EPA-660/3-74-027
DECEMBER 1974
Ecological Research Series
Pharmacokinetics of Toxic Elements
in Rainbow Trout
UJ
CO
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
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2. Environmental Protection Technology
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This report has been assigned to the ECOLOGICAL RESEARCH STUDIES
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assessed for their long- and short-term influences. Investigations
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fate of pollutants and their effects. This work provides the technical
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This report has been reviewed by the Office of Research and
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EPA-660/3-74-027
December 1974
PHARMACOKINETICS OF TOXIC ELEMENTS IN RAINBOW TROUT
I. Uptake, Distribution and Concentration of
Methylmercury by Rainbow Trout
(Salmo gairdneri) Tissues
II. The Mechanism of Methylmercury Transport
and Transfer to the Tissues of
the Rainbow Trout (Salmo gairdneri)
No. 3
by
Edward J. Massaro
Grant No. 800989
Program Element 1BA022
Project Officer
George Gardner
National Marine Water Quality Laboratory
South Ferry Road
National Environmental Research Center
Narragansett, Rhode Island 02882
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
Fo' Sale by the National Technical Information Service
U.S. Department of Commerce, Springfield, VA 221S1
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ABSTRACT
203
Hg-methylmercury (MeHg) was administered intragas-
trically in a single dose (0.5 mg Hg/kg=3.3yCi/kg) to trout
(av. wt. 250g). The fish were sacrificed from 1.0 hr. to 290
days postadministration. Twenty tissues were analyzed for
MeHg content by gamma scintillation spectrometry. The label
was taken up rapidly by blood, gills, spleen, liver and kidney
and more slowly by muscle, brain and lens. After 290 days:
(a)blood, spleen, kidney, liver and lens had concentration
factors (C.F.=tissue Hg conc./Hg dose x final tissue wt./
initial tissue wt.)=1.0;(b)in general, C.F.s had dropped
off by at least 2/3 from their maxima except for muscle,
brain and lens in which the C.F.s remained=their maxima;
(c)~64% of the dose still remained in the fish and skeletal
muscle (comprising ~55% of body weight) contained >40% of
the residue. Assuming MeHg excretion to be a first order
process, there are at least two rates of excretion—a rapid
initial rate resulting in a biological half-retention time
(HRT) of ~200 days and a slower, subsequent rate yielding an
HRT of >1000 days which, apparently, is governed by the rate
of release of MeHg from the skeletal muscle.
Hemoglobin (Hb) is the main methylmercury (MeHg) trans-
port protein in trout blood. In vitro, MeHg is taken up rapidly
in 3 minutes by red blood cells. MeHg binding in the RBC is
reversible in vitro as demonstrated by the efflux of Hg from
ii
-------
RBCs suspended in protein solutions. MeHg binding in the
RBC also is reversible in vivo as gel filtration chromato-
graphy of liver soluble proteins yielded identical elution
profiles for MeHg administered as the free salt or bound in
RBCs. The number of reactive sulfhydryl (-SH) groups per
molecule of Hb was found to be 4 by amperometric titration
with MeHgCi. The reactive -SH concentration in the RBC was
calculated to be at least 20mM, A mechanism for the efflux
of MeHg from the RBC is proposed involving the dissociation
of MeHg from Hb initiated by -SH groups outside the RBC and
migration of MeHg across the membrane as MeHgCi.
This report was submitted in fulfillment of Grant No.
800989 by the State University of New York at Buffalo under
the sponsorship of the Environmental Protection Agency. Work
was completed as of June 30, 1974.
iii
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CONTENTS
Section Page
I . Conclusions 1
II Recommendations 3
III Uptake, Distribution and Concentration
of Methylmercury by Rainbow Trout (Salmo
gairdneri) Tissues
Introduction
Materials and Methods
Results and Discussion
IV The Mechanism of Methylmercury Transport 14
and Transfer to the Tissues of the Rainbow
Trout (Salmo gairdneri)
Introduction
Materials and Methods
Results and Discussion
References 28
iv
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FIGURES
No. Page
3-1 Relative Abilities of Trout Tissue to 7
Concentrate MeHg
3-2 MeHg Uptake and Elimination Pattern of 8
Blood, Heart and Gonads
3-3 MeHg Uptake and Elimination Pattern of 9
Blood, Brain and Lens
3-4 MeHg Uptake by Skeletal Muscle 11
3-5 MeHg Dose Contained in Total Weight of 12
Each Tissue
4-1 The in vivo Removal of Me20^Hg from 21
Washed, Intact, Rainbow Trout RBCs by
Protein Solutions
4-2 The Decrease in Hg Concentration Factor 22
in Rainbow Trout Whole Blood after the
Intracardiac Injection of Me2°3HgCl
(O.Smg Hg/kg body weight)
4-3 Gel Filtration Elution Profiles of Rain- 23
bow Trout Soluble Liver Proteins 8 Days
after Intracardiac Injection of Me203HgCl
(O.Smg Hg/kg body weight)
4-4 Determination of the Number of Sulfhydryl 25
Groups of Dialyzed Rainbow Trout Hemolysate
(0.40 x 10~3 mmoles) by Amperometric Titra-
tion with Me203HgCl
4-5 Proposed Mechanism of the Transfer of MeHg 27
from Trout RBCs to Tissues
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ACKNOWLEDGMENTS
This research was supported by grants from the Federal
Water Pollution Control Administration (No. 18050 DRJ),
Environmental Protection Agency, pursuant to the Federal
Water Pollution Control Act; the Bureau of Sport Fisheries
and Wildlife (14-16-0008-623); the Food and Drug Administra-
tion, U.S. "Public Health Service (No. 5R01 FD0466-02) and
funds from a General Research Support Grant (No. 5 SO 1
RR05400-12) from the General Research Support Branch, Divi-
sion of Research Resources, National Institutes of Health.
We are grateful to Mr. Robert H. Griffiths, Superintendent
of Fish Culture, New York State Department of Environmental
Conservation and to Mr. Donald Longacre of the New York State
Fish Hatchery at Caledonia, New York, for the rainbow trout.
We thank Dr. K.K, Sivasankar Pillay and Mr. Charles Thomas, Jr.,
of the Western New York Nuclear Research Center for preparation
203
of the Hg-labeled methylmercury compound and for advice
concerning the isotope experiments. We are also grateful to
Dr. Gustavo Cudkowicz for the use of the gamma spectrometer;
to Mr. Philip Herzbrun for aiding in maintaining the fish and
for preparing the computer program employed in compiling the
data and to Dr. Stanley Bruckenstein, Department of Chemistry,
SUNY at Buffalo and his laboratory for the use of their equip-
ment and their assistance with the amperometric titrations.
vi
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SECTION I
CONCLUSIONS
Following administration of a single, intragastric
dose (O.Smg Hg/kg = 3.3yCi/kg) of MeHg to the rainbow trout,
maximum concentration factors (C.F.s) were reached in the
gills after one hours, and the skeletal muscle, brain and
lens after 34, 56 and > 290 days, respectively. Maximum
C.F.s were reached in most other tissues at ~7 days. Skeletal
muscle appeared to function as a reservoir for MeHg and ac-
cumulated "50% of the dose from 34 to 100 days postadministra-
tion. At 290 days postadministration, skeletal muscle con-
tained > 40% of the dose. MeHg accumulation in the brain never
rose above 0.1% of the dose. The rate of mercury excretion
appeared to be biphasic as a result of a slow elimination
from the skeletal muscle relative to the other tissues. As-
suming excretion to be a first order process, the half-reten-
tion time (HRT) for MeHg for the first 100 day postadministra-
tion was calculated to be ~200 days. Over the last 190 days
(days 100-290), a HRT of > 1000 days was obtained.
MeHg is taken up rapidly, in vitro and in vivo, by trout
RBCs. The uptake of MeHg by the RBC is dependent, to a large
extent, on the number of reactive -SH groups per hemoglobin
molecule which was found to be 4 for the trout. In the trout
-------
RBC, MeHg binds almost exclusively to hemoglobin. The binding
within the cell is freely reversible, in vitro and in vivo, by
-SH groups located outside the cell. The ability of MeHg to
rapidly penetrate the RBC membrane and to reversibly bind to
hemoglobin is responsible for its rapid transport and transfer
to tissues.
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SECTION II
RECOMMENDATIONS
Experience has shown that:
i. Mercury in any form can be converted in the natural
aqueous environment into a readily bioavailable,
highly toxic organic form, methyl mercury.
ii. The biological half-life of methyl mercury in fishes
is extremely long (> 1000 days in the case of the
rainbow trout, Salmo gairdneri),
iii. There is no comprehensive, systematic knowledge of:
a. the biological effects in man of low-level,
long-term (environmental) exposure to any single
toxic or potentially toxic environmental pollu-
tant.
b. the synergistic effects of multiple environmental
pollutants on living systems.
Therefore:
i. Environmental release of toxic or potentially toxic
substances should be monitored and controlled rigidly
to prevent:
a. (potentially irreversible) damage to aquatic
food webs and, therefore, man's dwindling food
food supply.
b. the reoccurrance of Minamata-like disasters:
episodes of environmentally derived human intox-
ication of epidemic proportions.
ii. The EPA should undertake the development of a compre-
hensive program of extramural support of basic research
in environmental toxicology: especially in those areas
dealing with low-level, long-term; synergistic; and
behavioral effects of pollutants.
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SECTION III
UPTAKE, DISTRIBUTION AND CONCENTRATION OF METHYtMERCURY
BY RAINBOW TROUT (SALMO GAIRDNERI) TISSUES
INTRODUCTION
It has been known for some time that fishes are
able to take up organic mercury from their environment
and concentrate it in their tissues (1,5,8,11,12,15,20)
to levels that are toxic to humans (4,6,14,17,18).
Our research is directed toward elucidating the mechanisms
of organic mercury concentration in the tissues of the
rainbow trout, Salmo gairdneri. Presently our attention
is focused on establishing the relative affinities of the
various tissues of this species for methylmercury (MeHg);
that is, the capacity of the tissues to take up, concen-
trate and store MeHg, and the half-retention time of this
compound in the tissues.
MATERIALS AND METHODS
Hatchery reared rainbow trout (Salmo gairdneri) of
similar genetic background were obtained from the New York
State Hatchery at Caledonia, New York. The natural mercury
content of their skeletal muscle was found to be —0.OSppm
by neutron activation analysis (10). The average weight of
the fish at the beginning of the experiment was 250 g, A
weight increase of up to 59% was recorded during the course
of the experiment (290 days).
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Maintenance of Fish
The fish were maintained in aged, aerated tap water in
200 gallon galvanized steel tanks in a temperature controlled
room. Water temperature fluctuated between 5 and 9°C. The
water was continuously filtered through activated charcoal
and cotton and was replaced with fresh water every other day.
The fish were fed a diet of commercial fish food pellets (Strike
Fish Feed: Agway, Inc., Syracuse, New York).
ortO
^ Hg labeled MeHg was obtained by isotope exchange between
20-*Hg labeled mercuric nitrate (radiochemical purity 98%:
ICN, Waltham, Mass.) and methyl mercuric chloride (assay > 95%:
obtained as the hydroxide from Alfa Chemical Co., Beverly, Mass.,
and subsequently dissolved in 1 N HC1). It was administered
to the fish in an accurately measured, single, intragastric
dose. This was accomplished via a stomach tube constructed
from small bore polyethylene tubing attached to a calibrated
glass syringe. The fish were starved for two days prior to
the MeHg feeding and were anesthetized with tricaine
methanesulphonate (MS 222: Sigma Chemical Col, St. Louis, Mo.)
prior to intubation. Each fish was given 0.5 mg Hg/kg body
weight corresponding to a radioactive dose of 3.3yCi/k body
weight. It was determined that the fish lost less than 5%
of the dose by regurgitation.
At least two fish were sacrificed at 15 different time
periods ranging from one hour to 290 days after the MeHg
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feeding. Approximately 20 different tissues were isolated
each time, placed in preweighed plastic vials and counted in
a Packard Model 5319 Gamma Scintillation Spectrometer. Tissue
MeHg content was determined by comparison with a MeHg standard
counted along with the samples.
The term "Concentration Factor,"
ng Hg / g wet wt. final tissue wt.
C.F. =
500 ng Hg / g initial tissue wt.
is employed to express the data. It reflects the ability of
the tissues to concentrate MeHg on a gram per gram wet weight
basis. Since the fish increase in weight significantly over
290 days, tissue MeHg concentrations appear to decrease even
in the absence of MeHg excretion. To account for this, the
first term of the equation is multiplied by a factor equal to
the final tissue weight divided by the initial weight.
RESULTS AND DISCUSSION
Fig. 3-1 illustrates the relative abilities of the trout
tissues to concentrate MeHg on a gram per gram basis (2,15,19).
The open bars represent the maximum C.F. found for each tissue
over the 290 day period. The solid bars represent the MeHg
C.F. at the end of the experiment. As can be seen, the max-
imum C.F.s for the blood and spleen were at least twice as
-------
HQ cone
Factor
10.0-
9.0-
80-
7.0-
6.0-
5.0-
4.0-
3.0-
2.0-
1.0-
0
BLOOD
open bar: maximum Hg concentration factor from
I hr to 290 days after administration
sold bar Hg concentration factor 290 day*
after administration
SPLEEN
KIDNEY
UVER
HEART
POSTERIOR
INTESTINE
STOR/3E
SWN LIPID
Figure 3-1. Relative Abilities of Trout Tissue to
Concentrate MeHg
high as those of any of the other tissues. The kidney and the
liver also were able to concentrate MeHg to a high degree. The
lowest concentrations of MeHg throughout the study were found
in the skin and storage lipid. Due to its high lipid solubility,
it might be expected that MeHg would be concentrated in the stor-
age lipid of the fish as is DDT, but this was not observed to be
the case.
-------
At the end of the 290 day study, the only tissues having
MeHg C.F.s greater than 1.0 were the blood, spleen, kidney,
liver and lens. In general, most of the tissue MeHg C.F.s had
decreased by at least 2/3 from their maximum values. However,
this was not true for the skeletal muscle, brain and lens, where
the MeHg concentrations after 290 days were nearly equal to the
maximum values.
Fig. 3-2 is a log-log plot of the MeHg uptake and elimina-
tion pattern exhibited by the blood, heart and gonads. Each
point represents the average value for at least two different
fish. The shape of the patterns is essentially identical, and
Hg Cone.
1.0-
0.1
0.01
BLOOD
HEART
GONADS
0.01
I 10 100
Days after Mercury Administration
1000
Figure 3-2. MeHg Uptake and Elimination Pattern of
Blood, Heart and Gonads
8
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similar patterns, differing only in their maxima, were exhibited
by the liver, kidney, spleen, swim bladder and bile. Thus, for
most of the tissues investigated, the maximum MeHg concentration
was reached approximately seven days after the MeHg feeding. This,
in turn, was followed by an approximately linear decrease in con-
centration over the next 100 days and a leveling-off of the rate
of decrease.
A distinctly different type of uptake and elimination pat-
tern was exhibited by the brain and lens. As illustrated in Fig. 3-3,
the brain of the trout was observed both to accumulate and release
Hg Cone
Foctor
10.0
1.0
O.I-
0.01
MUSCLE
0.01
O.I
I 10 100
Days after Mercury Administration
1000
Fig. 3-3, MeHg Uptake and Elimination Pattern of
Blood, Brain and Lens
-------
MeHg at a very slow rate (2,5,19). The maximum MeHg concentra-
tion was reached 50 days later in the brain than in the blood.
During the last 200 days of the experiment only a very slight
decrease was observed in the brain MeHg levels. Methylmercury
in the brain never rose above a C.F. of 1.0. It may be that the
blood-brain barrier of the trout is able to retard, to a certain
degree, the influx of MeHg. However, once MeHg has entered the
brain, transport out is very slow.
The slowest initial uptake of any of the trout tissues
was exhibited by the lens of the eye (Fig. 3). The lens also
was unique in that it exhibited no peak MeHg concentration.
Throughout the duration of the experiment the concentration
continued to increase even as the blood MeHg level declined.
The continuous uptake of MeHg by the lens may be related to the
high sulfhydryl content reported for this tissue and to the re-
ported occurrence of cataracts in MeHg intoxicated fishes (5).
Skeletal muscle, which comprises the bulk of the edible
portion of the trout, accounts for ^55% of its total body
weight. Fig. 4 illustrates the uptake of MeHg by the skeletal
muscle. The pattern is similar to that of the brain. Maximum
concentrations are reached -^-34 days after administration and
then decline only very slightly over the next 250 days. The
point at which uptake plateaus represents the storage of -~ 50%
of the MeHg dose. The slow rate of release of MeHg from skel-
etal muscle will govern, to a large extent, the rate of excre-
tion of MeHg from the whole fish.
10
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Hg Cone
Factor
IO.O
1.0-
0.1
0.01
BLOOD
0.01
O.I
I 10 100
Days after Mercury Administration
1000
Figure 3-4. MeHg Uptake by Skeletal Muscle
The percentage of the MeHg dose contained in the total
weight of each tissue 100 days after MeHg administration is
illustrated in Fig. 5. Each value was obtained by multiplying
the concentration of MeHg in the tissue by the weight of the
tissue, and dividing by the weight of MeHg originally adminis-
tered to that particular fish. Each bar is the average value
for four different fish. The significance of the skeletal
muscle as a storage reservoir for MeHg in the rainbow trout
is obvious. The skeletal muscle binds 50% of the MeHg dose
100 days after administration. This amounts to ~ 70% of the
MeHg present in the entire fish.
11
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60
50-
4O-
Pwcvrt of Hg dot* conloirad in
total wtlght of «och tissu* after 100 days
Sum of all tiwMf 7334*3.36%
HEAD
AND
FINS '
BLOOD
NTES
TNES
SPLEEN
HEART
BRAN
GONADS
KDNEY
GLLS
Figure 3-5. MeHg Dose Contained in Total Weight
of Each Tissue
Other tissues, some of which had relatively high C.F.s,
contributed very little to the total amount of MeHg stored.
An example is the spleen, which had a C.F. twice as high as
that of muscle after 100 days;but, due to it-s relatively small
mass, it contained only 0.1% of the total MeHg dose. It is
of interest to note that the brain also accumulated only 0.1%
of the dose. The intestinal contents were found to contain
0.05% of the dose with over 95% of this amount located in the
posterior intestine (that portion of the intestine leading from
12
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the stomach). This suggests that defecation may be an
important route of exretion of MeHg from the fish (3,5).
The total amount of MeHg measured in the whole fish
after 100 days amounted to -^-73% of the dose. The biolog-
ical half-retention time (HRT) for MeHg was calculated for
the first 100 days. Assuming excretion to be a first order
process and that 100% of the dose was retained at day 0
and 73% was retained at day 100, an HRT of ~ 200 days
(7,9) was obtained.
Total MeHg in the fish also was measured after 290 days,
At this time ~ 64% of the dose was present, amounting to a
decrease of ^12% over the last 190 days of the experiment.
The HRT calculated for this period (day 100 to day 290) was
>1000 days (7). Apparently there are at least two rates
of excretion of MeHg: a rapid initial rate, followed by a
much slower rate (7,13,16), which apparently is governed by
the rate of release of MeHg from the skeletal muscle.
13
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SECTION IV
THE MECHANISM OF METHYLMERCURY TRANSPORT AND TRANSFER
TO THE TISSUES OF THE RAINBOW TROUT (SALMO GAIRDNERI)
INTRODUCTION
The blood of the rainbow trout (Salmo gairdneri) concen-
trates methylmercury (MeHg) to a greater extent than any other
tissue (24). This has been reported to occur in a wide variety
of other species and, apparently, is a general phenomenon. The
erythrocyte (RBC) is the blood element responsible for concen-
trating MeHg (30,31). Relatively little is known concerning the
binding of MeHg within the RBC and the transfer of Hg from the
RBC to tissues. Recently, White and Rothstein (33) have demon-
strated, in vitro, the reversibility of the binding of MeHg to
human and rat RBCs. The studies we describe were undertaken
to elucidate the mechanism of distribution of MeHg in the rain-
bow trout; in effect, to determine the extent of binding of MeHg
to trout hemoglobin and the mechanism of MeHg transport from the
blood into the tissues/organs.
MATERIALS AND METHODS
Animals, tissue sampling, Hg analysis; Hatchery reared
rainbow trout were obtained from the New York State Hatchery
at Caledonia, New York and maintained as described previously
(24). The collection of tissue samples and the determination
of their Hg content by gamma scintillation spectrometry also
14
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have been described (24),
Reagents; 203Hg-labeled MeHgCl, > 95% radiochemical
purity, was obtained from New England Nuclear, Boston, Mass.
MeHgOH, 100.8% by titration, was obtained from Alfa Inorganics,
Beverly, Mass.
Collection and fractionation of whole blood; The fish
were anesthetized with ethyl m-aminobenzoate methanesulfonate
(MS-222; Sigma Chemical Co., St. Louis, Mo.), the caudal fin
was severed and a volume of blood was collected in an equal
volume of modified Alsever's solution (22). The RBCs, isolated
by centrifugation for 10 minutes at 2000g, were washed once
by resuspension in 2 volumes of Alsever's solution and lysed
in 2 volumes of distilled water. The lysate was incubated
for 6 hours at 5 C with stirring and the stroma removed by
centrifugation at 40,000g for 10 minutes. The stroma was dis-
carded except in the study in which its Hg content was measured.
In that case, it was freed of residual hemoglobin by washing
4 times in 5 volumes of 0.001 M NaH2P04/Na2HP04 buffer, pH 7.3
containing 0.1% NaCl.
Tissue soluble protein extract; Livers were homogenized
in a Virtis Model 60K Homogenizer (Virtis Co., Inc., Gardiner,
N.Y,) at 45,000 rpm for 2 minutes. The tissue:buffer ratio
was 1:1.5 (wt:vol). The buffers employed were 0.1 M Tris-citrate,
pH 6.8, in preparation for gel filtration chromatography and
0.02 M NaH2P04/Na2HP04, pH 7.3, containing 0.14 M NaCl, for the
study of the efflux of MeHg from the RBCs, A soluble protein
15
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extract was prepared by incubating the homogenate at 5°C for
10 minutes and centrifuging for 20 minutes at 40,000g.
Protein concentration measurements: Total soluble protein
concentration was measured by the colorimetric procedure of
Lowry, et^ al^. (9) . Stromal protein concentration was calculated
from total nitrogen values determined by the Kjeldahl method
(21). The concentration of hemoglobin was determined spectro-
photometrically employing the absorbance of cyanmethemoglobin
at 540nm. (23). This method assumes a molecular weight for
hemoglobin of 66,000.
MeHg administration: MeHg was administered intragastri-
cally by stomach tube (24) to fish averaging 350g in weight.
The dose solution (203Hg-MeHg plus carrier MeHg in 0.14 M
NaCl) contained 4,0 mg Hg/kg body weight (B.W.) and 3.3yCi/
kg B.W.
Uptake of MeHg by KBCs in vitro; One ml of a solution
of 203Hg-labeled MeHg in 0.14 M NaCl containing 50yg Hg and
3)iCi was added to 10 ml of whole blood in an equal volume of
Alsever's solution. The suspension was incubated at 3°C
with occasional stirring. At various time intervals, duplicate
aliquots were taken and separated into RBCs and plasma by
centrifugation. The RBCs were washed and the Hg content of
the RBCs and plasma was measured as described (vide supra).
Efflux of MeHg from RBCs in vitro; RBCs containing MeHg
were prepared as described (vide supra). They were isolated
16
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by centrifugation, washed twice with Alsever's solution and
suspended in 0.02 M NaH^PO,/Na2HPO,, pH 7.3, containing
O.lAMNaCl. The suspension contained 85mg hemoglobin/ml
and 5.7yg Hg (0.3yCi)/ml (equivalent to 13yg Hg/ml RBCs
packed at 2000g). Efflux of Hg from the KBCs was investi-
gated by adding 3ml of the suspension to 10ml of a 75mg/ml
solution of trout liver soluble protein in the same buffer.
The RBC suspension added to buffer alone served as the con-
trol. At various intervals, 0.5ml aliquots were withdrawn
and the RBCs isolated by centrifugation and washed. The
Hg content of the RBCs was determined and calculated as the
percentage of the total Hg in the aliquot. There was no
indication that the RBCs had lysed in the liver protein solu-
tion (no leakage of hemoglobin into solution).
The identical procedure was followed using trout hemolysate
(made 0.14 M in NaCl) containing 55mg/ml of hemoglobin in
place of the liver protein solution. In this case, 0.6ml
of the RBC suspension was added to 9.5ml of hemolysate.
Efflux of MeHg from RBCs in vivo: Me203Hg labeled RBCs
were prepared as described (vide supra). The RBCs were sus-
pended in Puck's saline solution (Grand Island Biological
Co., Grand Island, N.Y.) in preparation for injection into
the fish. The MeHg concentration of the suspension was
»
20yg Hg/ml.
17
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Fish averaging 485g in weight were employed. They
were anesthetized, tagged and injected into the heart in
groups of three g-ither with: (i) the KBC bound MeHg to
provide a MeHg dose of O.OSmg Hg/kg B.W, (3.5uCi/kg B.W.)
or (ii) an identical dose of free Me203Hg in Puck's saline.
Whole blood (0.2ml) was withdrawn from the heart of
each fish at various intervals after injection arid analyzed
for Hg concentration. Each sample was corrected to account
for the slight amount of Hg removed from the blood in prior
samplings.
RESULTS AND DISCUSSION
Uptake of MeHg by trout blood components; In vitro,
RBCs take up MeHg very rapidly. Approximately 3 minutes
after the addition of 5ppm MeHg to whole blood in an equal
volume of Alsever's solution, 84% of the Hg was found in
the RBCs. This figure reached 89% after 1 hour and remained
constant for the next 2 hours.
In vivo, < 2% of whole blood Hg was found in the plasma
OQT
2 weeks after an intragastric dose of Me HgCl (Table 1).
Almost 95% was found in the soluble contents of the RBC and
< 4% was bound to the stroma. In a volume of whole blood,
the hemolysate was shown to contain 7 times more protein than
the plasma and to have a Hg binding capacity 10 times that
of plasma on a mg Hg/g protein basis. These factors impart
18
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TABLE 4-1
Methylmercury and Protein Content of Fractions of Rainbow Trout Blood 14 Days After
A Single Intragastric Dose (4 mg Hg/kg Body Weight) of Me20%gCl
Fraction
Hg concentration
mg/100ml
whole blood
% of total
whole blood Hg
Protein
concentration0
g/100ml whole blood
Binding
capability
mg Hg/g protein
Plasma3
Stroma-free
0.07
4.70
1.40
94.80
1.43
9.67
0,05
0.49
' hemolysateb
Stroma
0.18
3.80
1.57
0.12
a. Includes RBC washes
b. Includes stroma washes
c. Calculated from the average of duplicate Kjeldahl analyses by multiplication by 6.25 (1.)
-------
to the hemolysate the ability to accumulate 70 times as much
MeHg as plasma.
Stroma-free hemolysate from the in vivo study (vide supra)
was fractionated by gel filtration chromatography employing
Bio-Gel 0.5m agarose. The protein fraction corresponding to
203
hemoglobin was found to contain 95% of the Hg present in
the hemolysate.
90^
Efflux of MeHg from the RBC; Me JHgCl labeled KBCs were
suspended in protein solution (ref. "Materials and Methods")
to investigate, in vitro, the reversibility of the MeHg-hemo-
globin bond. Figure 4-1 shows that the bond is reversible.
In a 75mg/ml solution of trout liver protein 21% of the RBC
Hg was removed in a 6.5 hour incubation period. Trout hemolysate,
containing 55mg/ml of hemoglobin, removed 36% of the total Hg
in a 12 hour incubation period. Control KBCs place in buffered
saline showed < 4% loss of Hg after 12 hours of incubation.
The rates of disappearance of MeHg from whole blood in
the experiment in which MeHg was injected into the rainbow
trout heart as free MeHgCl or as MeHg bound within washed,
intact RBCs were identical (Figure 4-2). Since MeHg is
taken up rapidly and almost exclusively by RBCs, the rate
at which it is lost from whole blood depends on the net rate
of its efflux through the RBC membrane plus the net rate of RBC
removal from the circulation (RBC replacement). Gel filtration
chromatography of the liver soluble proteins (which contained
20
-------
-I-
\
2 4 6 8 10
Hours After Addition
12
Figure 4-1. The in vitro removal of Mejig frOm washed,
intact, rainbow trout RBCs by protein solu-
tions. Buffer: 0.02 M NaPC^, pH 7.4, con-
taining 0.14 M saline. RBC MeHg concentra-
tion: 13yg Hg/ml packed cells.
85% of the liver Hg) showed identical elution profiles for MeHg
administered as the free salt or bound in RBCs (Figure 4-3).
Each profile had 4 main peaks of radioactivity plus a number of
21
-------
I
24
20
16
8
Days After Hg Injection
8
Figure 4-2. The decrease in Hg concentration factor in
rainbow trout whole blood after the intra-
cardiac injection of Me203HgCl (O.OSmg Hg/kg
body weight). Each value is the average
for 3 different fish. Injection of M
bound in washed RBCs: -o-o-o Injection of
Me HgCl in saline solution: -o-o-o
minor peaks. The Hg was found mainly in the void volume, the
hemoglobin fraction (at an elution volume of 300ml) and two other
fractions of molecular weight < 60,000 (based on molecular weight
22
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0.8 r
400
UN"
250 350
Elution Volume,mis
Figure 4-3.
Gel filtration elution profiles of rainbow
trout soluble liver proteins 8 days after
intracardiac injection of Me203HgCl (O.OSmg
Hg/kg body weight). CPM:
Absorbance at 280nm:
Injection of Me2°3HgCl in saline solution: A
Injection of Me203HgCl bound in washed RBCs: B
Bio Gel A 0.5m; column dimensions: 1.8 x 160cm;
flow rate: 13ml/hr; buffer: 0.1M Tris-citrate,
pH 6.8.
calibration of the column with standard proteins). The distri-
bution of radioactivity in these elution profiles was fundamen-
23
-------
tally identical to that obtained following intragastric admin-
istration of MeHg (Giblin and Massaro, unpublished data).
The demonstration, in vivo and in vitrot of the efflux
of MeHg from RBCs is significant because it is the reversibil-
ity of the binding of MeHg within the RBC which allows for the
transfer of Hg to the tissues.
Determination of hemoglobin -SH content; The number of
-SH groups per molecule of rainbow trout hemoglobin was deter-
mined by amperometric titration at the dropping mercury elec-
trode (28). MeHg is a highly specific reagent for protein -SH
groups (25) and was used to titrate the hemoglobin of freshly
prepared hemolysates which had been dialyaed for 20 hours against
2 changes of 100 volumes each of distilled water. Titrations
were performed in 0.1 M Tris-HCL, pH 7.4, and in the same buffer
made 8 M in urea. The values obtained were 3.7 and 4.3 moles
-SH per mole hemoglobin, respectively (Figure 4-4). Titrations
of standard solution of glutathione and bovine serum albumin
yielded literature -SH values. Since the rainbow trout hemo-
globin consists of 3 major and approximately 9 minor types
(27,32), the -SH value obtained for hemolysates must be taken
as the average of all the types.
When MeHg is added to a solution of rainbow trout hemoglo-
bin near neutral pH the solution becomes cloudy at a concentra-
tion of approximately 3 moles of MeHg per mole hemoglobin. This
does not occur in 8 M urea at the same pH. Except for making the
24
-------
1
A
04
0.3
0.2
0.1
-
* /
/
•
/ 3.7MoleSH/Mole
• ••••• \* Hemoqlobin
\
0.4
0.3
0.2
B
4.3 Mole SH/Mole
Hemoglobin
0.4
-I-
1.6
20
mlsCH3HgCI(2x103M)
Figure 4-4.
Determination of the number of sulfhydryl
groups of dialyzed rainbow trout hemolysate
(0.40 x 10~3 mmoles) by amperometric titra-
tion with Me203HgCl. Temperature: 22°C;
potential: -0.6V (vs. saturated calomel
electrode); total volume: 19 ml; solutions
contained 0.015% gelatin and 0.04 ml octanol;
MeHgCl dissolved in a 25% solution of di-
methylformamide in water.
0.1 M Tris-HCl, pH 7.4, made 8 M in urea: A
0.1 M Tris-HCl, pH 7.4: B
end point less sharp, the clouding did not appear to affect the
-»
amperometric data. The number of -SH groups obtained with and
25
-------
without 8 M urea was approximately 4 which would indicate that,
under physiological conditions, all of the -SH groups of trout
hemoglobin are available to bind MeHg.
The existence of 4 reactive -SH groups per hemoglobin
molecule makes the trout RBC at least 20mM in reactive -SH
(based on a measured RBC hemoglobin content of 300-350 mg/ml
RBCs packed at 2000g). It is this highly localized concentra-
tion of -SH which draws MeHg into the interior of the RBC and
binds it.
As we have demonstrated (vide supra), a high concentra-
tion of -SH outside the RBC induces the efflux of MeHg from
the cell and forms the basis for the transfer of MeHg from the
RBC to tissues. A possible mechanism for the efflux is illus-
trated in Figure 4-5. The mechanism is based on the fact
that a small, but definite proportion of MeHg in the presence
of excess thiol will be associated with halide as dictated
by the Mass Law (26). Highly lipid soluble MeHgCl traverses
the cell membrane to -SH groups outside the cell. Abundant
membrane -SH groups (11) facilitate transport through the
membrane. The exit of MeHgCl causes a shift in the equili-
brium inside the cell which is reestablished by disassocia-
tion of MeHg from hemoglobin. Eventually a dynamic MeHg equil-
ibrium is established based on the relative concentrations of
hemoglobin, RBC membrane, plasma and tissue -SH groups.
26
-------
RED
BLOOD
CELL
TISSUE
CELL
Hb02-S-HgMe
IK
*f
MeHgCl
Membrane protein-S-HgMe
MeHgCl
It
Membrane protein-S-HgMe
MeHgCl
Protein-S-HgMe
Figure 4-5, Proposed mechanism of the transfer of MeHg
from trout RBCs to tissues.
27
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SECTION V
REFERENCES
1. Ackefors, H. Mercury Pollution in Sweden with Special
Reference to Conditions in the Water Habitat. Proc.
Roy. Soc. London. _177_: 365-387, 1971.
2. Backstrom, J. Distribution Studies of Mercuric Pesti-
cides in Quail and Some Freshwater Fishes. Acta Pharmacol.
Toxicol. 27_, Suppl. 3:1-103, 1969.
3. Berglund, R. and M. Berlin. Rish of Methylmercury Cumu-
lation in Man and Mammals and the Relation Between Body
Burden pf Methylmercury and Toxic Effects. In: Chemical
Fallout> Current Research on Pesticides, Miller, M.W. and
Berg, G.G. (eds.). Springfield, C.C. Thomas, 1969. p.
258-273.
4. Birke, 6., A.6. Johnels, L.-O. Plantin, B. Sjostrand and
T. Westermark. Mercury Poisoning through Eating Fish?
Lakartidningen. 64:3628-3637, 1967.
5. Hannerz, L. Experimental Investigations on the Accumulation
of Mercury Compounds in Water Organisms. Rep. Inst. Fresh-
water Res. Drottningholm. 48_: 120-176, 1968.
6. Irukayama, K. The Pollution of Minamata Bay and Minamata
Disease. Advan. Water Poll. Res. 2k 153-180, 1966.
7. Jarvenpaa, T., M. Tillander, and J.K. Miettinen. Methyl-
mercury: Half-time of Elimination in Flounder, Pike and Eel.
Suom. Kemistilehti B. 43_: 439-442, 1970.
8. Johansson, F., R. Ryhage, and G. Westoo. Identification and
Determination of Methylmercury Compounds in Fish Using Com-
bination Gas Chromatograph-Mass Spectrometer. Acta Chem.
Scan. £4:2349-2354, 1970.
9. Miettinen, V., Y. Ohmomo, M. Valtonen, E. Blankstein, K.
Rissanen, M. Tillander, and J.K. Miettinen. Preliminary
Notes on the Distribution and Effects of Two Chemical Forms
of Methyl Mercury on Pike. Fifth RIS (Radioactivity in
Scandinavia) Symposium, Department of Radioactivity, Univ.
of Helsinki. 1969.
10. Pillay, K.K.S., C,C. Thomas Jr., J.A. Sondel, and C.M. Hyche.
Determination of Mercury in Biological Environmental Samples
by Neutron Activation Analysis. Anal. Chem. 43:1419-1425,
1971.
28
-------
11. Raeder, M.G. and E. Snekvik. Mercury Determinations in
Fish and Other Aquatic Organisms. Kgl. Nor. Vidensk.
Selskabs. Forh. 21:102-109, 1949.
12. Methyl Mercury in Fish. A Toxicologic-Epidemiologic
Evaluation of Risks. Report from an Expert Group. Nord.
Hyg. Tidskr. 1971. Suppl. 4, 364 p.
13. Rothstein, A. and A. Hayes. The Metabolism of Mercury
in the Rat Studied by Isotope Techniques. J. Pharmacol.
Exp. Ther. 13£:166-176, 1960.
14. Skerfving, S., A. Hansson, and J. Lindsten. Chromosome
Breakage in Human Subjects Exposed to Methyl Mercury through
Fish Consumption. Arch. Environ. Health. 2^:133-139, 1970.
15. Stock, A. and F. Cucuel. Die Verbreitung des Quecksilbers.
Naturwiss. 2^:390-393, 1934,
16. Swensson, A. and U. Ulfvarson. Distribution and Excretion
of Mercury Compounds in Rats Over a Long Period after a
Single Injection. Acta Pharmacol. Toxicol. 26:273-282,
1968.
17. Takeuchi, T. Pathology of Minamata Disease. In: Minamata
Disease, Study Group of Minamata Disease, Kutsuna, M (ed.).
Kumamoto, Kumamoto Univ., 1968. p. 141-228.
18. Takeuchi, T. Biological Reactions and Pathological Changes
of Human Beings and Animals Under the Condition of Organic
Mercury Contamination. International Conference on Environ-
mental Mercury Contamination. Ann Arbor, Michigan. 1970.
19. Tsurga, H. Tissue Distribution of Mercury Orally Given to
Fish. Full. Jap. Soc. Sci. Fish. 29^403-409, 1963.
20. Wetb'o, G. and K. Noren. Mercury and Methylmercury in Fish.
Var fo'da. JLO: 138-178, 1967.
21. Bradstreet, R.B. The Kjeldahl Method for Organic Nitrogen.
New York, Academic Press, 1965.
22. Bukantz, S.C., C.R. Rein and J.F. Kent. Studies in Comple-
ment Fixation. J. Lab. Clin. Med. ^:393-404, 1946.
23. Cannon, R.K. Proposal for a Certified Standard for Use in
Hemoglobinometry-Second and Final Report. J. Lab. Clin. Med.
'52:471-476, 1958.
29
-------
24. Giblln, F.J. and E.J. Massaro. Pharmacodynamics of Methyl-
mercury in the Rainbow Trout (Salmo gairdneri): Tissue Uptake,
Distribution and Excretion. Toxicol. Appl. Pharmacol. 24:
81-91, 1973.
25. Hughes, W.L. A Physicochemical Rationale for the Biological
Activity of Mercury and Its Compounds. Ann. N.Y. Acad. Sci.
£5_:454-460, 1957.
26. Hughes, W.L. Protein mercaptides. Cold Spring Harbor Symp.
Quant. Biol. U.: 79-84, 1950.
27. luchi, K. and K. Yamagomi. Electrophoretic Pattern of Larval
Haemoglobins of the Salmonid Fish, Salmo gairdneri irideus.
Comp. Biochem. Physiol. _28_:977-979, 1969.
28. Leach, S.J. The Reaction of Thiol and Bisulphide Groups with
Mercuric Chloride and Methylmercuric Iodide. Aust. J. Chem.
jJ:520-546, 1960.
29. Lowry, O.K., N.J. Rosenbrough, L.A. Farr and R.J. Randall.
Protein Measurement with Folin Reagent. J. Biol. Chem.
193:265-275, 1951.
30. Nordberg, G.F. and S. Skerfving. Metabolism of Mercury. In:
Mercury in the Environment, Friberg, L. and Vostal, J. (eds.).
Cleveland, CRC Press, 1972. p. 29-90.
31. Passow, H. The Red Blood Cell: Penetration, Distribution
and Toxic Actions of Heavy Metals. In: Effects of Metals on
Cells, Sub-cellular Elements and Macromolecules, Maniloff, J.,
Coleman, J. and Miller, M. (eds.). Springfield, Charles C.
Thomas, 1970. p. 291-344.
32. Tsuyuki, H. and R,E. Gadd. The Multiple Hemoglobins of Some
Members of the Salmonidae Family. Biochem. Biophys. Acta
21:219-221, 1963.
33. White, J.F. and A. Rothstein. The Interaction of Methylmercury
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1973.
30
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-660/3-74-027
4. TITLE AND SUBTITLE
PHARMACOKINETICS OF TOXIC ELEMENTS IN RAINBOW TROUT
6. PERFORMING ORGANIZATION CODE
3. RECIPIENT'S ACCESSIONING.
5. REPORT DATE
December, 1974 (issue)
7. AUTHOR(S)
Massaro, Edward J.
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORG \NIZATION NAME AND ADDRESS
SUNY at Buffalo
Buffalo, New York 14214
10. PROGRAM ELEMENT NO.
1BA022
11. CONTRACT/GRANT NO.
800989
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
IB. SUPPLEMENTARY NOTES
18. ABSTRACT ^'JJHg-methylmercury (MeHg) was administered intragastrically in a single
dose (O.Smg Hg/kg=3.3uCi/kg) to trout (av. wt. 250g). The fish were sacrificed from
1.0 hr. to 290 days postadministration. Twenty tissues were analyzed for MeHg content
by gamma scintillation spectrometry. The label was taken up rapidly by blood, gills,
spleen, liver and kidney and more slowly by muscle, brain and lens. After 290 days:
(a)blood, spleen, kidney, liver and lens had concentration factors (C.F.=tissue Hg
conc./Hg dose x final tissue wt./initial tissue wt.)=l-0;(b)in general, C.F.s had
dropped off by at least 2/3 from their maxima except for muscle, brain and lens in
which the C.F.s remained=their maxima; (c)~64% of the dose still remained in the fish
and skeletal muscle (comprising "55% of body weight) contained >40% of the residue.
Assuming MeHg excretion to be a first order process, there are at least two rates of
excretion—a rapid initial rate resulting in a biological half-retention time (HRT) of
200 days and a slower, subsequent rate yielding an HRT of> 1000 days which, apparently,,
is governed by the rate of release of MeHg from the skeletal muscle.
Hemoglobin (Hb) is the main methylmercury (MeHg) transport protein in trout blood.
In vitro. MeHg is taken up rapidly in 3 minutes by red blood cells. MeHg binding in
the RBC is reversible jln vitro as demonstrated by the efflux of Hg from RBCs suspended
in protein solutions. MeHg binding in the RBC also is reversible in vivo as gel fil-
tration chromatography of liver soluble proteins yielded indentical elution profiles
tor MeHg administered as the free salt or bound in RBCs. The number of reactive sulf-
tiydryl (-SH) groups per molecule of Hb was found to be 4 by amperometric titration with
MeHgCl. The reactive -SH concentration in the RBC was calculated to be at least 20triM0
A mechanism for the efflux of MeHg from the RBC is proposed involving the dissociation
of MeHg from Hb initiated by -SH groups outside the RBC and migration of MeHg across
. t_ > _ _ ts C\J \*i/~* n r\«* A m r-i r^*-\r*t it«r-**-rjiKijtnrurt... ^
the membrane as MeHgCl.
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