.1874 g/cm3 at
20oC
Oxidation
+1, +2
+2
+1
+2
+2
state
Source: National Research Council Committee on the Toxicological Effects of Methylmercury. (2000).
Toxicological effects of methylmercury. Washington, DC: National Academy Press.
Mercuric mercury has a high affinity for sulfhydryl (thiol) groups, especially for the
anion R-S-, and can jump rapidly from one thiol group to another (Clarkson & Magos, 2006).
Its high affinity for sulfhydryl groups, which results in the poisoning of essential enzymes, is
the main cause for methylmercury toxicity (Junghans, 1983). Another factor for its
biological activity is due to oxidation-reduction reactions (Aschner & Aschner, 1990).
Animal experiments suggest that inorganic and methylmercury form a complex with
glutathione in bile. Chelating agents containing thiol groups are the only effective means in
removing mercury from the body. Small molecules containing thiol groups, such as cysteine
and glutathione, aid in disposition and transportation of mercury. Mercuric mercury also
binds readily to the selenide anion, Se2-. Insoluble mercuric selenide is formed, and resides
in tissues (Clarkson & Magos, 2006).
EXPOSURE ASSESSMENT
During the mid-19th century, methylmercury was first recognized by chemists as
dimethylmercury. The majority of current human exposure is in the form of
9
monomethylmercury through fish, marine mammals, and crustacean consumption, with fish
consumption as being the most important source. Exposure to methylmercury from non-fish
sources is very low. However, terrestrial animals fed with fish meal and consuming foods
grown in mercury-contaminated areas are negligible sources of methylmercury.
Methylmercury exposure can vary with individual fish consumption needs, and types of fish
consumed. Limited data also suggests that coastal regions have higher fish consumption
rates. Ethnic and cultural populations, along with recreation fishermen, may have increased
susceptibility to methylmercury exposure (Clarkson & Magos, 2006).
Elemental mercury exposure is due to mercury vapor released by dental amalgams.
Most amalgams used in the past contained 50% mercury. When vapor is released and enters
into tissues, it is oxidized to inorganic mercury. Other exposure sources include accidents at
chloralkali plants, religious ceremonies, children’s toys, and wastewater (National Research
Council Committee on the Toxicological Effects of Methylmercury, 2000). Other sources of
methylmercury, with the exception of fish and seafood consumption, have virtually
disappeared due to health warnings by international agencies. Since the 1970’s, local
pollution have been regulated; however, global cycling of mercury continues due to
anthropogenic sources. Health agencies also regulate mercury levels in fish. Mercury levels
of greater than 1 ppm are not sold in commercial markets (Clarkson & Magos, 2006).
Methylmercury binds to fish tissues through the thiol group of cysteine residues
found in fish protein, and also to free amino acids. Methylmercury in fish tissues cannot be
removed by cooking, or cleaning that does not destroy muscle tissues. The methylmercury
species accounts for majority of total mercury in fish tissues (National Research Council
Committee on the Toxicological Effects of Methylmercury, 2000).
10
TOXICOKINETICS
Biological methylation and bioaccumulation within the food chain demonstrates that
ingestion is the most common method of methylmercury intake. Methylmercury
consumption is almost entirely absorbed into the bloodstream. 94-95% of methylmercury
ingested from fish is absorbed into the GI tract. Within the stomach, low pH and high
chloride concentrations favor the formation of uncharged and lipid-soluble MeHgCl,
allowing easy transportation across gastric walls, and easily penetrates placental and bloodbrain barriers.
Methylmercury readily distributes throughout the body since it is a lipophilic
compound (Table 1). Methylmercury forms a complex with the amino acid cysteine,
becoming highly mobile. This complex mimics methionine, a neutral amino acid, and enters
into cells through a neutral amino acid carrier. It exits cells by forming a complex with
reduced glutathione and attaches to a membrane carrier. Majority of mercuric ions within
biological systems are bound to molecules with free sulfhydryl groups. Mercuric ions rarely
exist in an unbound state. About 95% of methylmercury in fish ingested by humans readily
absorbs from the GI tract and into the bloodstream where 90% binds to cysteine residues
present on hemoglobin molecules within the red blood cells, while the remainder binds to
plasma proteins. Methylmercury enters into the bloodstream 15 minutes after ingestion, and
peaks within 3-6 hours. Distribution is completed within 4 days of exposure. It takes 1-2
days longer for methylmercury to peak within the brain, in comparison to other tissues. At
this point, the brain contains 6% of the dose, which is 10% of the total body’s bioburden, and
slowly demethylates to inorganic mercuric mercury. Methylmercury transports across the
blood-brain barrier as a meHg-L-cysteine complex through the L-system amino acid carrier.
This complex releases from a meHg-glutathione complex through gamma-
11
glutamyltransferase and dipeptidase, suggesting that glutathione plays an indirect role in
transporting methylmercury into endothelial cells (WHO, 1990). Methylmercury is
distributed between blood and extravascular tissues. A portion of methylmercury
administered, converts to inorganic mercury in all organs except the kidney. The kidney and
liver have the highest concentration of methylmercury. Humans ingesting methylmercury
daily have inorganic mercury present in whole blood, plasma, breast milk, liver, and urine at
7%, 22%, 39%, 16-40%, and 73%, respectively. Liver, kidney medulla, and kidney cortex
contain total mercury concentrations around the range of hundreds of ng/g, with 33%, 15%,
and 11%, respectively, of mercury as methylmercury. Cerebrum, cerebellum, heart, and
spleen contain total mercury concentrations around the range of tens of ng/g. 80% of
mercury detected in those organs is in the form of methylmercury (National Research
Council Committee on the Toxicological Effects of Methylmercury, 2000). The blood-brain
barrier limits the transport of organic mercury into hair. Hair has a high affinity for mercury,
and once it is incorporated, demethylation no longer occurs. In addition, mercury is
irreversibly bound to hair and is removed only when the hair is shed. A study by Farris,
Dedrick, Allen, and Smith (1993), observed that after an oral tracer dose of 203Hg-labeled
methylmercury chloride to a growing rat, by day 21, almost 90% of the mercury in the skin is
integrated into hair. Mercury concentrations peak on day 28, then slowly declines. At the
end of day 98, over 12% of the original dose and approximately 90% of the body burden is
found within hair.
A case study of methylmercury toxicity involving a family that consumed
methylmercury contaminated pork shows that the cerebrum and cerebellum are especially
susceptible to methylmercury. An autopsy performed on the female member of the family
12
showed brain-damage related to mercury concentrations in different regions of the brain.
Inorganic mercury comprised 82-100% of total mercury, implying that most of the
methylmercury had been converted to inorganic mercury through demethylation by bacteria
found in the intestinal flora of the GI tract. Macrophage cells that are present in the spleen,
also convert methylmercury to inorganic mercury. Mercury concentrations were isolated to
the cerebrum and cerebellum. MRI’s on other members of the family depicted damage
around the calcarine and parietal cortices, and cerebellum, which are sections of the brain
controlling coordination, balance, and sensations (Davis et al., 1994).
Approximately 1% of methylmercury body burden excretes daily through the bile and
feces. The conversion of methylmercury to inorganic mercury may be a key factor in
methylmercury excretion. Biliary methylmercury reabsorbs and complexes with glutathione
and eliminates through the bile. Fecal elimination includes biliary excretion of
methylmercury and inorganic mercury, then complexing with glutathione and sulfhydryl
peptides. Poor absorption of inorganic mercury across intestinal walls from biliary secretion
passes directly into feces. Methylmercury secreted into the intestines reabsorbs back into the
bloodstream, and contributes to a secretion-reabsorption cycle, or the enterohepatic
circulation cycle. This occurrence accounts for increased amounts of methylmercury passing
through intestinal walls, and provides a continuous supply of substrate for intestinal
microflora, which converts methylmercury to inorganic mercury. 10% of the converted
inorganic mercury is absorbed back into the bloodstream and is transported to tissues,
plasma, breast milk, bile, and urine. 90% of an ingested dose excretes through the feces as
mercuric Hg in humans. After a single exposure of methylmercury, feces contain 65%
inorganic mercury and 15% methylmercury. Urine contains 1% inorganic mercury and 4%
13
methylmercury. Using whole-body measurements, the half-life of methylmercury is 70-80
days (WHO, 1990).
Biochemical mechanisms of methylmercury toxicity may be due to several factors,
such as mitochondrial changes, lipid peroxidation, microtubule disruption, and disrupted
protein synthesis. The primary mechanism of methylmercury neurotoxicity is through
disruption of protein synthesis. Mitotic arrest is a sensitive indicator of methylmercury
exposure in mice. The ratio of late to total mitotic figures is substantially reduced in the
cerebellum of exposed mice, indicating mitotic arrest. A study by Sarafian and Verity (1991)
shows that methylmercury intoxication causes membrane peroxidation in nerve cells,
specifically free-radical induced lipid peroxidation, since antioxidants, like selenium and
vitamin E, protect against methylmercury neurotoxicity. Oxidative stress is another
mechanism of methylmercury toxicity. Glutathione is the major antioxidant of the cell, and
after exposure to methylmercury, glutathione concentrations decline, then increase (Miura &
Clarkson, 1993). Cells resistant to methylmercury toxicity have an increase rate of efflux of
methylmercury, and a proportional increase of glutathione concentrations than normal cells.
Another mechanism is methylmercury disruption of microtubules in the neuronal
cytoskeleton. Mercury binds to thiols present in tubulin, which is the subunit that forms
microtubules, and blocks polymerization and depolymerization of microtubules. Cellular
process is disrupted since polymerization and depolymerization of microtubules is required
for cell functions. Methylmercury is slowly transformed to inorganic mercury within the
brain. It is unclear whether methylmercury toxicity is directly due to the parent compound,
due to its metabolite inorganic mercury, or free radicals that are generated from metabolizing
14
methylmercury to inorganic mercury (National Research Council Committee on the
Toxicological Effects of Methylmercury, 2000).
Methylmercury is highly toxic due to its hydrophobic nature. Hydrophobicity allows
the compound to easily transport across membrane barriers, and is able to quickly attack
organs distal to the point of ingestion, such as the brain. Methylmercury binds to thiol
containing molecules, such as glutathione (GSH); it is the main nonprotein thiol in rat
cerebrum that binds to methylmercury. Hirayama (1980) added another thiol molecule, Cys,
with methylmercury, which resulted in increased uptake into capillary endothelial cells of the
blood-brain barrier. Cys S-methylmercury conjugates (CH3Hg-S-Cys) are substrates
transportable for a neutral amino acid transporter into the capillary endothelium of the bloodbrain barrier. Also, Landner (1971) and Jernelov (1973) suggested that structural similarities
between the amino acid methionine and CH3Hg-S-Cys allows methylmercury to cross the
membrane barrier, possibly by a transport protein, system L.
TRANSPORT OF METHYLMERCURY TO TARGET ORGANS
Following the consumption of food contaminated with methylmercury, uptake into
intestinal enterocytes is dependent upon the enzyme, γ-glutamyltransferase, and the
involvement of proteins from the OAT family. Methylmercury is readily absorbed when it is
conjugated with byproducts from glutathione catabolism. When the activity of γglutamyltransferase is inhibited, transporting methylmercury-glutathione conjugates into
enterocytes decrease by 50%. Within the intestinal enterocytes, the methylmercury
conjugate is further degraded at the luminal plasma membrane to yield CH3Hg-S-CysGly.
Di- and tripeptide transporters transport amino acids. The structural resemblance of CH3HgS-CysGly to a small peptide allows the conjugate to cross the luminal membrane of
15
enterocytes. Another form of a methylmercury conjugate, CH3Hg-S-Cys, is the primary
species that is secreted into the intestines from bile, and absorbs rapidly by enterocytes
(Bridges & Zalups, 2010).
Methylmercury is transported out of the enterocytes into the systemic circulation.
The uptake of methylmercury-glutathione S-conjugates into erythrocytes is facilitated by the
OAT family of proteins (Bridges & Zalups, 2010). Foulkes (1993) suggested that
intracellular concentrations of glutathione regulate the basolateral efflux of methylmercury
from enterocytes into the circulation. A glutathione transporter may aid in the basolateral
efflux due to the structural similarities between glutathione and CH3Hg-S-G.
After absorption through the intestines, methylmercury is delivered to the liver from
portal blood through amino acid carriers and a glutathione transporter. Transporting
methylmercury from hepatocytes into the canalicular membrane involves glutathione. After
secretion into the bile, CH3Hg-S-G is hydrolytically catabolized by plasma membrane
enzymes to yield CH3Hg-S-Cys, which is reabsorbed into cells lining the bile ducts and
enterocytes within the intestines (Bridges & Zalups, 2010).
BLOOD-BRAIN BARRIER
The cationic form of methylmercury is conjugated to thiol-containing biomolecules.
When methylmercury takes the form of CH3Hg-S-Cys, it mimics the structure of methionine
which allows this species to cross the blood-brain barrier through an amino acid carrier. This
amino acid carrier is localized in the apical and basolateral plasma membranes of endothelial
cells lining the blood-brain barrier (Bridges & Zalups, 2010).
The endothelial cells aid in the regulation of the CNS milieu by regulating ion
composition, nutrient entry, and the elimination of wastes. The barrier restricts the passage
16
of solutes without carrier transport systems, and only allows specific physical properties to
crossover from the blood to the brain (Aschner & Aschner, 1990).
NEUROCHEMICAL EFFECTS OF MERCURIALS
Methylmercury is a potent neurotoxin that affects the central nervous system (CNS)
and induces clinical symptoms such as tremor, ataxia, and mental disturbances. The
neurochemical process in which methylmercury generates neuropsychological effects
involves the inhibition of cholinergic neurotransmission within the brain in vitro. These
stages include choline uptake, choline acetyltransferase (ChAT), muscarinic, and nicotinic
receptor binding. The synthesis of acetylcholine (ACh) is a sensitive stage according to in
vitro studies due to the inhibition of ChAT. In addition, after long-term administration of
methylmercury to adult rats, ChAT decreases, and ACh synthesis rate and concentrations
also slightly decrease. Following cessation of methylmercury exposure, ACh levels begin to
return to normal (Komulainen & Tuomisto, 1987).
Methylmercury inhibits in vitro enzymes responsible for metabolizing catecholamines
and their byproducts. In mouse brain homogenate, high concentrations of methylmercury
hinder the enzymes tyrosine hydroxylase (TH), DOPA decarboxylase, monoamine oxidase
(MAO), and catechol-O-methyltransferase (COMT). In vivo, a continuous administration of
methylmercury during postnatal development of the nervous system causes a permanent
defect in DA and NA neurons. Additionally, at micromolar concentrations, methylmercury
inhibits dopamine (DA) and noradrenaline (NA) uptake into synaptosomes in vivo and in
vitro. As DA uptake decreases, DA and NA turnover are increased, and NA levels elevate.
In chronic high-dose administration to adult rats, levels of NA increase concomitantly with
DA (Komulainen & Tuomisto, 1987).
17
In regards to serotonergic transmission, at micromolar concentrations, methylmercury
inhibits the uptake of 5-hydroxytryptamine (5-HT) and stimulates spontaneous release into
hypothalamic synaptosomes and blood platelets. Imipramine binding in platelet membranes
is also inhibited, which suggests a blockade of the 5-HT uptake carrier (Komulainen &
Tuomisto, 1987).
Administration of methylmercury to brain tissue in vitro inhibits the uptake of amino
acids, such as gamma-aminobutyric acid (GABA), glycine, and glutamate. In addition, their
release is stimulated, and ligand binding to glycine and benzodiazepine receptors is inhibited
in vitro. Compared to monoaminergic or cholinergic transmission, GABAergic transmission
in the rat brain is not as sensitive to methylmercury, especially since methylmercury
poisoning is not necessarily convulsive (Komulainen & Tuomisto, 1987).
Pharmacokinetic factors play a role in determining neurochemical toxicity, especially
with the distribution of methylmercury within the brain. There are other factors that
determine the effectiveness for methylmercury toxicity. A threshold concentration must be
achieved before methylmercury becomes toxic. In addition, the binding of methylmercury to
multiple proteins decreases the effective fraction acting on critical sites. Lastly, toxicity is
dependent upon the total cumulated dose rather than on the exposure time (Komulainen &
Tuomisto, 1987).
Several mechanisms affect neurons. First, methylmercury has a high affinity for SHgroups; therefore, processes where these groups are present are disturbed at a certain
exposure concentration. The inhibition of neuronal protein synthesis contributes
significantly to the observed effects. Decreased protein synthesis and the inhibition of axonal
transport lead to the decrease in enzyme activity in nerve terminals. Secondly, mitochondria
18
regulate the concentrations of free intracellular calcium. Therefore, methylmercury
depresses the mitochondrial glycolytic pathway, oxidative phosphorylation, and tissue
respiration, which results in the disturbance of energy-requiring processes. In addition,
intracellular calcium homeostasis is also impaired (Komulainen & Tuomisto, 1987).
NEUROPHYSIOLOGICAL EFFECTS OF MERCURIALS
Methylmercury poisoning has a long latency period lasting several months (WHO,
1990). However, following the latency stage, methylmercury exposure causes selective
restricted and focal cerebral lesions to the granule cells of the cerebellum and neurons in the
visual cortex in adults (Yokoo et al., 2003). Manifestations of methylmercury poisoning are
paresthesia (abnormal sensation or numbness) along the skin of the hands, feet, and mouth.
At increased concentrations, ataxia (effect in coordination or gait) and visual disorders
(constriction of visual fields and blurring) are observed. As methylmercury exposure
increases, symptoms of dysarthria (difficulty in speech), generalized motor function
impairment, and loss of muscle power occurs. Severe poisonings result in deafness,
blindness, myoclonic jerks (involuntary muscle spasms), general physical and mental
debilitation, paralysis, or death. Damages to the brain are localized to the visual cortex, and
the primary sensory and motor areas. If exposure is moderately severe, gradual
improvements in muscle power, ataxia, and dysarthria is possible after methylmercury blood
levels decrease. However, visual functions are the last to improve (Junghans, 1983).
According to Yorifuji, Tsuda, Takao, Suzuki, and Harada (2009) an obvious doseresponse relationship was observed between hair mercury concentrations and perioral
memory loss, which was substantially increased (p = 0.03) for exposure levels above 20 to 50
ug/g. Additionally, hair mercury concentrations below 50 ug/g were associated with
19
neurobehavioral disturbances to visual (chromatic discrimination, contrast sensitivity and
peripheral fields) and psychomotor functions (tremor, dexterity, grip strength, complex
movement sequences, hand-eye coordination, and rapid alternating movements). These
outcomes from low chronic levels of methylmercury exposure suggested that a guidance
level of 50 ug/g in hair may not be a protective criteria (Yokoo et al., 2003).
Neurodevelopmental studies using animal models exposed to methylmercury inutero, early postnatally, or as an adult perpetuated toxicity effects related to dose, which
included sensory, sensorimotor, and cognitive development. Experimental studies using
monkeys, rodents, and cats reported similar adverse neurological effects consistent with adult
Minamata Disease. Neurotoxic signs among adults reflected regional specificity of
neuropathological effects. Indications of ataxia, constriction of the visual field, and sensory
disturbances were associated with lesions present in the calcarine cortices, dorsal root
ganglia, and the cerebellum (National Research Council Committee on the Toxicological
Effects of Methylmercury, 2000).
CARDIOTOXICITY
Mercury accumulates in the heart; therefore, exposures to mercury have been
associated with hypertension and abnormal cardiac function. Toxic and subtoxic exposure to
elemental and organic forms of mercury in children and adults alter blood pressure
regulation.
Exposure to organic mercury was associated with cardiovascular changes as observed
in three clinical case reports and two epidemiological investigations. The first evidence of
cardiovascular abnormalities was provided by Jalili and Abbasi’s 1961 study describing
patients who were hospitalized during the Iraqi grain poisoning epidemic. Those patients
20
who were severely poisoned exhibited irregular pulse as well as electrocardiograms showing
ventricular ectopic beats, prolongation of the Q-T interval, depression of the S-T segment
and I inversion. Similarly, in a Cinca, Dumetrescu, Onaca, Serbanescu, and Nestorescu,
(1979) study, electrocardiograms of four family members who consumed ethylmercury
contaminated pork revealed similar findings, which included abnormal heart rhythms with ST segment depression and T-wave inversions. Two children within this family died from
cardiac arrest, and their autopsies revealed myocarditis.
Oxidation of low-density lipoprotein (LDL) is a key factor for the development of
artherosclerosis, with oxidized LDL particles present within lesions in the arterial wall. As
LDL plasma concentrations increase, there is an increased deposition of LDL particles into
the arterial wall, allowing the arterial walls to be susceptible to oxidative damage. There is a
positive relationship between oxidized LDL levels within circulatory blood and intima-media
thickness (IMT) associated with the progression of atherosclerosis in carotid arteries and
plaque formation in the carotid and femoral arteries of men (Virtanen, Rissanen, Voutilainen,
& Tuomainen, 2007).
The first incident of observing cardiovascular disease (CVD) from methylmercury
exposure was through several studies with the Kuopio Ischemic Heart Disease Risk Factor
(KIHD) study cohort which was initially published by Salonen et al. (1995) (Virtanen et al.,
2007). Salonen et al. (1995) conducted a 7-year study examining the relation of mercury
from dietary fish intake and the risk of acute myocardial infarction (AMI) and death from
coronary heart disease (CHD) among Finnish men, through analyzing hair and urine. The
role of lipid peroxidation, pro-oxidative minerals, and antioxidants in artherosclerosis and
CHD was also assessed in methylmercury toxicity. Men who consumed at least 30 g. of fish
21
per day or had a hair mercury concentration of greater than 2 ppm had a higher risk of
suffering an AMI. Men with greater than 2.0 ug/g of hair mercury had a two-fold increased
risk of developing AMI, when adjusting for age, examination year, ischemic exercise ECG,
and maximal oxygen uptake. Men who also consumed > 30 grams of fish per day had 56%
higher mean mercury hair levels than men who consumed less than 30 grams of fish per day.
There was an increased risk of coronary heart disease with increased hair mercury levels
among Finnish men who consumed fish from contaminated lakes. High mercury
concentrations in hair and high urinary mercury excretion, coupled with elevated titers of
immune complexes containing oxidized lipoprotein support that mercury elevated the risk of
AMI through promoting lipid peroxidation.
Later studies conducted by Salonen, Seppanen, Lakka, Salonen, and Kaplan (2000),
among the same Finnish population depicted an accelerated progression of carotid
artherosclerosis through hair mercury concentrations, coupled with ultrasonographic
assessment of the IMT of the carotid arteries. High mercury hair concentrations were
independently associated with accelerated progression of carotid atherosclerosis and were a
strong risk factor for an increase in carotid IMT. Previous findings from the 1995 study
associating high mercury concentrations in hair with increased lipid peroxidation and risk of
myocardial infarction provided evidence of mercury toxicity as an etiology of atherosclerosis
and CVD. Cell culture studies established that mercury exposure promoted reactive oxygen
species (ROS) through the cleavage of methylmercury, thus causing the progression of lipid
peroxidation (Park, Lim, Chung, & Kim, 1996).
In a 2000 follow-up study by Rissanen et al. (as cited in Virtanen et al., 2007),
utilizing the same KIHD cohort, authors observed that high mercury content in hair
22
attenuated the beneficial effects of fatty acids from fish associated with the risk of acute
coronary events. The latest follow-up study conducted by Virtanen et al. (2005), found that
men with > 2.0 ug/g of mercury in hair had a 1.60-fold risk of acute coronary event, 1.68fold risk of CVD, 1.56-fold risk of CHD and 1.38-fold risk of any death, compared to men
with less than 2.0 ug/g of mercury in hair. Additionally, for each microgram of mercury in
hair, the risk of acute coronary event increased by 11%, risk of CVD death increased by
10%, the risk of CHD death increased by 13%, and the risk of any death increased by 5%.
A study by Choi et al. (2009) examined long term methylmercury exposure among
forty-two Faroese whaling men. Mercury analysis was conducted on hair, blood, and toenail
samples, as well as including mercury hair results from seven years earlier. Findings
suggested that increased methylmercury exposure promoted CVD due to increased blood
pressure and IMT. Adverse effects on CVD were observed at lower methylmercury levels
than that associated with neurotoxicity. These studies suggested that methylmercury may be
a risk factor for CVD; however, results were inconsistent.
OTHER HEALTH EFFECTS
Chronic exposure animal studies assessed the carcinogenic potential of
methylmercury. Many studies did not achieve the maximum tolerated dose (MTD), and
failed to demonstrate carcinogenic effects. Additionally, genotoxicity studies were
inconclusive correlating genetic damage to methylmercury exposure in humans. Several
studies that reported higher rates of chromosomal aberrations after mercury exposure may be
caused by confounding factors that affected chromosomal aberrations, such as age or
influence of other toxicants (National Research Council Committee on the Toxicological
Effects of Methylmercury, 2000). However, three studies exhibited a dose-response effect
23
after oral exposure to methylmercury. Also, the International Agency on Research of Cancer
(IARC) categorized methylmercury as a Group 2B compound (possibly carcinogenic to
humans), based on sufficient evidence in experimental animals exposed to methylmercuric
chloride. A study by Mitsumori, Maita, Saito, Tsuda, and Shirasu (1981) fed 120 ICR mice
(60 males, 60 females), diets containing methylmercury at doses of 0, 1.6, or 3.1 mg/kg/day
for 78 weeks. At the highest dose, majority of the mice died by week 26. Majority of males
in the low-dose group had significantly greater onsets of renal epithelial adenocarcinomas
and renal adenomas, compared to controls. Renal tumors were not observed in females.
Hirano, Mitsumori, Maita, and Shirasu (1986) conducted a study as a follow-up to Mitsumori
et al. (1981) study. ICR mice (60 per sex) were fed methylmercury chloride at lower doses
for 104 weeks. Males were fed the following doses: 0, 0.03, 0.15, or 0.73 mg/kg per day.
Females were fed the following doses: 0, 0.02, 0.11, or 0.6 mg/kg per day. Males
demonstrated increased incidence of renal epithelial tumors. No renal tumors were exhibited
in females. Another study by Mitsumori, Hirano, Ueda, Maita, and Shirasu (1990) also
resulted in an increased incidence of renal tumors among male B6C3F mice after chronic
methylmercury exposure. Males were fed 0, 0.03, 0.14, or 0.69 mg/kg per day, and females
0, 0.03, 0.13, or 0.60 mg/kg per day of methylmercury chloride for 104 weeks. The MTD
was reached for males in the mid-dose range, and high-dose range for females. Chronic
exposure studies exhibited that methylmercury exposure increased renal tumors in male
mice, but not for female mice. Also, tumors were secondary to cell damage and repair, and
were primarily observed in doses causing kidney toxicity.
Epidemiological studies have not demonstrated an association between
methylmercury exposure and cancer rates. However, two studies from Janicki, Dobrowski,
24
and Krasnicki (1987) and Kinjo et al. (1996) show an association between methylmercury
exposure and acute leukemia. A case-control study conducted by Janicki et al. (1987),
collected 47 hair samples from leukemia patients and found a statistically significant increase
in Hg concentrations compared to 52 healthy subjects. Data was also analyzed for specific
types of leukemia, and patients with acute leukemia had higher mercury hair concentrations
compared to other types of leukemia. Kinjo et al. (1996) compared cancer death rates
between Minamata Disease (MD) survivors to a control population without the disease. Both
populations consumed fish daily and lived in the same region of Japan. Based on five
observed deaths, MD survivors were eight times more likely than the control population to
die from leukemia.
Despite having no epidemiological data on methylmercury effects on immune
function, the immune system appeared sensitive to methylmercury. Animal studies
demonstrated that methylmercury affected immune-cell ratios, cellular responses, and
immune system development. Through oral exposure to methylmercury, Ilback (1991) found
altered ratios of lymphocyte subpopulations, increased lymphoproliferation due to B- and Tcell mitogens, and lowered natural killer cell activity in mice. Female Balb/c mice orally
exposed to 3.9 ppm methylmercury (equivalent to 0.5 mg/kg per day for 12 weeks) exhibited
decreased thymus weight and cell volume. Lymphoproliferation due to B- and T-cell
mitogens increased, and natural killer cell activity decreased. Red-blood cell counts were
also higher in exposed vs. unexposed mice, however, white blood cell counts were
unaffected.
Mice exposed to 0, 3, or 10 ppm for 4 weeks exhibited altered splenocyte and
thymocyte subpopulations, and caused a dose-dependent decrease in splenocyte glutathione
25
concentrations and mitogen-stimulated calcium flux (Thompson, Roellich, Grossmann,
Gilbert, & Kavanagh, 1998).
Reproductive human studies were not identified following exposure to
methylmercury. However, animal studies reported abortion and decreased litter size. Preand post-implantation losses were exhibited in rats, mice, guinea pigs, and monkeys. Fuyuta,
Fujimoto, and Hirata (1978) fed an oral dose of 7.5 mg/kg methylmercury to rats during
gestational days 7-14. Increased fetal deaths and incidence of malformations were observed.
A dose of 5 mg/kg was also administered and associated with increased incidence of
malformations and reduced fetal weight. Lee and Han (1995) gave oral doses of
methylmercuric chloride at 10, 20, and 30 mg/kg to Fischer 344 rats on gestation day 7.
Fetal survival decreased by 19.1%, 41.4%, and 91.1% for each dose level, respectively. In
addition, implantation sites in each dose group decreased by 5.9%, 13.7%, and 22.5%,
respectively.
Female Macaca fascicularis monkeys were exposed to methylmercury hydroxide at
50, 70, or 90 ug/kg per day for four months (Burbacher, Mohamed, & Mottett, 1988).
Reproductive problems, such as decreased conception rates, early abortions, and stillbirths
were observed. Number of conceptions decreased as dose concentration increased.
Conceptions for each dose were 93% for controls, 81% for a dose of 50 ug/kg per day, 71%
for a dose of 70 ug/kg per day, and 57% for a dose of 90 ug/kg per day. Reproductive effects
were observed at a maternal blood concentration greater than 1.5 ppm. Following prolonged
exposure of ½ to over 1 year, maternal toxicity was observed in 70 and 90 ug/kg per day for
dosings of greater than 2 ppm. Maternal toxicity was not observed at an exposure
concentration of 50 ug/kg per day.
26
Paternal exposure to methylmercury and its effects on reproduction were also studied.
Khera (1973) exposed male rats with 5 to 7 doses of 1, 2.5, or 5 mg/kg of methylmercury
chloride daily, prior to mating with unexposed females. A dose-related increase in postimplantation losses and reduced litter size was observed. Mohamed, Burbacher, and Mottet
(1987) examined testicular function after male Macaca fascicularis monkeys were fed
methylmercury hydroxide at 50 or 70 ug/kg per day for 20 weeks. There was no decrease in
sperm count; however, a decrease in the percentage of motile sperm, reduction in sperm
speed, and increase in abnormal sperm were observed. Sperm motility returned to normal
after the removal of methylmercury exposure, but sperm morphology remained abnormal.
Renal toxicity was observed through inhalation exposure to metallic mercury.
However, organic mercury was rarely reported in human studies to induce renal toxicity.
Cases confirming renal toxicity occurred following neurological symptoms. Animal studies
demonstrated methylmercury induced renal toxicity. Fibrosis in the renal cortex of female
rats was observed following 12 weeks of methylmercury exposure at a dose of 0.84 mg/kg
per day (Magos & Butler, 1972). Rats that were fed with methylmercury chloride at 0.1
mg/kg per day for 2 years had increased kidney weights and decreased proximal convoluted
tubule enzymes (Verschuuren et al., 1976). Continual exposure to methylmercury to rats and
mice resulted in nephrosis (Mitsumori et al., 1990; Solecki, Hothorn, Holzweissig, &
Heinrich, 1991). Proximal tubule degeneration was observed in mice given 0.11 mg/kg per
day of methylmercury chloride for 2 years (Hirano et al., 1986). Similarly, the Mitsumori et
al. (1990) study also exhibited epithelial degeneration and regeneration of proximal tubules
and interstitial fibrosis in methylmercury fed male and female mice after 2 years of exposure
to methylmercury at a dose of 0.2 mg/kg per day. Lastly, Yasutake, Hirayama, and Inouye
27
(1991) demonstrated that a single oral dose (16 mg/kg) hindered renal function due to
increased plasma creatinine concentrations and swelling of tubular epithelium with
exfoliation of cells into the tubular lumen.
Methylmercury is highly toxic since exposure can affect multiple organ systems
throughout the lifespan of a person. Studies on carcinogenicity are inconclusive; however,
renal tumors may be evident at doses above the MTD of methylmercury. Exposure to
methylmercury could increase human susceptibility to autoimmune disorders and infectious
diseases by damaging the immune system. Although reproductive effects are not clearly
elucidated, studies on nonhuman primates show that methylmercury causes reproductive
defects. Primary targets for methylmercury toxicity are the cardiovascular and neurological
systems (National Research Council Committee on the Toxicological Effects of
Methylmercury, 2000).
Animal studies suggested that hematological changes, such as anemia and clotting
disorders, ensued following mercury exposure. Munro, Nera, Charbonneau, Junkins, and
Zawidzka (1980) exposed rats to 0.25 mg/kg of mercury every day, for 26 days. Males
exhibited decreased hematocrit and hemoglobin production, as well as neurotoxicity and
increased mortality, compared to controls.
Blood coagulation following methylmercury exposure was measured in a 1989 study
by Kostka, Michalska, Krajewska, and Wierzbicki. A single dose of methylmercury chloride
at 17.9 mg/kg per day or five consecutive dosings at 8 mg/kg per day were administered to
rats. Blood coagulation was measured 1, 3, and 7 days after the single dosing, or 24 hours
after five consecutive days of dosing. In both dosing groups, a reduction in clotting time and
an increase of fibrinogen concentrations in plasma were observed.
28
ORAL REFERENCE DOSE (RFD)
Threshold levels are defined as the exposure level below the dose in which no adverse
symptoms are observed; it is a safe exposure concentration of a chemical. Acceptable levels
are calculated using the threshold levels with a safety factor included. A safety factor is
designed to allow rare sensitivities which are not factored during the experimental process
(Junghans, 1983).
Three supporting epidemiological studies were applied for the derivation of the RfD.
The nervous system, specifically developmental neuropsychological impairment, was the
most sensitive target organ to use as an endpoint for calculating the RfD. EPA used
longitudinal, developmental studies that were conducted in the Seychelles Islands, the Faroe
Islands, and New Zealand. The Seychelles study was composed of 779 mother-infant pairs
from a population that consume fish. Infants were followed from birth to 5.5 years of age,
and neuropsychological endpoints were assessed at various ages. The Faroe Islands study
was a longitudinal study that consisted of 900 mother-infant pairs. Mercury from cord blood
and maternal hair was measured. In addition, children at the age of seven were tested on
various behavioral tasks. The New Zealand study was a prospective study in which 38
children with maternal hair concentrations that were greater than 6 ppm during pregnancy
were matched with children whose mothers had lower mercury hair levels. The Faroe
Islands and New Zealand studies obtained dose-related effects for various
neuropsychological endpoints (U.S. Environmental Protection Agency [USEPA], 1987).
The BMD was used to quantify the dose-effect relationship. Using cord blood data at
steady state from the Faroese study, the one-compartment model was applied to convert dose
to RfD. The concentration in blood corresponded to the BMDL05. An absorption factor of
0.95 was employed since methylmercury uptake was reported to be 95%. Swartout and Rice
29
(2000) used data from a cohort of 20 pregnant Nigerian women and adjusted for five liters of
blood with a log-triangular distribution. This blood volume was applied for V. The
elimination constant as cited by Stern (1997) was 0.014 days-1. As recommended in the EPA
Methodology, the body weight for pregnant women used in the dose conversion was 67 kg.
A modifying factor was not included (MF=1) since no issues were identified. Two factors
were considered in the intraspecies uncertainty factor of 10, which accounted for a factor of 3
for interindividual toxicokinetic variability in ingested dose estimation and an additional
factor of 3 for pharmacodynamic variability and uncertainty. Dose conversion using the onecompartment model was:
d = BMDL05 ug/L x 0.014 days-1 x 5L x 10 x 1
0.95 x 0.059 x 67 kg
producing an RfD of 0.1 ug/kg/day (USEPA, 1987).
HAIR AS A BIOMARKER OF EXPOSURE
Biomarkers indicate signaling events in biological systems or samples, and are
classified as markers of exposure, effect, and susceptibility. Blood and urine samples are
biomarkers of exposure. A biomarker of exposure is the toxicant or its metabolite or the
product of an interaction between the toxicant and a target cell. Several factors are
confounders for the utilization and interpretation of biomarkers of exposure. The body
burden of the contaminant can be the cause from exposure to more than one source. The
measured toxicant may be a metabolite from another type of toxicant (U.S. Department of
Health and Human Services, 1999).
In order to assess the absorbed dose or concentrations within the brain, levels of total
mercury in blood or scalp hair are selected as biomarkers. Blood gives an estimate of recent
exposure of one to two half-lives, while hair shows the average exposure over the growth
30
period of the hair segment. Hair is an excellent way of determining methylmercury exposure
because it is easy to collect, has high methylmercury content so analysis is easy to perform
since hair contains 250-300 times more methylmercury than blood, and is a noninvasive
collection protocol. Additionally, hair is stable for storage, easily accessible, and each
centimeter of hair represents a month of methylmercury exposure. A time-line of previous
mercury exposures can be established depending on hair length, and is a biomarker of longterm exposure. When mercury is incorporated within hair, it remains unchanged.
Methylmercury hair concentrations give reliable information on the internal dose when
methylmercury is consumed within the diet, since majority of the concentration found in hair
is in the form of methylmercury. In addition, a collection of studies have established a strong
correlation between the amount of fish consumed, along with mercury concentrations in the
fish and hair (U.S. Department of Health and Human Services, 1999; Zareba, Cernichiari,
Goldsmith, & Clarkson, 2007).
A 1993 study conducted by Suzuki et al., determined that mercury concentrations in
hair correlated with body-burden amounts in target organs from analyzing 46 human
autopsies in Tokyo, Japan. He concluded that mercury concentrations in hair were
significantly correlated with mercury levels in the cerebrum, cerebellum, heart, spleen, liver,
kidney cortex, and kidney medulla. Cernichiari et al. (1995) also confirmed a correlation
between hair mercury concentrations and concentrations present within the brain. Maternal
hair, maternal blood, fetal blood, and fetal brain levels were compared among the Seychelles
Islands cohort. Total mercury concentrations within infant brains were highly correlated
with maternal hair levels among four measurements. Correlations existed between maternal
hair to maternal blood (r=0.82), and infant brain concentrations (r=0.6-0.8). Additionally,
31
maternal blood concentrations correlated to infant blood (r=0.65); infant blood
concentrations correlated to infant brain (r=0.4-0.8).
Cosmetic hair treatments have been considered to remove mercury from hair.
However, a comprehensive study by McDowell et al. (2004) assessed mercury levels in
human hair among U.S. children and women of childbearing age and found no effects from
cosmetic treatment on mercury hair levels. Thirty-seven percent of women reported using
hair treatment, and the geometric mean for total mercury levels in hair were similar between
treated and untreated hair groups. Results were also consistent with a 1974 study by
Giovanoli-Jakubczak, Greenwood, Smith, and Clarkson that determined total and inorganic
mercury in hair through flameless atomic absorption, and methylmercury through
chromatography. The authors indicated that there were no appreciable differences in total
and inorganic mercury before and after washing, dyeing, or bleaching of hair.
The deposition of methylmercury into hair is purported to occur through a large
amino acid carrier, methylmercury-cysteine. Amino acids are a major substrate and in high
demand for proteins, especially keratin for hair growth, since they play a role in protein
synthesis. Methylmercury concentrations in hair are a measure of the methylmercurycysteine complex within the plasma, which is the same complex that is transported into the
brain. Methylmercury is directly integrated into the hair during the growth phase (Clarkson
& Magos, 2006).
According to Cernichiari et al. (1995), mercury in hair following methylmercury
exposure consists of 80% to 98% methylmercury, with the remainder in the form of
inorganic mercury. Inorganic mercury poorly accumulates in hair, and suggests that the
inorganic fraction is due to the conversion of methyl to inorganic mercury within the hair
32
follicle, which is consistent to a report by Lindberg, Bjornberg, Vahter, and Berglund (2004),
indicating that total mercury concentrations within hair reflects organic mercury, and not
inorganic mercury exposure.
ANALYTICAL METHODS FOR ANALYZING
METHYLMERCURY
Analyzing biological and environmental samples are difficult due to the presence of
inorganic and organic species of metals. Therefore, when analyzing for mercury within a
sample, all forms are reduced to the elemental state. Additionally, mercury is also relatively
volatile and sample loss can occur during sample preparation and analysis. Due to
inadvertent cross-contamination, glass or Teflon containers are cleaned and acid-leached
prior to trace-level analysis (U.S. Department of Health and Human Services, 1999). The
analytical process of determining methylmercury in solid samples consists of extraction,
separation and detection. The appropriate instrumentation is dependent upon the nature of
the sample and mercury concentration (Torres, Frescura, & Curtius, 2009).
Analytical methods used to determine methylmercury in different matrices include
cold vapor atomic absorption spectrophotometer (CVAAS), gas chromatography (GC),
neutron-activation analysis (NAA), atomic fluorescence spectrometry (AFS), or directmercury analyzer (DMA) (U.S. Department of Health and Human Services, 1999). CVAAS
is the most common method since it is highly sensitive and easy to operate. A reducing agent
is necessary to convert the oxidation state of mercury 2+ to elemental mercury for detection
(Torres et al., 2009). GC is utilized to selectively measure between mercury species, and is
widely applied to quantify methylmercury in fish tissues. NAA is the most accurate and
sensitive method and used as the reference method. Total and inorganic mercury is analyzed,
and organic method concentrations are obtained from the difference (WHO, 1990). AFS
33
offers a suitable alternative to CVAAS due to its detection within the part per trillion (ppt)
range, along with its excellent precision and recovery (U.S. Department of Health and
Human Services, 1999). DMA is an alternative to classical analytical methods. It requires
no sample preparation and can deliver results in six minutes per sample, which is
significantly efficient than traditional chemistry techniques (USEPA, 2007). For purposes of
this research, the Milestone Direct Mercury Analyzer (DMA) is the instrument of choice for
the analysis of mercury in hair.
As referenced by USEPA Method 7473, which was published in 2007, analytical
procedures for the DMA do not require the conversion of mercury to mercuric ions. Solid
and liquid sample matrices are analyzed without preliminary acid digestion or sample
preparation. It incorporates thermal decomposition, catalytic conversion, amalgamation, and
atomic absorption spectrophotometry. The sample is first dried and thermally decomposed
through controlled heating stages, then is introduced into a quartz tube. A continuous flow of
oxygen carries the decomposed products through a hot catalyst bed in order to trap halogens,
nitrogen, and sulfur oxides. Mercury species are also reduced to elemental mercury.
Reaction gases then carry the byproducts to a gold amalgamator where mercury is selectively
trapped, while non-mercury vapors and other decomposed products are flushed from the
system. The amalgamator is heated in order to release the trapped mercury through a single
beam, fixed wavelength atomic absorption spectrophotometer of 253.7 nm. A study
conducted by Milestone comparing fish and biological sample results analyzed through ICPMS/CVAAS and DMA determined that both results were in excellent agreement with each
other.
34
CHAPTER 3
MATERIALS AND METHODS
SUBJECTS
A cross sectional study was employed to assess mercury exposure among locals of
Oahu, Hawaii in order to determine risk from fish consumption. A total of 110 subjects (57
men, 53 women) were selected from each of the five regions of Oahu– Central Oahu, the
Leeward Coast, Southeast Oahu, Windward Oahu, and the North Shore. In order to give an
overall representation of the entire Oahu community, an average of twenty people were
recruited from public locations throughout each of the five districts from December 2010 –
January 2011.
Participants were questioned during a screening process prior to survey and hair
collection. Subject’s eligibility was based upon the following criteria: spoke English, older
than 18 years of age, non-pregnant women, and resided in Hawaii. This study was assessing
methylmercury toxicity primarily caused by fish ingestion within vulnerable populations,
such as residents of Oahu, Hawaii. Therefore, visitors of Hawaii were excluded from the
study. Consent forms were given to participants and debriefed with complete information
regarding the study. The study was conducted after participant agreed to terms and signed
the consent form.
The protocol was reviewed by San Diego State University’s Institutional Review
Board in order to protect participant’s rights and welfare according to federal regulations and
SDSU’s Federal Wide Assurance. IRB approved the protocol as minimal risk since the study
involved a prospective collection of hair samples from participants through noninvasive
35
techniques, and the surveys did not disclose sensitive and private information. Participation
was voluntary. If a subject refused to participate, any attempt at recruitment will end. If they
gave permission to become a subject, the appropriate information was presented prior to
continuing the interview. This included introducing the investigator, thorough explanation of
the study being conducted, assuring their anonymity, allowing them to read the informed
consent document, and answering any questions and concerns from the participant. Before
signing the consent form, participants were reassured that they can withdraw from the
experiment at any time without any repercussions. Participant information was kept
confidential by storing paperwork and samples in a locked file cabinet belonging to the
investigator. Subject’s identity was identified through numerics matching the hair sample to
survey. Consent forms, questionnaires, surveys, and data were retained for a minimum of
three years.
INDIVIDUAL DIETARY SURVEYS
A questionnaire was presented to individuals present at public areas throughout Oahu,
Hawaii (see Appendix). The questionnaire included demographic information about the
participant, their fish consumption habits, and their general knowledge of methylmercury.
Demographic questions included gender, ethnicity, age, education, income, city of residency,
length of residency, occupation, as well as other exposures to mercury, not due to
consumption. Fish consumption questions included whether fish was caught or purchased,
type of fish consumed, how many times a week fish was consumed, how did participant
normally prepare the fish, what parts of fish was consumed, and approximate portion size
eaten (in lb.). General questions on knowledge of methylmercury included any awareness of
fish consumption advisories, medium presenting knowledge on fish consumption awareness,
36
if participant was concerned about methylmercury toxicity, would consumption decrease if
participant knew fish was contaminated with methylmercury, if participant felt fully
informed about risk and benefit of fish consumption, and suggestions on public awareness.
SUBJECTS AND BIOMARKER ASSESSMENT
Untreated hair samples were cut from the occipital region, one-centimeter from the
scalp with clean, unused razor blades, and placed in a sealable polyethylene bag with the
appropriate identifier to match the survey to the appropriate hair sample. Samples were kept
at room temperature pending analysis, and were analyzed within 28-days of sample
collection, since many mercury species can volatilize.
Reagents were interference free by using high purity acetone and double distilled
water. Supplies and sample containers were also washed with detergents followed by an
acid-wash to remove metal residues. Nickel sample boats were also autoclaved at 650 oC for
eight hours following general cleaning in order to remove any residual mercury present
following analysis. As applied by Kwaansa-Ansah, Basu, and Nriagu (2010), and Carneiro
et al. (2011), the following cleaning and drying procedures were utilized to prepare hair
samples for analysis.
Approximately 0.5 grams of the hair sample were removed from polyethylene bags
using clean forceps. Small plastic weigh boats were labeled with designations from
polyethylene bag. Double distilled (DDI) water was added to weigh boat to remove
impurities accumulated on the outer hair follicle, and placed on a mechanical rocker
(Scientific Industries Inc., New York). DDI water was then decanted from the weigh boat
and HPLC grade acetone (CAS #67-64-1, > 99.9% purity, lot # 072500) from Fisher
Scientific (Pittsburg, PA) was added. After the acetone was decanted, hair samples air dried
37
then was placed on a Fisher Scientific hotblock (Pittsburg, PA). Hair samples were dried
until constant weight.
ANALYTICAL PROCEDURE
Methylmercury was analyzed through the Milestone Direct Mercury Analyzer
(DMA) (Milestone, Shelton, CT). The theory of its operation utilized thermal
decomposition, amalgamation, and atomic spectrophotometry which followed EPA method
7473 from the "Test Methods for Evaluating Solid Waste, Physical/Chemical Methods" (SW846). Since samples were thermally decomposed, acids were not used in sample preparation
and therefore, hazardous wastes were not generated.
The operation of the DMA was automated. The dried hair sample was placed into a
nickel boat and weighed using a Sartorius balance (Sartorius, Germany), and the weight was
recorded into the DMA. Then the sample was placed into the autosampler, and a robotic arm
removed the sample and transferred it into the oxygenated quartz dry and decomposition
furnace where the sample thermally decomposed at 650oC. Within the furnace, the sample
was dried, then thermally and chemically decomposed in order to free mercury from the hair
sample. The decomposed products were carried by a high-purity carrier gas, oxygen, into the
metal oxide catalyst furnace. The catalyst reduced all mercury species to elemental mercury
at 615 oC. The sample vapor moved into the amalgamator where mercury was selectively
collected onto a gold amalgamator. When the remaining byproducts from the decomposed
sample passed through the amalgamator with the help of the carrier gas flow, the
amalgamator was heated to 170 oC to release the mercury vapor. The mercury vapor then
moved through absorbance cells positioned into the light path of a single-wavelength
spectrophotometer into dual cell cuvettes, and the absorbance (peak height) of mercury was
38
measured at 253.7 nm. A heating unit maintained the spectrophotometer and cuvette at 125
o
C to prevent condensation and to minimize mercury contamination due to carry-over from
prior sample analysis. Mercury concentration results were recorded as micrograms (ug) of
mercury per gram (g) of hair. Dual cell cuvettes analyzed hair samples as low as single digit
parts per billion (ppb) to high level parts per million (ppm) without having to dilute the
sample. One sample cuvette specifically measured the hair sample at a low range of 0-35 ng,
and the second cuvette measured at a high range of 35-500 ng. The DMA was highly
sensitive with an instrument detection limit of 0.005 ng (Environmental Protection Agency,
SW-846 Method 7473).
CALIBRATION AND STANDARDIZATION
A calibration curve was created and stored prior to first time use of the DMA. Unless
any major component within the DMA had to be changed, the stored calibration curve was
utilized for subsequent analyses. However, a Certified Reference Material (CRM) was
analyzed before and after analyzing hair samples in order to ensure acceptable mercury
recovery with the calibration curve and method employed. Dogfish Liver Certified
Reference Material for Trace Metals (DOLT-4) (National Research Council, Canada), was
utilized as a calibration check, and has a mass fraction value of 2.58 + 0.22 mg/kg. The
DMA needed to be recalibrated if results for the DOLT-4 were 10% higher or 10% lower
than the expected value. The equation used to calculate percent recovery (%R) was:
%R = (observed Hg concentration of DOLT-4 /
expected concentration of DOLT-4) x 100%
Recoveries were acceptable and ranged from 93-108%.
39
The calibration curve was created by diluting 1000 ppm Hg stock standard. The
stock standard was checked for expiration prior to application. The solvent used to dilute the
stock standard was 2% diluted hydrochloric acid. The following equation was used to
calculate working standards:
Working standard (ppm) = Stock solution (ppm) x
(Volume of stock solution (mL) / Total volume (mL))
A working standard of 5 ppm was created by pipetting 500 uL from the 1,000 ppm
stock standard, and diluting up to 100 mL. Low level working standards were stored in
brown glass bottles and stored at 5-10 oC. Standards were minimally exposed to air to
prevent decomposition. A 100 ppb working standard was created by taking 2 mL from a 5
ppm working standard, and diluting it to 100 mL. From the 100 ppb working standard, a
calibration curve was fashioned using the equation:
Hg (ng) = Hg concentration in the sample (ppm) x
Sample weight (mg)
The following working standard range was created by pipetting the following
volumes from the 100 ppb working standard into a quartz boat (Table 2). Standards were
analyzed from low concentration to high concentration to avoid carry-over. The sample boat
was at room temperature before introducing the standard into the boat.
Table 2. Working Hg Standard Concentrations Used for 5-Point
Calibration Curve
Working Hg standard concentration
Volume to pipet for desired ng
0.5 ng
5 uL of 100 ppb
1 ng
10 uL of 100 ppb
5 ng
50 uL of 100 ppb
10 ng
100 uL of 100 ppb
20 ng
200 uL of 100 ppb
40
A regression equation was created from a 5-point calibration curve in order to
determine mercury concentrations within the hair samples. A least-squares parabola was the
line of best fit, with the equation:
A= (-0.0068) + (0.0571 x Hg) – (0.0008 x Hg2)
The equation had a correlation coefficient value of 0.9998.
For quality control purposes, a calibrated 20 mg. Cahn weight was weighed using the
Sartorius prior to weighing hair samples. Percent recovery was calculated using the
following equation:
%R = (observed weight / expected weight) x 100%
Acceptance criteria were +/- 10% of the expected value. Recoveries were acceptable
and ranged between 98-102%. Empty nickel boats were analyzed until a peak height of <
0.0010 was achieved since negligible amounts of mercury contamination can significantly
affect low level mercury results. A method blank with mercury-absent all purpose flour was
also analyzed prior to the sequence run in order to assure that samples were free from
mercury contamination that could be introduced within the system and samples. The
acceptance criteria for method blanks were < 0.0010 peak height. In order to minimize
deviations during the sequence run, a coefficient of variation was conducted by analyzing a
CRM before and after every sequence run. The equation used to calculate the coefficient of
variation (CV) was:
CV = standard deviation x 100%
Acceptance criteria were +/- 10% between both CRM measurements. Coefficient of
variation ranged between 1.0-9.4%. A low level laboratory control standard (LCS) was
analyzed prior to analyzing hair samples in order to ensure that low concentrations of
41
mercury can be detected with the DMA. DOLT-4 was mixed with all purpose flour to dilute
the mercury content by 75% of the total sample weight. The equation used to calculate
percent recovery was:
%R = (observed Hg concentration /
expected concentration) x 100%
Acceptance criteria were +/- 10% of the expected value. Recoveries were acceptable
and ranged from 93-108%.
With every batch run of approximately ten hair samples, a duplicate hair sample was
analyzed to determine the precision of results. Relative percent difference (RPD) was
calculated as:
RPD = (|difference of original and duplicate concentration| /
average of original and duplicate concentration) x 100%
Acceptance criteria was set as < 10% between the original and duplicate sample.
RPD’s were acceptable and ranged from 0.95-5.2%.
CALCULATION OF RISK INDICES
Risk indices were calculated with the following equations, where daily dietary intake
(ug meHg/kg body weight/d) was the average daily dose of MeHg and RfD was the tolerable
daily intake (ug/kg/d) according to the denomination given by USEPA. In order to convert
hair mercury concentrations to average daily dose of MeHg, a one-compartmental model was
applied assuming that biological parameters were at steady state. According to USEPA
(1997), a ratio of 250:1 converted hair mercury concentrations (mg of Hg/kg of hair) to blood
mercury concentrations (mg of Hg/L of blood) prior to calculating the daily dietary intake.
Hazard Index (HI) should be 1 or less to ensure no risk for populations.
42
d=CxbxV
A x f x bw
•
d = daily dietary intake (micrograms of MeHg per kilogram of body weight per day)
•
C = Concentration in blood (ug/L)
•
b = elimination constant (0.014 days-1)
•
V = volume of blood in the body (5 L)
•
A = absorption factor (expressed as unitless decimal fraction of 0.95)
•
f = fraction of daily intake taken up by blood (unitless, 0.05)
•
bw = body-weight default of 60 kg for an adult female, and 70 kg for an adult male
Hazard Index = Daily Intake Dose
RfD
STATISTICAL ANALYSIS
Mercury hair concentrations were matched to participant’s survey. Survey answers
were designated with a numerical code to find associations between mercury in hair along
with variables that may contribute to higher mercury exposures. Statistical analyses were
performed using SPSS (version 13.0 SPSS, Inc). P-values of 0.05, 0.01, and 0.001 were used
for statistical significance. Median, minimum, maximum, 75th, and 95th percentiles were
calculated and reported for data analysis. Data were not normally distributed; therefore, the
Mann-Whitney nonparametric test analyzed for significance of mercury hair levels between
variables for each category. A non-parametric equivalent of ANOVA, the Kruskal-Wallis ksample test, determined the significance of mercury hair levels for ordinal or nominal
variables.
43
CHAPTER 4
RESULTS
DEMOGRAPHIC FACTORS IN RELATION TO HAIR
MERCURY LEVELS
Table 3 depicts the median, 75th and 95th percentiles, minimum and maximum hair
mercury levels among men and women, which were further subcategorized to 45 years old
and younger, and 46 years old and older. The median mercury hair level for all 109 men and
women was 0.97 ug/g, the minimum mercury hair level was 0.02 ug/g and the maximum was
23.34 ug/g. Ninety-five percent of the population had mercury hair levels within 5.45 ug/g,
and seventy-five percent of the population within 2.02 ug/g. Men had a median mercury hair
level of 1.19 ug/g, with the minimum and maximum as 0.23 ug/g and 7.04 ug/g, respectively.
The 95th percentile was 5.75, and the 75th percentile was 2.74 ug/g. Younger men (< 45
years) had a median mercury hair level of 0.97 ug/g, the minimum mercury hair level was
0.23 ug/g and the maximum was 7.04 ug/g. The 95th percentile was 5.93 ug/g, and the 75th
percentile was 2.23 ug/g. Older men (> 46 years) had a median mercury hair level of 2.00
ug/g, the minimum mercury hair level was 0.53 ug/g and the maximum was 6.66 ug/g. The
95th percentile was 6.49 ug/g, and the 75th percentile was 2.92 ug/g. Women had a median
hair mercury level of 0.69 ug/g, with the minimum and maximum as 0.02 ug/g and 23.34
ug/g, respectively. The 95th percentile was 3.03 ug/g, and the 75th percentile was 1.58 ug/g.
Younger women had a median hair mercury level of 0.57 ug/g, with the minimum and
maximum as 0.02 ug/g and 2.40 ug/g, respectively. The 95th percentile was 2.21 ug/g, and
the 75th percentile was 1.31 ug/g. Older women had a median hair mercury level of
44
Table 3. Median, 75th and 95th Percentiles, Minimum and Maximum Hair Mercury
Levels (ug/g) of Hair Sampled on December 2010 to January 2011 from Residents
Living Throughout Oahu, Hawaii. Hair Mercury Levels are Categorized
According to Gender, and Subcategorized by Age (Younger than, and Including 45
Years of Age; Older than, and Including 46 Years of Age)
N
Median
109
0.97
57
1.19 *
All ages
< 45 years
35
0.97
> 46 years
22
2.00
52
0.69
Women
All ages
< 45 years
39
0.57
> 46 years
13
1.22
* men significantly higher than women, p < 0.05
All subjects
Men
75th
2.02
2.74
2.23
2.92
1.58
1.31
2.05
95th
5.45
5.75
5.93
6.49
3.03
2.21
N/A
Min
0.02
0.23
0.23
0.53
0.02
0.02
0.05
Max
23.34
7.04
7.04
6.66
23.34
2.40
23.34
1.22 ug/g, with the minimum and maximum as 0.05 ug/g and 23.34 ug/g, respectively. The
75th percentile was 2.05 ug/g. Older men and women have higher hair mercury in
comparison to their younger counterparts.
Table 4 summarizes demographic variables as reported by residents, in relation to the
median, minimum, and maximum hair levels. Median hair levels were reported in this study
since hair mercury concentrations were not normally distributed. Significant variables in
univariate analysis included gender and region of residency (p < 0.05), as well as years of
residency (p < 0.001). Men exhibited increased hair mercury concentrations compared to
women. The median hair mercury concentrations among men were 1.19 microgram of
mercury per gram of hair (ug/g); median hair mercury concentrations for women were 0.69
ug/g. Residents who lived along the North/West regions of Oahu had significantly higher
median hair mercury levels of 1.13 ug/g, in comparison to median hair mercury
concentrations of 0.73 ug/g from residents who resided in other regions (south, windward,
east, and central) of Oahu. Subjects who lived on Oahu for less than ten years, had
significantly lower median mercury hair levels of 0.42 ug/g, compared to median hair
mercury levels of 1.13 ug/g from residents who lived on Oahu for more than ten years. Other
45
Table 4. Demographic Variables as Reported by Residents in Relation to Median,
Minimum, and Maximum Hair Mercury Levels
VARIABLE
N
18-25
34
26-35
24
36-45
16
46-55
26
> 56
10
Male
57
Female
53
MEDIAN HAIR Hg (ug/g)
(min – max)
Age, years
0.66
(0.02 – 7.04)
0.95
(0.06 – 5.65)
0.81
(0.10 – 3.26)
1.82
(0.05 – 23.34)
1.35
(0.33 – 6.66)
Gender
1.19
(0.23 – 7.04)
0.69
(0.02 – 23.34)
*
1.13
(0.02 – 7.04)
0.73
(0.05 – 23.34)
*
North/West Vs. Other Regions
North/West
33
All Others
74
Residency, years (< 1 – 10 Vs. 11 – 40)
< 1 – 10
15
11 – 40
89
Asian
39
Hispanic/Latino
5
Native Hawaiian/Pacific Islander
29
White/Caucasian
17
Mixed Race
18
0.42
(0.06 – 1.84)
1.13
(0.02 – 23.34)
***
Ethnicity
1.11
(0.05 – 23.34)
0.49
(0.10 – 1.01)
0.97
(0.02 – 7.04)
0.95
(0.06 – 5.65)
0.95
(0.16 – 2.57)
(table continues)
46
Table 4. (continued)
VARIABLE
N
Grade School
2
High School
34
Associates/Trade Degree
15
Current College
20
College Degree
28
Graduate Degree
11
MEDIAN HAIR Hg (ug/g)
(min – max)
Education
2.08
(0.98 – 3.18)
0.99
(0.05 – 23.34)
0.73
(0.02 – 2.07)
0.77
(0.14 – 2.57)
1.00
(0.13 – 5.40)
1.13
(0.06 – 6.66)
Income
< 25000
23
25000 – 39999
13
40000 – 49999
11
50000 – 75000
20
> 75000
23
No Response
19
Yes
82
No
28
0.75
(0.06 – 23.34)
1.11
(0.14 – 7.04)
0.95
(0.02 – 3.27)
1.64
(0.10 – 5.40)
1.60
(0.05 – 6.66)
0.68
(0.31 – 4.51)
Fillings
* significantly higher, p < 0.05
***significantly higher, p < 0.001
1.01
(0.02 – 23.34)
0.71
(0.06 – 5.40)
47
demographic variables measured were age, ethnicity, education, income, and presence of
amalgam fillings but were not significantly associated with hair mercury levels.
FISH CONSUMPTION FACTORS IN RELATION TO HAIR
MERCURY LEVELS
Fish consumption was a significant contributing factor to increased hair mercury
concentrations. As depicted in Table 5, significant variables included frequency of fish
consumption (p < 0.01), portion size of fish meal (p < 0.001), frequency of fish consumption
in conjunction with portion size (p < 0.001, p < 0.05), amount of fish parts consumed (p <
0.001), and whether or not target organs were consumed (p < 0.001). Residents who
consumed fish at a frequency of less than 1 day/week had lower hair mercury levels (0.67
ug/g) compared to residents who consumed fish at a higher frequency of one to greater than
six days per week (1.21 ug/g). Those who consumed ¼ to ½ pound of fish with each meal
also had greater mercury concentrations within their hair (1.51 ug/g), compared to those who
consumed less than ¼ pounds of fish (0.62 ug/g). Residents who consumed one pound, and
greater than one pound of fish per meal each week had the highest hair mercury
concentrations, 1.73 ug/g and 1.08 ug/g, respectively, compared to residents who consumed a
half a pound of fish per meal or less, each week (0.92 ug/g and 0.62 ug/g). Those who
consumed greater than three fish parts (meat, skin, eyes, brain, head, heart) had hair mercury
levels of 1.60 ug/g compared to residents who ate one to two fish parts (0.74 ug/g).
Residents who consumed target organs (brain, head, heart) had increased hair mercury
concentrations (1.90 ug/g), in comparison to those who did not consume target organs (0.73
ug/g). Additionally, when assessing the consumption of different fish parts in relation to
median hair levels as portrayed in Table 6, those who consumed the brain, head, and heart
had the highest hair mercury concentrations (1.98 ug/g, 1.90 ug/g, and 2.01 ug/g,
48
Table 5. Fish Consumption Factors as Reported by Residents in Relation to
Median, Minimum, and Maximum Hair Mercury Levels
VARIABLE
N
Store
70
Fishing
14
Store and Fish
20
Other
4
< 1 day/week
51
MEDIAN HAIR Hg (ug/g)
(min – max)
Source
0.97
(0.05 – 23.34)
1.11
(0.14 – 5.40)
0.91
(0.02 – 5.65)
0.20
(0.06 – 1.36)
Frequency
1 - > 6 days/week
59
0.67
(0.02 – 23.34)
1.21
(0.10– 7.04)
**
Portion
< ¼ lb.
47
¼ - ½ lb.
51
> ½ lb.
12
0.62
(0.02 – 23.34)
1.51
(0.12 – 6.66)
1.51
(0.10 – 5.65)
***
Frequency and Portion
¼ lb./week
34
½ lb/week
24
1 lb/week
30
> 1 lb/week
22
0.62
(0.02 – 23.34)
0.92
(0.12 – 5.52)
1.73
(0.17 – 6.66)
1.08
(0.10 – 7.04)
***
*
Amount of Fish Parts Consumed-Grouped Together
<1–2
70
3–>6
39
0.74
(0.02 – 7.04)
1.60
(0.10 – 23.34)
***
If Target Organs (Brain, Head, Heart) Are Consumed
Yes
36
No
73
* significantly higher, p < 0.05
** significantly higher, p < 0.01
***significantly higher, p < 0.001
1.90
(0.10 – 23.34)
0.73
(0.02 – 7.04)
***
49
Table 6. Consumption of Different Fish Parts in Relation to Median, Minimum, and
Maximum Hair Levels as Reported by Residents
VARIABLE
n
MEDIAN HAIR Hg (ug/g)
(min – max)
Meat
Yes
105
No
3
Yes
65
No
43
Yes
27
No
82
Yes
22
No
87
Yes
10
No
98
Yes
36
No
73
0.98
(0.02 – 7.04)
0.13
(0.06 – 0.16)
**
1.19
(0.10 – 7.04)
0.69
(0.02 – 4.51)
**
1.60
(0.31 – 5.65)
0.83
(0.02 – 23.34)
*
1.98
(0.31 – 23.34)
0.82
(0.02 – 7.04)
**
Skin
Eyes
Brain
Heart
2.01
(0.10 – 5.17)
0.94
(0.02 – 23.34)
Head
* significantly higher, p < 0.05
** significantly higher, p < 0.01
***significantly higher, p < 0.001
1.90
(0.14 – 23.34)
0.73
(0.02 – 7.04)
***
50
respectively), compared to individual consumption of fish meat, skin, and eyes (0.98 ug/g,
1.19 ug/g, and 1.60 ug/g respectively). Table 7 portrayed that the type of method employed
to cook a fish played a role in mercury levels found in hair. Those who consumed raw fish
had significant hair mercury levels of 1.01 ug/g, compared to those who did not consume raw
fish (0.52 ug/g). Other cooking methods (boiled, oven, grilled, fried, barbecue) were not
contributing factors to increased hair mercury concentrations.
Table 7. Various Fish Cooking Methods as Reported by Residents in Relation to
Median, Minimum, and Maximum Hair Mercury Levels
VARIABLE
N
Yes
96
No
14
Yes
36
No
74
Yes
73
No
37
Yes
48
No
62
Yes
34
No
76
MEDIAN HAIR Hg (ug/g)
(min – max)
Raw
1.01
(0.02 – 23.34)
0.52
(0.06 – 1.69)
*
Oven
0.94
(0.02 – 7.04)
0.97
(0.05 – 23.34)
Grilled
1.05
(0.02 – 23.34)
0.75
(0.05 – 5.40)
Fried
1.09
(0.02 – 23.34)
0.92
(0.06 – 4.51)
Barbecue
1.09
(0.02 – 23.34)
0.92
(0.05 – 5.52)
* significantly higher, p < 0.05
HAZARD INDEX AND CARDIOTOXICITY
Hazard Index (HI) is exposure expressed as a noncarcinogenic risk and establishes
whether methylmercury contaminated fish endangers human health. It is the ratio of
exposure, in terms of dietary intake (ug/kg/day), and risk value (RfD) in order to estimate
51
risk. HI values exceeding one suggest that potential adverse health effects are present within
a population. As exposure increases above the reference dose, probability of adverse health
effects also increases.
Among 52 women residing on Oahu, the average HI value was 0.92; the average HI
value among 57 male residents was 1.61, which is above the safety criteria of one. Among
the entire population, 56% of the population produced HI values that were < 1, 21% were
approximately one, 15% were approximately two, 5% were approximately four, and 2% were
approximately five; HI values ranged from as low as 0.02, to as high as 5.93. 38% of women
were above the HI of one; 53% of men were above the HI of one.
The study population was separated into two groups, individuals who had HI’s > 1
and HI’s < 1 and significant variables were further scrutinized to observe trends that
contribute to at-risk populations having an HI > 1. Table 8 depicts the percent of at-risk and
no-risk populations based on factors, such as age, gender, region of residency, length of
residency (in years), if they have amalgam fillings, frequency of fish consumption (in days
per week), portion size per meal (in pounds), and if target organs were consumed. The
majority of the at-risk population was comprised of older men that consumed a higher
frequency of fish. In addition, majority of the at-risk population lived in the north or west
regions of Oahu (58%) and resided in Oahu for eleven or more years (53%); 60% of the norisk population lived in other regions and 93% resided in Oahu for ten years or less.
Additionally, majority of the at-risk population (71%) consumed target organs compared to
the no-risk population that did not consume target organs (67%). Majority of the no-risk
population were younger than 45 years of age, and 62% were female. The frequency of the
52
Table 8. Percent of At-Risk (HI > 1) and No-Risk (HI < 1) Populations Based on Age,
Gender, Region of Residency, Length of Residency, Presence of Amalgam Fillings,
Frequency of Fish Consumption, Portion Size per Meal, and Consumption of Target
Organs
VARIABLE
n
HI < 1
%
POPULATION,
HI < 1
n
HI > 1
%
POPULATION,
HI > 1
18-25
26-35
36-45
46-55
> 56
25
15
9
8
2
74
63
60
31
22
9
9
6
18
7
26
38
40
69
78
47
62
29
20
53
38
42
60
18
31
58
40
93
47
1
47
7
53
53
57
38
12
47
43
70
41
15
34
30
59
76
38
42
11
31
7
24
62
58
67
29
24
25
33
71
Age, years
Gender
Male
26
Female
33
North/West Vs. Other Regions
North/West
13
All Others
46
Residency, years (< 1 – 10 Vs. 11 –
40)
< 1 – 10
14
11 – 40
41
Fillings
Yes
43
No
16
Frequency
< 1 day/week
35
1 - > 6 days/week
24
Portion
< ¼ lb.
35
¼ - ½ lb.
19
> ½ lb.
5
If Target Organs (Brain, Head, Heart) Are Consumed
No
Yes
48
10
53
at-risk population consuming fish of > 1 day/week was greater (59%) for the at-risk
population than the no-risk population who consumed fish at a frequency of > 1 day/week;
the no-risk population who consumed a fish meal < 1 day/week was appreciably less (70%)
than the at-risk population consuming a fish meal of < 1 day/week. In addition, there was a
decreasing trend in portion size consumed for the no-risk population, and majority of the
population (76%) consumed < ¼ pound of fish per meal; majority of the at-risk population
(62%) consumed ¼ - ½ pound of fish per meal. There was not an observable difference
between the at-risk and no-risk populations for the presence of amalgam fillings. Adverse
effects were assessed according to gender-specific sensitive endpoints. Based on studies
conducted by Salonen et al. (1995) and Virtanen et al. (2005), cardiotoxic effects were
exhibited by men with > 2.0 ug/g hair mercury concentrations. Among the male population
residing in Oahu, 37% presented hair mercury levels above 2.0 ug/g, while 63% of the male
population had hair mercury levels below 2.0 ug/g. Among the female population,
developmental neuropsychological impairment was the most sensitive endpoint measured,
and the established RfD was the measurement used to determine whether toxicity was
acquired. Among the female population residing in Oahu, 37% contained hair mercury
levels above the RfD, while 63% of the female population had hair mercury levels below the
RfD.
54
CHAPTER 5
DISCUSSION
RELATIONSHIP BETWEEN DEMOGRAPHIC FACTORS AND
HAIR MERCURY LEVELS
This present study addressed methylmercury using hair mercury concentrations as an
indication of exposure among a population of healthy adults, which is uncommon for
methylmercury studies since susceptible populations, such as women of childbearing age and
children, are usually examined (Salonen et al., 1995). In this study among residents of Oahu,
Hawaii, older men appeared to exhibit the highest methylmercury exposure and risk for
methylmercury toxicity compared to their younger counterparts, and women. Among
women, the average HI value was 0.92, however, among women of childbearing age, 38%
had a HI > 1.0, indicating that both men and women were potentially at risk.
Age was a significant factor in increased hair mercury levels when categorized as less
than 45 years vs greater than 45 years. However, age was not significant when categorized
in more groups as depicted in Table 4, although when the age of the population increased,
median hair mercury levels also increased; similarly, Table 3 exhibited that those youngest
within the population had the lowest median hair mercury levels. Table 3 also showed that
the median values of individuals > 46 years of age exceeded the National Research Council
and EPA’s established reference dose of an equivalent hair mercury concentration of 1 ug/g
(Elhamri et al., 2007; National Research Council Committee on the Toxicological Effects of
Methylmercury, 2000; Steckling et al., 2011). An investigation by Taiwan’s EPA also
reported that subjects older than 40 years of age had higher mercury hair concentrations than
55
subjects younger than 20 years old, which was concurrent with studies from other countries
that increasing age correlated with higher hair mercury concentrations. Since mercury is not
easily excreted, the body burden increases with age (Chien, Gao, & Lin, 2010).
Hair mercury levels for those who lived within the rural (north and west) regions of
Oahu were higher than those who lived in urban regions; hair mercury concentrations were
significantly higher for those who resided in Oahu for longer than ten years versus
individuals who resided in Oahu for less than ten years (Table 4). This follows a 1995 study
from Salonen et al. stating that despite a low percentage of men living in rural areas, these
men had a 39.3% higher mean hair mercury concentrations compared to men living in highly
populated areas. Kruzikova, Kensova, Blahova, Harustiakova, and Svobodova (2009) found
that there was a significant relationship between mercury levels in hair coupled with the
study region. The author found that people living in bigger towns, such as Prague, had
healthier lifestyles which caused a greater consumption of fish. In this present study, there
was a significant relationship between length of residency and hair mercury levels, unlike the
Yokoo et al. (2003) study reporting that length of residency was not significantly associated
with hair mercury levels. The discrepancy may be due to the amount of categories
statistically analyzed; the Yokoo et al. (2003) study included 5 categories while two
categories were assessed in this study.
RELATIONSHIP BETWEEN FISH CONSUMPTION FACTORS
AND HAIR MERCURY LEVELS
High mercury hair levels were significantly associated with fish consumption
variables, especially for frequency (Table 5). Table 8 displayed that the at-risk population
consumed fish at a greater frequency than the no-risk population, which is consistent with
past studies showing that exposure to methylmercury was higher among people who
56
regularly ate fish, compared to those who occasionally or never consumed fish (Black et al.,
2011; Fakour, Esmaili-Sari, & Zayeri, 2010; McDowell et al., 2004; National Research
Council Committee on the Toxicological Effects of Methylmercury, 2000; U.S. Department
of Health and Human Services, 1999).
Increased hair mercury levels were associated with older men (Table 3), and the atrisk population consisted of a majority of older men that consumed a high frequency of fish
(Table 8). Yorifuji et al. (2009) also found that high exposures were associated with older
male fishermen. According to Elhamri et al. (2007), male subjects had higher hair mercury
concentrations due to an increased fish consumption frequency (measured in days/week).
Similar to findings in this present study, a distinct trend was evident in males that hair
mercury levels accumulated with increasing age. The dependence of hair mercury levels on
age is due to greater fish consumption for men, and also results in an imbalance between
intake and excretion rates since there is no mechanism to actively secrete methylmercury;
therefore, consequently causing age related bioaccumulation. Also supporting this finding is
the NHANES 2010 study which demonstrated that among the fish consumers surveyed, the
average consumption of fish (in g/day) is highest among males older than 21 years (17.86
g/day) (Tran, Barraj, & Bi, 2010).
Initially, it was hypothesized that ethnicity and cultural practices of fish consumption
may play a role in methylmercury toxicity. Ethnicity was not a significant factor for
increased hair mercury levels (Table 4). However, cultural and lifestyle factors, such as
consumption of different fish parts, especially target organs which includes the brain, head,
and/or heart, resulted in high hair mercury levels in comparison to those who did not
consume target organs (Table 5). Majority of the at-risk population also consumed target
57
organs, while majority of the no-risk population did not consume target organs (Table 8).
Judd et al. (2004) discussed how some communities are more susceptible to contaminant
exposure through the consumption of different fish parts that the general U.S. population
would otherwise avoid, and is caused by contaminants concentrating in different tissues.
MEN AS A SUSCEPTIBLE POPULATION TO
CARDIOTOXICITY
The median hair level for women were below the 1 ug/g established RfD (Table 4),
and 38% of the female population had mercury hair levels above the RfD. However, 38% is
still significant, and the finding of a high value in a young woman is concerning, as one of
the greatest risks is to the fetus. Women seem to have lower body burdens of
methylmercury. This might be due to transferring methylmercury body burdens to breast
milk as well as the fetus, aiding as routes of elimination, however, the number of offspring
for women of childbearing age was not assessed, so the contribution of number of children to
body burden of methylmercury cannot be ascertained here. The at-risk population was
comprised of majority male, and the no-risk population was majority female (Table 8). The
median hair concentration for men was also above the established RfD (Table 4). Since men
appeared as a susceptible population, it is of interest that recent studies have established
cardiotoxicity as a novel endpoint to determine adverse health effects from methylmercury
exposure for the male population. In previous studies, men with the highest hair mercury
levels of > 2.0 ug/g had an adjusted 1.60-fold risk of an acute coronary event, 1.68-fold risk
of cardiovascular disease (CVD), 1.56-fold risk of coronary heart disease (CHD), and 1.38fold risk of any death compared to men in the lower two-thirds of exposure levels. In
addition, for each microgram of mercury in the hair, the risk of acute coronary event
increased by 11%, the risk of CVD death increased by 10%, the risk of CHD death increased
58
by 13%, and the risk of any death increased by 5% (Choi et al., 2009; Salonen et al., 1995;
Salonen et al., 2000; Virtanen et al., 2007).
Methylmercury induced cardiotoxicity is a consequence of the promotion of lipid
peroxidation. Lipid peroxidation is an autocatalytic event that is initiated by free radicals,
which is produced from aerobic metabolism. Since transition metal ions contain one or more
unpaired electrons, they can react with other molecules, create new radical molecules, and
further catalyze the reaction. Lipid peroxidation occurs due to lipid damage from free
radicals. Polyunsaturated fatty acids in cell membranes (phospholipids in LDL) undergo
degradation through a chain reaction. The presence of oxidized LDL attracts monocytes into
the arterial wall where they differentiate into macrophages. Within the arterial wall,
macrophages scavenge for oxidized LDL and accumulate intracellular lipids, which induce
the macrophages to release proinflammatory cytokines, and further perpetuate monocyte
recruitment and accumulation of lipid-rich macrophages. Studies show that oxidized LDL is
one of the most common cell types in early atherosclerotic lesions. Antioxidants and
antioxidative enzymes scavenge for free radicals and protect from LDL peroxidation
(Virtanen et al., 2007).
Mercury promotes lipid peroxidation through a variety of mechanisms. First,
mercury is a transition metal and act as a catalyst for Fenton-type reactions that produce free
radicals. Cleavage of methylmercury generates free radicals that lead to lipid peroxidation.
A number of studies confirmed mitochondrial dysfunction, the production of hydrogen
peroxide of reactive oxygen species (ROS), glutathione or thiol depletion, and apoptosis after
exposure to micromolar concentrations of methylmercury. Secondly, since mercury has a
high affinity for sulfhydryl groups, mercury inactivates antioxidative thiolic compounds such
59
as glutathione. Sulfhydryl groups account for 10% to 50% of the antioxidative capacity of
plasma in plasma membranes. Glutathione is a key player in the regeneration of the
tocopheroxyl radical to tocopherol. Mercury poisoning causes increased lipid peroxidation in
the liver and kidneys, and inactivates two important enzymes, superoxide dismutase and
catalase that scavenge hydrogen peroxide. Paraoxonase is another extracellular antioxidative
enzyme that is inactivated. A genetic defect for this enzyme lowers the paraoxanase activity,
and decreases its antioxidative defense efficacy, and increases the risk of individuals for
acute myocardial infarction (AMI). Third, mercury either binds to selenium to form an
insoluble compound, mercuric selenide, or mercury reduces the bioavailability of selenium.
In the case of mercury binding, selenium is inactivated and cannot complex with glutathione
peroxidase, which is an important scavenger for hydrogen and lipid peroxides. This
promotes lipid peroxidation, and subsequently, artherosclerosis (Salonen et al., 1995;
Salonen et al., 2000).
BENEFITS OF FISH CONSUMPTION
Fish is an essential food source, especially for coastal populations. Scientific studies
confirm that fish is a superior protein source. Indices of the amino acid profile and the ability
to support growth is higher for fish proteins, than for beef, pork, and chicken proteins.
Approximately 50% of the fatty acids in lean fish and 25% in fatty fish are polyunsaturated
fatty acids. In contrast, beef only contains 4-10% polyunsaturated fatty acids. Fish is a good
source of niacin and vitamin B12, and better sources in vitamins D and A, compared to beef,
pork, or chicken (WHO, 2008). Despite fish containing harmful toxicants, such as
methylmercury, they are also a source of beneficial nutrients, such as long chain omega-3
fatty acids, eicosapentanoic acid, and docosahexanoic acid, which can help prevent chronic
60
diseases like cardiovascular disease and have positive effects on systems that are adversely
affected by methylmercury. In some studies, omega-3 fatty acids promote beneficial
neurologic development; however, results are inconsistent. The American Heart Association
recommends that fatty fish is consumed two times a week in order to reap the health benefits
without excessive toxicant exposure. Additionally, some studies suggest that seafood
containing high levels of micronutrients, such as selenium and vitamin E can protect against
mercury toxicity without specifically altering methylmercury absorption or excretion
(Mergler et al., 2007; Virtanen et al., 2007).
STUDY CONFOUNDERS AND LIMITATIONS
Several issues can affect the hair mercury results in this study. The growth rate of
hair varies among individuals due to variations during the cycle of hair growth, transition,
and terminal resting; it is the growth phase in which methylmercury is incorporated into the
hair follicle. In this study, multiple hairs were sampled and reflected hair that recently
incorporated methylmercury. However, ten to thirty percent of its follicles were in the
terminal resting phase, which reflected less-recent exposure. Also, an average growth rate of
1.1 centimeters per month is generally assumed in order to chronologically track
methylmercury exposure. However, interindividual variability in hair growth rates can
deviate from this standard, and is dependent on age, race, gender, and season. Additionally,
as the hair emerges from the scalp, the proximal end of the shaft is used as a benchmark to
relate measurements of length and time. Depending where the hair shaft is cut, may cause
variation in hair mercury concentrations. In this study, the length of hair varied for each
individual, since some had long or short hair; some men had only an inch of hair available to
61
sample. The length of hair sampled is also a confounder in this study (National Research
Council Committee on the Toxicological Effects of Methylmercury, 2000).
In addition, the persons sampled were not a random sample of the Hawaii population,
be rather approached at public areas. This makes the results difficult to extrapolate to the
population of Hawaii. In addition, utilization of questionnaires can be associated with
systematic errors, misclassification, and different types of biases. Recall or reporting bias is
common in studies focusing on study habits over long periods of time. Other studies
document that people generally overreport healthy or positive habits, while underreporting
lifestyle activities that have a negative health effect (Noisel, Bouchard, Carrier, & Plante,
2010).
The relatively small number of cases does not allow much extrapolation for different
variables assessed in this study. However, other methylmercury studies using hair as a
biomarker of exposure for a population report a sample size of approximately 100
individuals. In addition, despite the small sample size, results are significant and indicate
clear relationships between hair mercury levels and several demographic and fish
consumption factors.
Although it was not a measured variable in this study, alcohol consumption could
have also biased results. Ethanol is an inhibitor of the enzyme catalase, which reduces the
oxidation of mercury vapor into ionic mercury within the blood. This causes an increased
quantity of non-oxidized mercury to cross the blood-brain barrier. Also, studies indicate that
a fatty degeneration of the liver from the result of alcohol abuse increases the concentration
of methylmercury within the liver, the kidney, and brain (Drasch, O-Reilly, Beinhoff, Roider,
& Maydl, 2001).
62
All possible risks and benefits of fish consumption were not considered. Other
contaminant exposures such as PAH, PCB, and dioxins are found in fish and may exert
adverse effects and react additively or synergistically with mercury; however, it would be
rather difficult and complex to integrate all possible factors contributing to human toxicity
from fish consumption.
Also of concern was a woman with a mercury hair concentration of 23.34 ug/g which
was much higher than the rest of the study population. This person did consume brains and
the head of fish, and consumed less than ¼ pound of fish each meal for less than one
day/week. Other factors that were not measured may have caused an abnormally high hair
mercury level in the individual.
Lastly, in order to calculate individual hazard indices, individual weights instead of a
standardized weight of 60 kg. for women and 70 kg. for men should have been utilized since
many of the hazard indices were borderline to one. Overall, despite these limitations, the
value of the results gave a general view of factors contributing to methylmercury toxicity
among residents of Oahu, Hawaii in relation to fish consumption.
HOW TO PROTECT THE PUBLIC FROM
METHYLMERCURY EXPOSURE
According to Table 9, that details sentiments of Oahu residents regarding
methylmercury exposure and fish consumption, 62% of the population is concerned with
methylmercury exposure, and 61% would cease fish consumption if they had knowledge that
methylmercury were present within fish. Despite the lack of knowledge of the population,
the state government has posted fish advisory signs since 58% have seen these advisories
throughout the island. 60% of the population does not feel fully informed about fish
63
Table 9. Percentage of Total Population Reporting Sentiments on Public Health
Information About Methylmercury Exposure Through Fish Consumption
N
PERCENT (%) OF TOTAL
POPULATION
NO
46
42
YES
63
58
NO
41
38
YES
68
62
SENTIMENT
If fish advisories seen around Oahu
If concerned about methylmercury exposure
Cease fish consumption if methylmercury present in fish
NO
38
39
YES
67
61
NO
66
60
YES
42
40
Fully informed about fish consumption
Fully informed about methylmercury exposure from fish consumption
NO
86
78
YES
21
22
consumption; an even greater percentage (78%) does not feel full informed about
methylmercury exposure through fish consumption.
Currently, federal government agencies, such as the FDA, has informed the public by
posting advice for susceptible populations, like children and pregnant women, and women of
childbearing age to limit shark and swordfish consumption to one meal per month, since
methylmercury is high in these predatory species. The FDA advises the general population
to consume shark and swordfish to seven ounces per week. For fish species containing 0.5
ppm of methylmercury, regular consumption should be restricted to 14 ounces per week
(U.S. Department of Health and Human Services, 1999). However, residents of Oahu do not
64
regularly consume high predatory fish species. By surveying Oahu residents, consumption of
the top three species of fish are reportedly salmon, ahi tuna, and mahimahi (see Table 10)
which are consistent to the top three species consumed by the U.S.
Table 10. Consumption of Individual Fish Species from Oahu, Hawaii in
Relation to Median, Minimum, and Maximum Hair Levels as Reported by
Residents
VARIABLE
n
MEDIAN HAIR Hg (ug/g)
(min – max)
Ahi
Yes
84
No
15
Yes
35
No
64
Yes
7
No
92
Yes
4
No
95
Yes
8
No
91
Yes
34
No
65
Yes
12
No
87
1.02
(0.02 – 23.34)
0.78
(0.39 – 5.40)
Salmon
0.96
(0.14 – 4.51)
0.98
(0.02 – 23.34)
Marlin
1.60
(0.23 – 7.04)
0.97
(0.02 – 23.34)
Tilapia
1.02
(0.10 – 23.34)
0.97
(0.02 – 7.04)
Opakapaka
0.69
(0.42 – 2.12)
0.98
(0.02 – 23.34)
Mahimahi
1.01
(0.12 – 6.66)
0.96
(0.02 – 23.34)
Aku
1.50
(0.05 – 7.04)
0.93
(0.02 – 23.34)
(table continues)
65
Table 10. (continued)
VARIABLE
n
MEDIAN HAIR Hg (ug/g)
(min – max)
Uhu
Yes
4
No
95
Yes
4
No
95
Yes
3
No
96
Yes
1
No
98
Yes
2
No
97
Yes
3
No
96
Yes
3
No
96
Yes
4
No
95
Yes
1
No
98
Yes
1
No
98
0.76
(0.50 – 5.40)
0.97
(0.02 – 23.34)
Kala
0.76
(0.31 – 3.26)
0.97
(0.02 – 23.34)
Papio
1.95
(1.01 – 2.36)
0.96
(0.02 – 23.34)
Moi
NA
0.97
(0.02 – 23.34)
Aholehole
1.89
(0.51 – 3.26)
0.97
(0.02 – 23.34)
Opah
0.69
(0.24 – 6.66)
0.98
(0.02 – 23.34)
Yellowtail
1.80
(0.62 – 3.28)
0.97
(0.02 – 23.34)
Mempachi
1.59
(0.57 – 2.73)
0.97
(0.02 – 23.34)
Ulua
NA
0.98
(0.02 – 23.34)
Otoda
NA
0.98
(0.02 – 23.34)
(table continues)
66
Table 10. (continued)
VARIABLE
n
MEDIAN HAIR Hg (ug/g)
(min – max)
Ono
Yes
6
No
93
Yes
1
No
98
Yes
5
No
94
Yes
4
No
95
Yes
4
No
95
Yes
1
No
98
Yes
1
No
98
Yes
3
No
96
Yes
1
No
98
Yes
1
No
98
1.34
(0.88 – 5.65)
0.95
(0.02 – 23.34)
Sea Bass
NA
0.97
(0.02 – 23.34)
Cod
1.31
(0.62 – 2.57)
0.96
(0.02 – 23.34)
Bangus
0.81
(0.39 – 2.86)
0.97
(0.02 – 23.34)
Butterfish
1.02
(0.78 – 1.32)
0.97
(0.02 – 23.34)
Saba
NA
0.97
(0.02 – 23.34)
Au
NA
0.97
(0.02 – 23.34)
Weke
1.95
(1.00 – 5.40)
0.96
(0.02 – 23.34)
Kumu
NA
0.97
(0.02 – 23.34)
Pompano
NA
0.97
(0.02 – 23.34)
(table continues)
67
Table 10. (continued)
VARIABLE
MEDIAN HAIR Hg (ug/g)
(min – max)
n
Onaga
Yes
4
No
95
Yes
1
No
98
Yes
1
No
98
Yes
1
No
98
Yes
1
No
98
Yes
4
No
95
Yes
5
No
94
0.64
(0.42 – 1.19)
0.98
(0.02 – 23.34)
Swordfish
NA
0.98
(0.02 – 23.34)
Sardines
NA
0.98
(0.02 – 23.34)
Ehu
NA
0.98
(0.02 – 23.34)
Catfish
NA
0.98
(0.02 – 23.34)
Uhu
0.76
(0.50 – 5.40)
0.97
(0.02 – 23.34)
Akule
2.07
(1.21 – 3.18)
0.94
(0.02 – 23.34)
*
* significantly higher, p < 0.05
population, with the exception of mahimahi (Tran et al., 2010). The methylmercury levels in
these species are generally less than 0.2 ppm, and consumption advice is not given for these
species (U.S. Department of Health and Human Services, 1999). Additionally, the EPA
Office of Water issues guidance to states regarding sampling and analysis procedures to use
for assessing health risks from consuming locally caught fish since these populations would
be susceptible to methylmercury and contaminant exposure. A screening value of 0.6 ppm
68
(wet wt.) in fillets is the criteria to establish fishable waters. There are no listed advisories
for the state of Hawaii (U.S. Department of Health and Human Services, 1999).
Advisories are voluntary recommendations about fish consumption and are not
subject to regulation. States have the main responsibility in protecting residents from
potential harm caused by fish caught in local waters, but may choose not to issue advisories.
Majority of the residents of Hawaii are concerned about methylmercury exposure through
fish, which is a staple food source, and are open to education regarding safe consumption.
In addition, majority of the population do not have much knowledge about
methylmercury exposure, along with the risks and benefits of consuming fish; despite the
additional health benefits, many are willing to remove fish from their diet due to the presence
of methylmercury.
In order to reduce the residents’ exposure to methylmercury in fish, policies and
effective communication regarding safe fish consumption must be utilized, and regulatory
policies and measures need to be implemented to reduce levels of methylmercury emissions
into the environment. Presently, guidelines are catered to susceptible populations, but more
emphasis should be placed on adults, specifically men and older individuals. Along with the
current listed frequency and portion size, and low-level methylmercury, non-predatory fish
species for safe consumption, it would be beneficial to also define safe cooking practices. It
should be explicitly stated to primarily consume fish meat, and to discard all other organs due
to high methylmercury content. Despite methylmercury and contaminants present in fish, the
benefits of fish consumption should also be highlighted, and that safe fish consumption
reduces the risks of methylmercury toxicity. To effectively reach the target audience, it is
important to identify the educational level and to transmit awareness through more than one
69
potential source of information. Hawaii fish advisories can be found through the internet;
however, some residents may not have access to technology in rural parts of the island.
Another effective tool in mitigating methylmercury toxicity is to emphasize a diet that is high
in vitamin E and selenium. Vitamin E acts as a radical scavenger and alters methylmercury
metabolism. It stabilizes membranes by interacting with fatty acid chains; therefore, free
radicals that breakdown methylmercury are scavenged, and can also react with methyl
radicals. Selenium slows the formation of methylmercury free radicals through the
decomposition of peroxides that aid in promoting methylmercury decomposition.
Additionally, certain selenium metabolites can complex with inorganic mercury formed
through methylmercury decomposition, so it is unavailable for ligand binding, and prevents
methylmercury from functioning as a radical initiator through univalent redox modifications
(Ganther, 1978).
70
CHAPTER 6
CONCLUSION
This study assessed the risk of methylmercury exposure through fish consumption
among residents of Oahu, Hawaii using hair as a biomarker of exposure. Age, gender, and
fish consumption habits were significant factors in increased susceptibility to methylmercury
exposure and increased risk to methylmercury toxicity. Specifically, older men tend to have
higher hair mercury concentrations in comparison to younger men and women of any age
group due to greater fish consumption frequency and age related bioaccumulation.
Additionally, fish consumption habits, such as consuming ¼ to ½ pound of fish per meal at a
frequency of greater than one day per week, along with the consumption of target organs
increased methylmercury exposure and risk to methylmercury toxicity.
Established neurological endpoints for methylmercury toxicity are based upon
epidemiological studies observing children and women of childbearing age. More studies
need to scrutinize methylmercury endpoints affecting the adult population, especially for
men. Cardiotoxicity studies, especially the Kuopio studies, are comprehensive in order to
establish an RfD value for men using cardiovascular disease and cardiotoxicity endpoints.
Since fish is an important staple for Oahu residents, proper guidelines for safe fish
consumption should include not only frequency and portion size, and listing low-level
methylmercury, non-predatory fish species, but it would also be beneficial to define safe
cooking practices. An example would be to explicitly state to primarily consume fish meat
and to discard all other organs due to high methylmercury content. The benefits of fish
consumption should also be highlighted, and that safe fish consumption practices reduce the
71
risks of methylmercury toxicity. Fish is a local and global commodity; therefore, this study
adds to the growing evidence that methylmercury exposure is present around the globe with
no geographical, social, economic, or cultural boundaries.
72
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79
APPENDIX
SURVEY ADMINISTERED TO OAHU RESIDENTS
80
Survey Administered to Oahu Residents Detailing Subjects’ Demographics, Fish
Consumption Habits, and General Knowledge on Methylmercury
(FOR OFFICIAL USE ONLY)
DATE:_________________________________________
TIME:_________________________________________
LOCATION:____________________________________
IDENTIFIER:____________________________________
Hello, my name is Alethea Ramos, and I am a graduate student in the Public HealthToxicology program at San Diego State University. I was also born and raised in
Hawaii. Do you speak English? Do you eat fish? Are you at least than 18 and a current
resident of Hawaii? If you are female, are you not pregnant? If you said “yes”, then
you are the right candidate for this study.
I am working on a thesis to assess methylmercury exposure from eating fish among the
locals of Oahu, Hawaii. If necessary, guidelines will be recommended to lessen the
exposure to methylmercury found in fish.
This survey will help find 1) if demographics (ie: gender, age, ethnicity, etc.) play a role
in methylmercury exposure, 2) information on how much fish you eat, and 3) level of
public awareness about methylmercury. Participation in this survey is voluntary and
confidential. You will receive a $3.00 gift certificate for participating in this study. The
survey should take around 5 minutes.
MAHALO FOR YOUR TIME AND ASSISTANCE!!!
81
A. DEMOGRAPHIC QUESTIONS
1. What is your gender?
a. Male
b. Female
c. Other/No Response
2. What is your ethnicity
a. American Indian or Alaskan Native
c. Black/African-American
e. Native Hawaiian or Other Pacific Islander
f. White/Caucasian
Other________________
b. Asian
d. Hispanic or Latino
g.
3. What is your job? _____________________________
4. What is the highest educational level you have completed
a. Grade School
c. Associates Degree/Trade School
e. College Degree
g. Post graduate
b. High School
d. Current college student
f. Graduate Degree
5. Approximate household income?
a. Less than $25,000 b. $25,000 - $39,999
c. $40,000 - $49,999
d. $50,000 – 75,000
e. Greater than $75,000
f. No Response
6. Do you feel comfortable reading and writing in English?
a. Yes b. No
7. How old are you?
a. 18-25
b. 26-35
c. 36-45
d. 46-55
e. 56-65
f. 66+
8. What city in Hawaii do you live in?__________________________
9. How long have you been living in Hawaii?_____________________________
10. Do you have amalgam fillings (silver dental fillings or caps)?
a. Yes b. No
11. Do you know if you have been exposed to mercury? If yes, how?______________________
a. Yes b. No
c. Not sure
82
B. FISH CONSUMPTION QUESTIONS
1. Please select where you primarily get your fish from. Specify where.
a. Store bought: ____________________
b. Fishing: _________________________
c. Fish auction:______________________
d. Other:___________________________
2. What types of fish do you eat (name top 3)?
_____________________________________________________________________
3. How many times a week do you eat fish?
a. Less than 1 day / week
c. 3-5 days / week
b. 1-2 days / week
d. Greater than 6 days / week
4. Approximately how much fish do you eat with each meal?
a. Less than 1/4 lb.
b. 1/4 -1/2 lb.
c. Greater than 1/2 lb.
5. How many times a month do you eat fish prepared the following ways?
a. Raw (poke/sashimi/sushi):______
b. Boiled:______
c. Oven:______
d. Grilled on stovetop: _______
e. Fried:_______
f. Barbecue on grill:________
g. Other:_______
6. How often do you eat the following fish parts?
a. Meat: a) Never b) Seldom c) Sometimes d) Often e) Always
b. Skin: a) Never b) Seldom c) Sometimes d) Often e) Always
c. Eyes: a) Never b) Seldom c) Sometimes d) Often e) Always
d. Brain: a) Never b) Seldom c) Sometimes d) Often e) Always
e. Head: a) Never b) Seldom c) Sometimes d) Often e) Always
f. Heart: a) Never b) Seldom c) Sometimes d) Often e) Always
g. Other organs: ________ : a) Never b) Seldom c) Sometimes d) Often e)Always
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C. GENERAL INFORMATION ON MERCURY (METHYLMERCURY)
1. Have you heard or seen advisories/warnings for methylmercury contamination in fish around
Oahu? If yes, name the source of advisory?
a. No
b. Yes, ____________________________
2. Are you concerned about methylmercury exposure?
a. Yes
b. No
3. If there was methylmercury in your fish, would you stop eating fish?
a. Yes
b. No
4. Do you feel fully informed of risks and benefits from eating fish?
a. Yes
b. No
5. Do you feel fully informed about methylmercury exposure from eating fish?
a. Yes
b. No
6. If you answered yes in question (5), how did you learn about methylmercury exposure from
eating fish?
________________________________________________________________________
7. Briefly state what you know regarding methylmercury exposure from eating fish.
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
____________________________________________________________
8.
Do you have any suggestions on how to make the public more aware of methylmercury exposure
from eating fish?
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
______________________________________________________________________________
____________________________________________________________
MAHALO FOR VOLUNTEERING IN THIS STUDY!