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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series.  This series describes research  on the effects of pollution
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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

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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

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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

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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.

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       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

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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

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      -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

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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

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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

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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

-------
                             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

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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
     with Erythrocytes.  Toxicol. Appl. Pharmacol.  26:370-384,
      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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