TOXICOL4XiYAND
APPLIEDPHARMACOLOGY
Time-Dependent zo3Hg in Mice
32,432-442(1975)
Tissue/Organ Uptake and Distribution Exposed to Multiple Sublethal Doses Methyl Mercury
of of
PAUL SALVATERRA,EDWARDJ. MASSARO,~JOHNB. MORGANTI AND BRADLEYA.LOWN State University of New York at Buflaio, Buffalo, New York 14214 and Department of Psychology, State University College at Buflalo, Buffalo, New York 14222 Received August 26,1974; accepted October 29,1974
Time-Dependent Tissue/Organ Uptake and Distribution of ‘03Hg in Mice Exposed to Multiple Sublethal Dosesof Methyl Mercury. SALVATERRA, P., MASSARO, E. J., MORGANTI, J. B. AND LOWN, B. A. (1975). Toxicol. Appl. Pharmacol. 32, 432442. Swiss-Webstermice were injected intraperitoneally with one to ten doses(2.5 mg Hg/kg) of 203Hg-labeled methyl mercury. Doses were administeredat 72-hr intervals. Maximal tissue/organ Hg uptake usually occurred 72 hr postinjection and was monitored at 3 and 6 days by gamma scintillation spectrometry. Most tissues/organsrequired five to six dosesto attain maximal Hg concentrations (the carcassrequired sevendoses;hair and fat, eight doses;lens,nine doses).Data for hair were highly variable. Except for hair (which required seven to eight doses),maximal tissue/organconcentration factors (CF) were reached 72 hr after the first dose. Kidney, liver, and hair attained CF values> 1. Exceptfor hair, all CF valuesdecreased with increasingdose number. Initial rates of decreasewere much greater for kidney, blood, spleen,muscle,and liver than lens, brain, and fat. Only hair exhibited a significantly higher Hg concentration at 6 days after each dosethan at 3 days. With increasing dose number, blood Hg; tissue/organHg ratios remainedrelatively constantfor liver, kidney, spleen,and muscle;decreased for lensand brain; and decreasedfor hair and fat after an initial increase. Methyl mercury (MeHg) is recognized as the major industrial pollutant responsible for casesof human poisoning in Minimata and Niigata, Japan (Expert Group Report, 1971; Friberg and Vostal, 1970). Fatalities and neurologic disturbances have been traced to repeated consumption of contaminated fish and shellfish (Takeuchi, 1972). Since this discovery, concern over high MeHg levels in fishesand the aquatic environment in general has spurred intensive research into the distribution of this potentially lethal compound in the human food chain (Backstrom, 1969; Berglund and Berlin, 1969; Westoii, 1969; Jernelov, 1969). However, very little effort has been expended on establishing the overall mechanism of toxic action (Berlin et al., 1965; Chang and Hartmann, 1972a, b; Cremer, 1962; Diamond and Sleight, 1972; Klein et al., 1972; Kuwahara, 1970; Patterson and Usher, 1971; Salvaterra et al., 1973; Yoshino et al., 1966a, b). 1 To whom all correspondence should be addressed. Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214. 432 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form reserved.
METHYLMERCURY
‘IISSUE/ORGAN
DlSTRIBUTION
433
Tissue/organ distribution and pharmacodynamic studies have been performed using a variety of species-eg., rats (Norseth, 1969, 1970; Swensson et al., 1959; Ulfvarson, 1969), cats (Kitamura, 1968), dogs (Swensson et al., 1959), guinea pigs (Iverson et ul., 1973), fish (Giblin and Massaro, 1973), and mice (ijstlund, 1969; Suzuki et al., 1963 ; Suzuki, 1969). Most of these studies monitored the fate only of single doses. Also tissue/organ uptake and release rates have been followed only for short time periods (Berlin and Ullberg, 1963; &tlund, 1969; Suzuki et al., 1963; Swensson and Ulfvarson, 1968). Moreover, where data are available, the Hg content of only a limited number of tissues/organs has been assessed. In this study, we administered repeated doses (up to 10) of MeHg and determined concentrations in 10 tissues/organs at intervals of 3 and 6 days following each dose. The dose level selected for multiple administration was approximately 20% of the single dose 7-day LD50. The time period between doses was 3 days since major symptoms of MeHgintoxication are related to CNS dysfunction (Expert Group Report, 1971, Takeuchi, 1972) and 3 days are required to achieve maximal Hg concentrations in the brain of the mouse after a single dose of MeHg (Salvaterra et al., 1973). METHODS Subjects Mice. Two hundred male Swiss-Webster mice (Carworth Breeding Laboratories, New York) which had a mean weight of 37 g at the beginning of the experiment and 42 g at the end served as subjects. They were housed individually in stainless steel cages with wire mesh bottoms and had free access to food and water. Materials MethyZ mercury.
MeHgOH (Alfa Inorganics, Beverly, Mass.) was dissolved in 0.14 M NaCl to yield a solution containing approximately 0.25 mg Hg/ml as MeHgCl.’ This was combined with 203Hg-labeled MeHgCl (New England Nuclear, Boston, Mass.) in 0.01 M Na&O, (4.97 mCi/ml) to produce solutions containing 0.25 mg Hg/ml with specific activities of approximately 3 and 10 &i/ml. The lo-&i/ml solution was employed in the initial five series of intraperitoneal (ip) injections and the 3-@/ml solution in the last five series, Procedure LD.50 determination.
25 mg Hg (as MeHg)/kg,
Groups of eight mice were injected ip with 5, 10, 15, 20, or respectively, of stable MeHgCl in NaCl solution. The LD50
z To check the stability of the isotope solutions and the identity of the anion associated with the MeHg, thin-layer chromatography was employed. Fresh samples and samples of I-mo-old injection solution, MeHgOH in H20, HgC12 in 0.14 M NaCl, MeHgCl in 0.14 M NaClandmixtureswerechromatographed on commercially prepared (Analtech, Inc., Newark, Delaware) cellulose and silica gel G thin layer plates (250 pm thick, 1 x 3.5 or 3.5 x 6 in.); 5 or 1Oq.d samples were applied with a Hamilton microliter syringe (Hamilton Co., Whittier, Calif.) and the plates developed at room temperature, in 3 M NH.+OH. After drying at room temperature, Hg-containing spots (a yellow spot on a green/brown background) were visualized by spraying with a 0.4% (w/v) benzene solution of dithiazone (diphenylthiocarbazone, Merck). The plates were then scored in l-cm sections, scraped into test tubes, and radioactivity determined by gamma counting. The results indicate complete conversion of the OHto the Cl- in 0.14 M NaCl and also indicated a radiochemical purity of >95% after storage of the solution for 1 mo.
434
SALVATERRA
ET AL.
and slope function, along with 95 % confidence limits, were determined according to Litchfield and Wilcoxon (1948). Dosing of animals. This study was designed so that the total quantity of MeHg administered would be fatal to all animals if administered in a single dose. One hundred animals received 2.5 mg Hg/kg (1.0 ml of injection solution/100 g body wt) ip, and an equal number of controls received 1.Oml of 0.14 M NaCl/lOO g. Tissue ‘03Hg concentrations were monitored 3 and 6 days after each dose by gamma spectrometry (de i@a). Three days after the first dose, ten animals were selected randomly from each group. Five animals from the MeHg-treated group were sacrificed immediately for determination of tissue concentrations of ‘03Hg and the remaining five were sacrificed 3 days later. The remaining experimental animals were reinjected with MeHg and the controls with saline. This experimental design ultimately generated two series (sacrificed at 3 and 6 days, respectively) consisting of ten groups of five mice each receiving a total of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, and 25.0 mg Hg/kg (as MeHgCl), respectively. The last group of 6-day postinjection animals contained only four experimentals and four controls since one animal of each group died before the completion of the experiment. Collection of tissue samples. The animals were sacrificed by cervical dislocation. Tissues were rapidly dissected, blotted dry and placed in tared, air-tight polyethylene vials, and weighed. Hair and fat samples were obtained from carcasses stored at -20°C for approximately 3 wk. A preliminary investigation had shown no alteration in hair and fat 203Hg contents of carcasses stored as long as 6 wk. Tissue and organ sampling proceeded as follows : The pericardial cavity was opened and whole blood was drawn from the heart by syringe (an aliquot of approx. 100 ~1 was monitored for radioactivity). A portion of the right lobe of the liver was removed (no significant differences were found in 203Hg content among the lobes or various areas of individual lobes of this organ). The spleen was dissected free of connective tissue and removed in toto. The left kidney was exposed and the posterior half removed (no significant differences in 203Hg concentrations were found between the anterior and posterior halves of this organ). The right hind leg was skinned and the gastrocnemius muscle removed. Skin was removed from the head and the whole brain was extirpated and dissected into cerebellum and cerebral cortex. Normally, both lenses were assayed together (no significant difference in 203Hg concentrations between the left and right lens was observed). Hair (approx. 10 mg) was shaved from the middle portion of the back just below the neck with a scalpel and small pieces of contaminating skin were removed by freehand dissection under a magnifying lens. Subcutaneous fat was scraped from the skin beneath the hair sampling site. Of all the data, those for hair were the most variable. An explanation of this phenomenon was sought. To assessthe contribution of possible surface contamination to this variability, hair samples were washed with detergent, rinsed with distilled water, oven dried, and counted. No difference between washed and unwashed hair was found, nor was 203Hg detected in hair of control animals. Hair from the hind limb of some animals also was sampled and the results obtained agreed with those for hair taken from the back. For determination of “whole body” concentrations of 203Hg, the frozen carcasses were fragmented and homogenized with an equal amount of water (w/v) at high speed
METHYLMERCURY
TISSUE/ORGAN
435
DISTRIBUTION
in a Waring Blendor until a smooth homogenate was obtained. An aliquot corresponding to approximately 2 g of original tissue weight was used for counting. Determination
of radioactivity
and calculation
of tissue/organ
Hg concentrations.
Tissue radioactivity was measured in a Packard gamma scintillation spectrometer (model 5319). In general, enough counts were collected for each sample to hold the counting error to less than 1%. Counting efficiency averaged 28.1 “//o. The total amount of mercury in each tissue was calculated by comparison of experimental samples with triplicate standards prepared each injection day from 10-/d aliquots of the injection solution which were placed in 0.3 ml of 10% bovine serum albumin (BSA) and sealed in glass ampules. No detectable loss of 203Hg from the MgHg-BSA standards or the injection solution occurred during the course of the experiment. Terminology. The ability of each tissue to concentrate Hg relative to the dose can be expressed by the term concentration factor (CF) (Giblin and Massaro, 1973). CF is calculated by dividing the tissue Hg concentration (pg Hg/g wet wt) by the total accumulated dose (mg Hg/kg). A number greater than 1 indicates the ability of that tissue/organ to accumulate mercury above the cumulative dose. RESULTS
The LD50 and 95 % confidence limits for 7 days (or 3 mo) was estimated to be 13.5 (9.3-19.6) mg Hg (as MeHg)/kg. The dose-response curve was rather steep with a slope function of 1.6 (1.3-2.0). Table 1 shows the survival rate of the mice for each dose TABLE SURVIVAL
RATE
Time after dose (days) 1 2 3 4 5 6 7
3mo
1
OF MICE AT INCREASING INJECTED INTRAPERITONEALLY
LEVELS
OF MeHg
5
Dose level (mg Hg/kg) ______10 15 20
25
gb 8 8 8 8 8 8 8
8 8 8 7 7 7 6 6
2 2 2 2 2 2 2 2
8 6 3 2 2 2 2 2
5 3 2 1 0 0 0 0
a As MeHgCl.
* Number of animals (out of eight) surviving at this time and dose level.
level. All animals which survived the first day, but eventually died, exhibited weight loss and certain CNS signs of MeHg intoxication (e.g., loss of muscular control and piloerection). However, no tremors were observed in any group.
436
SALVATERRA ET AL.
Maximal Hg concentrations occurred in most tissues/organs 3 days after administration of doses 5 or 6 (a total of 12.5-15.0 mg Hg/kg) and then decreased, though not statistically significantly (e.g., the tissue Hg concentrations had reached a plateau). This dose-response pattern is illustrated in Fig. 1 for the 3-day sampling period. It was similar for the 6-day period. For hair and fat (8 doses = 20 mg Hg/kg) and lens (9 doses = 22.5 mg Hg/kg) the plateau was shifted to the right (e.g., these tissues
1-1-1-1-1-1-1-1-1-1 LiverQluscle-,Lenso
3
6
/-,-1-,-f 9 12 15 18 21 24 27 30
Doys
FIG. 1. Total accumulated tissue/organ concentrations of mercury (Hg) 3 days after the terminal dose of a series of intraperitoneal doses of methyl mercury (MeHg). Each dose consisted of 2.5 mg Hg (as MeHg)/kg and was administered every 3 days. Each point represents the mean + SE for five animals.
Cumulative
Dose (mq Hq/kq)
2. Whole body concentrations of Hg 3 days after the terminal dose of a series of doses of MeHg administered ip. Each dose consisted of 2.5 mg Hg (as MeHg)/kg. The Hg concentrations were calculated from measurements of 203Hg in homogenates of five pooled carcasses. FIG.
required a higher dose and/or a longer time to reach a plateau). The “whole body” concentration of Hg was maximal after 7 doses (17.5 mg Hg/kg) as shown in Fig. 2. Since five animals were pooled before homogenization, no measure of individual variability is available. Six aliquots of homogenate were counted and gave standard errors of 2 % or less.
METHYLMERCURY
TISSUE/ORGAN
437
DISTRIBUTION
In Fig. 3 the CF for each tissue 3 and 6 days after each dose is plotted vs the total accumulated dose. The only tissues exhibiting CF values greater than 1 at any time were: kidney (max CF >9.6), liver (max CF >1.5), and hair (max CF >5.5). Hair differed from the other tissues with respect to the number of doses required to reach the maximum CF. For all tissues/organs except hair, the maximum CF was reached 3 days after the initial dose. For hair, the value of the maximum CF was variable and was reached only after 7 or 8 doses (17.5-20.0 mg Hg/kg; Fig. 3). All CF values except that of hair decreased as total dose increased. Moreover, it should be noted that, in relation to Hg uptake, hair does not appear to exhibit saturation kinetics (Fig. 1). Apparently, most tissues were not able to concentrate Hg at the same rate at which total dose was increased. This is not explained by the expected decrease
CF ?
CF
4-
B Spleen
I Kidney
Cerebellum
0
50
(00
150
200
250
Cumulative Dose ( mg Hg/kg I FIG. 3. Concentration factors (CF) for zo3Hg for tissues/organs of the mouse 3 and 6 days after the terminal dose of a series of doses of MeHg administered ip. Each dose consisted of 2.5 mg Hg (as MeHg)/kg. Each point represents the mean for five animals + SE.
438
SALVATERRA ET
AL.
in CF (as the total dose was increased) due to weight gain during the course of the experiment. That is, the (partial) correction for weight gain obtained by multiplying the CF by the ratio, average weight of each dose group over initial average weight of each group, makes no significant difference. Two other features of Fig. 3 should be noted. Initially, the rates of decrease of the CF values of certain tissues (kidney, blood, spleen, muscle, and liver) are relatively large while those of other tissues (lens, cerebellum, cortex, and fat) are considerably smaller. Also the 3- and 6-day data are of similar magnitude and, in some cases (lens, cerebellum, cortex, and fat), the curves intersect indicating occasional continued uptake through the 6-day sampling interval. For each tissue, the ratio, Hg concentration 6 days after each dose divided by Hg concentration 3 days after each dose, did not vary in any apparent systematic manner
FIG. 4. Averageof the ratio, Hg(tissue/organ) concentration6 daysafter eachdosedividedby Hg concentration3 daysafter eachdose,of a series of dosesof 203Hg-labeled MeHg (2.5 mgHg/kg) administered ip. BarsrepresentSE.
with dose. Therefore, the ratios of the individual tissues were averaged for all ten dose levels and are presented in order of decreasing magnitude in Fig. 4. The only tissue exhibiting a significantly higher concentration of Hg 6 days after each dose compared to 3 days was hair. Fat, lens, cortex, and cerebellum retained 75-100x of the 3-day values at 6 days while the remaining tissues retained only 70 % or less. The rapid tissue distribution of ip injected MeHg in animals indicates that it must be transported primarily by the blood (White and Rothstein, 1973). In order to assess any dose-dependent changes in the partitioning of Hg between the blood and the tissues, the blood Hg/tissue Hg ratios were calculated and plotted vs total dose in Fig. 5. For clarity, only the 6-day data are presented (the 3-day values are similar). A relatively constant ratio is observed for the liver, kidney, spleen, and muscle indicating a constant distribution of Hg between blood and these tissues. However, in the case of lens, cortex, and cerebellum the ratio decreases reflecting a greater proportion of Hg
METHYLMERCURY
IO 0 I-
TISSUE/ORGAN
439
DISTRIBUTION
\
O’
Fat Hair
.-J-~;~~
5
10
15
zo-
i5-
CumulativeDose(mg Hg /kg 1 FIG. 5. Blood-tissue ratios of 203Hg 6 days after the terminal dose of a series of 2.5 mg Hg (as MeHg)/kg doses administered ip. Each point represents the average of five animals.
in these tissues compared to blood as the total dose increased. Hair and fat show an initial increase in the ratio followed by a decrease. This initial increase reflects the fact that there is a much slower rate of uptake of Hg by these tissues compared to blood at the time of the first few sampling periods (Fig. 1). DISCUSSION
Saturation kinetics for MeHg uptake were exhibited by all tissues with the possible exception of hair. This was somewhat unexpected since saturating tissue concentrations have not been identified as such in the literature for MeHg administered in repeated doses (Friberg, 1959; Gage, 1964; Suzuki, 1969; Suzuki et al., 1963; Swensson et al., 1959; Ulfvarson, 1962). Possible explanations for this could include: (a) an insufficient time period between doses for the MeHg to reach a state of dynamic equilibrium among the tissues; (b) use of very large doses which caused extensive tissue necrosis; or (c) very small doses which did not. permit accumulation of sufficient tissue concentrations to show saturation kinetics; (d) insufficient number and/or distribution of sampling intervals. In this study nearly all tissues achieved maximal Hg uptake during the 3-day interval between doses (Fig. 4), indicating the establishment of dynamic equilibria. The only tissue which did not consistently attain a maximum Hg concentration between sampling intervals (3 and 6 days after each dose) was hair (Fig. 4). Since hair is metabolically inactive and concentrates Hg significantly, it may function as an important excretion route of this compound (i)stlund, 1969; Ulfvarson, 1970). Quantitatively [e.g., its large CF (Fig. 3) and continuous growth], hair may play a major role in the regulation of tissue MeHg concentrations in hairier species (e.g., mice, rats, and lower primates) vs relatively hairless animals such as man. The continuous uptake of MeHg by hair and its high capacity for this compound may explain, in part, why mice are able to tolerate a relatively large dose of MeHg administered in small repeated doses. It should be noted that none of the animals in this study developed any CNS signs characteristic of MeHg intoxication.
440
SALVATERRA ETA.
The individual variability in hair Hg concentrations seen in this study (Fig. 1) and in humans (Birke et al., 1972) may be related to individual differences in susceptibility to MeHg intoxication. Thus, hair of different composition may have a greater or lesser capacity to bind Hg, or assuming a finite rate of binding, the more rapid the turnover (growth and fallout) the less susceptible the animals to intoxication. Saturation kinetics for tissue MeHg concentrations could be explained by an increase in elimination rate, a decrease in absorption, or both. In this study, an apparent increase in elimination rate could be seen for the Hg eliminated via the hair. Other major excretion routes of MeHg such as the urine and feces (Friberg, 1959; Gage, 1964; &tlund, 1969) were not investigated in this study. Thus, no definite conclusions can be drawn concerning the effects of dose and time on the rate of Hg excretion via these routes. However, in other studies (unpublished observations) we have observed that fecal excretion plays an important role in MeHg elimination after a single dose. In contrast, only a minor quantity of Hg is excreted via the urine. A decrease in the rate of absorption of the MeHg may not be ruled out, however, since a thickening of the peritoneum was observed in some experimental as well as a few control animals and probably represents a side effect of the ip route of administration. No grossly visible peritonitis or loss of subcutaneous fat was observed as has been reported for rats receiving weekly ip doses of 10 mg Hg/kg (Diamond and Sleight, 1972). Thickening of the spleen capsule also was observed in most animals receiving six or more doses of MeHg (not in controls). The constant blood/tissue Hg ratios for kidney, liver, spleen, and muscle (Fig. 5A) indicate a simple distribution equilibrium. On the other hand, a decrease in this value as seen for the lens, cerebellum, and cortex, indicates a more complex partitioning mechanism. Thus, in contrast to previous suggestions (Expert Group Report, 1971), this observation indicates that blood may not be the ideal tissue for estimating Hg content of brain after repeated exposure. Moreover, the greater ability of the lens, cerebellum, and cortex than the blood to bind Hg (Fig. 4) on repeated exposure (Fig. 5B) may explain the apparent target nature of these tissues in MeHg poisoning (Backstriim, 1969; Suzuki, 1969). Metallothionein (Jakubowski et al., 1970; Pulido et al., 1966) may be responsible for the ability of the kidney and, to some extent, the liver, to concentrate Hg (Fig. 1, 3). This study provides data on the dynamics of MeHg in a model system after repeated exposure to sublethal doses. These data may be of value in the understanding of the biochemical and behavioral changes observed after repeated exposure to MeHg.
REFERENCES studies of mercuric pesticides in quail and some freshwater fishes. Acta Pharmacol.Toxicol. 27 (Suppl. 3), l-103. BERGLUND, F. AND BERLIN, M. (1969). Risk of methylmercury cumulation in man and mammals and the relation between body burden of methylmercury and toxic effects. In ChemicalFallout (M. W. Miller and G. G. Berg, eds.), pp. 258-273. Thomas, Springfield. BERLIN, M., JERKSELL, L. G. AND NORDBERG, G. (1965). Accelerated uptake of mercury by brain caused by 2,3-dimercaptopropanol (BAL) after injection into the mouse of a methylmercuric compound. Acta Pharmacol.Toxicol. 23, 312-320. BACKSTR~M, J. (1969). Distribution
METHYLMERCURY TISSUE/ORGAN DISTRIBUTION BERLIN,
M.
AND
ULBERG,
441
S. (1963). Accumulation and retention of mercury in the mouse.
Arch. Environ. Health 6, 610-616.
G., JOHNELS, G., PLANTIN,L.-O., SJ~STRAND, B., SKERFVING, S. ANDWESTERMARK, T. (1972). Studieson humansexposedto methylmercury through fish consumption. Arch. Environ. Health 25, 77-91. CHANG, L. W. AND HARTMANN, H. A. (1972a).Ultrastructural studieson the nervoussystem after mercuryintoxication. I. Pathologicalchangesin the nervecell bodies.Acta Neuroparhol. 20, 122-138. CHANG, L. W. AND HARTMANN, H. A. (197213).Blood-brain barrier dysfunction in experimental mercury intoxication. Acta Neuropathol. 21, 179-184. CREMER, J. E. (1962).The action of triethyl tin, triethyl lead,ethyl mercury and other inhibitors on the metabolismof brain and kidney slicesin vitro using substrateslabeled with 14C. BIRKE,
J. Neurochem.
9,289-298.
S. D. (1972).Acute and subchronicmethyl mercury toxicosis in the rat. Toxicol. Appl. Pharmacol. 23,197-207. EXPERT GROUP REPORT (1971).Methyl mercury in fish: A toxicologic-epidemiologic evaluation of risks. Nord. Hyg. Tidskr. Suppl. 4, 364 pages. FRIBERG, L. (1959). Studieson the metabolismof mercuric chloride and methyl mercury dicyandiamide.AMA Arch. Industr. Health 20,42-49. FRIBERG, L. AND VOSTAL, J. (eds.)(1970).Mercury in the environment: An Epidemiological and Toxicological Appraisal. ChemicalRubber Co. Press,Cleveland,Ohio. GAGE, J. L. (1964). Distribution and excretion of methyl and phenyl mercury salt. Brit. J. Industr. Med. 21, 197-202. GIBUN, F. J. AND MASSARO, E. J. (1973).Pharmacodynamicsof methyl mercury in the rainbow trout (Salmo gairdneri) : Tissueuptake, distribution andexcretion. Toxicol. Appl. Pharmacol. 24, 81-91. IVERSON, F., DOWNIE, R. H., PAUL, C. ANDTRENHOLM, H. L. (1973).Methyl mercury: Acute toxicity tissuedistribution and decay profiles in the guineapig. Toxicol. Appl. Pharmacol. DIAMOND,
S. S. AND SLEIGHT,
z&4,545-554.
JAKUEZ~WSKI, J., PIOTROWSKI, J. ANDTROJANOWSKA, B. (1970).Binding of mercury in the rat : Studiesusing203HgC12 and gel filtration. Toxicol. Appl. Pharmacol. 16, 743-753. JERNELBV, A. (1969).Conversionof mercury compounds.In Chemical Fallout (M. W. Miller and G. G. Berg, eds.),pp. 68-74. Thomas,Springfield. KITAMURA,S. (1968).Determination of mercury content in bodiesof inhabitants,cats,fishes, and shellsin Minamata district and in the mud of Minamata Bay. In Minamata Disease (M. Kutsuna, ed.), pp. 257-266.Kumamato University, Japan. KLEIN, R., HERMAN,S. P., BRUBAKER, P. E., LUCIER,G. W. ANDKRIGMAN, M. R. (1972). A model of acute methylmercury intoxication in rats. Arch. Pathol. 93, 408-418. KUWAHARA, S. (1970).Effect of mercurialson the function of the brain. J. Kumamoto Med. sot. 44,214221. LITCHFIELD, J. T., JR. AND WILCOXON, F. (1948).A simplifiedmethodof evaluatingdose-effect experiments.J. Pharmacol. Exp. Ther. 96,99-l 13. NORSETH, T. (1969). Studies on the Biotransformation of Methylmercury Salts in the Rat. Ph.D. thesis,the University of Rochester,Rochester,New York. NORSETH, T. (1970). Studies on the biotransformation of ‘03Hg-labelledmethyl mercury chloride in rats. Arch. Environ. Health 21, 717-727. ~STLUND, K. (1969). Studieson the metabolismof methylmercury and dimethylmercury in mice. Acta Pharmacol. Toxicol. 27 (Suppl. l), 1-132. PATTERSON, R. A. AND USHER, D. R. (1971).Acute toxicity of methylmercury on glycolytic intermediatesand adeninenucleotidesof rat brain. Life Sci. 10 (Part II), 121-128. PULIDO, R., KAGI, J. H. R. AND VALLEE, B. L. (1966).Isolation and somepropertiesof human metallothionein.Biochemistry 5, 1768-1777. SALVATERRA, P., LOWN, B., MORGANTI, J. AND MASSARO, E. J. (1973).Alterations in neurochemicaland behavioral parametersin the mouseinducedby low dosesof methyl mercury. Acta Pharmacol. Toxicol. 33, 177-l 90.
442
SALVATERRA
ET AL.
SUZUKI, T. (1969). Neurological symptoms from concentration of mercury in the brain. In Chemical Fallout (M. W. Miller and G. G. Berg, eds.), pp. 245-257. Thomas, Springfield. SUZUKI, T., MIYAMA, T. AND KATSUNUMA, H. (1963).Comparativestudy of bodily distribution of mercury in miceafter subcutaneousadministrationof methyl, ethyl and n-propyl mercury acetates.Jap. J. Exp. Med. 33, 177-282. SWENSSON, A., LUNDGREN, K.-D. AND LINDSTR~M, 0. (1959). Retention of various mercury compoundsafter subacuteadministration. Arch. Industr. Health 20, 467-472. SWENSSON, A. AND ULFVARSON, U. (1968).Distribution and excretion of mercury compounds in rats over a long period after a singleinjection. Acta Pharmacol.Toxicol. 26, 273-283. TAKEUCHI, T. (1972). Biological reactions and pathological changesof human beingsand animalsunder the condition of organic mercury contamination. In EnvironmentalMercury Contamination(R. Hartung and B. D. Dinman, eds.), pp. 247-289. AM Arbor Science, AM Arbor. ULFVARSON, U. (1969).The effect of the size of the doseon the distribution and excretion of mercury in rats after intravenousinjection of various mercury compounds.Toxicol. Appl. Pharmacol.15, 517-524. ULFVARSON, U. (1970).Transportation of mercury in animals.StudiaLaborisSalutis6, l-63. ULFVARSON, U. (1962). Distribution and excretion of somemercury compoundsafter long term exposure.Znt. Arch. Gewerbepath.19,412-422. WEST@ G. (1969).Methylmercury compoundsin animalfoods. In ChemicalFallout (M. W. Miller and G. G. Berg, eds.),pp. 75-93. Thomas,Springfield. WHITE, J. F. AND ROTHSTEIN, A. (1973).The interaction of methylmercury with erythrocytes. Toxicol. Appl. Pharmacol.26, 310-384. YOSHINO, Y., MOZAI, T. AND NAKAO, K. (1966a).Distribution of mercury in the brain and its subcellularunits in experimentalorganic mercury poisonings.J. Neurochem.13, 397-406. YOSHINO, Y., MOZAI, T. AND NAKAO, K. (1966b).Biochemicalchangesin the brain in rats poisonedwith alkylmercury compound, with specialreferenceto the inhibition of protein synthesisin brain cortex slices.J. Neurochem.13, 1223-1230.
APPLIEDPHARMACOLOGY
Time-Dependent zo3Hg in Mice
32,432-442(1975)
Tissue/Organ Uptake and Distribution Exposed to Multiple Sublethal Doses Methyl Mercury
of of
PAUL SALVATERRA,EDWARDJ. MASSARO,~JOHNB. MORGANTI AND BRADLEYA.LOWN State University of New York at Buflaio, Buffalo, New York 14214 and Department of Psychology, State University College at Buflalo, Buffalo, New York 14222 Received August 26,1974; accepted October 29,1974
Time-Dependent Tissue/Organ Uptake and Distribution of ‘03Hg in Mice Exposed to Multiple Sublethal Dosesof Methyl Mercury. SALVATERRA, P., MASSARO, E. J., MORGANTI, J. B. AND LOWN, B. A. (1975). Toxicol. Appl. Pharmacol. 32, 432442. Swiss-Webstermice were injected intraperitoneally with one to ten doses(2.5 mg Hg/kg) of 203Hg-labeled methyl mercury. Doses were administeredat 72-hr intervals. Maximal tissue/organ Hg uptake usually occurred 72 hr postinjection and was monitored at 3 and 6 days by gamma scintillation spectrometry. Most tissues/organsrequired five to six dosesto attain maximal Hg concentrations (the carcassrequired sevendoses;hair and fat, eight doses;lens,nine doses).Data for hair were highly variable. Except for hair (which required seven to eight doses),maximal tissue/organconcentration factors (CF) were reached 72 hr after the first dose. Kidney, liver, and hair attained CF values> 1. Exceptfor hair, all CF valuesdecreased with increasingdose number. Initial rates of decreasewere much greater for kidney, blood, spleen,muscle,and liver than lens, brain, and fat. Only hair exhibited a significantly higher Hg concentration at 6 days after each dosethan at 3 days. With increasing dose number, blood Hg; tissue/organHg ratios remainedrelatively constantfor liver, kidney, spleen,and muscle;decreased for lensand brain; and decreasedfor hair and fat after an initial increase. Methyl mercury (MeHg) is recognized as the major industrial pollutant responsible for casesof human poisoning in Minimata and Niigata, Japan (Expert Group Report, 1971; Friberg and Vostal, 1970). Fatalities and neurologic disturbances have been traced to repeated consumption of contaminated fish and shellfish (Takeuchi, 1972). Since this discovery, concern over high MeHg levels in fishesand the aquatic environment in general has spurred intensive research into the distribution of this potentially lethal compound in the human food chain (Backstrom, 1969; Berglund and Berlin, 1969; Westoii, 1969; Jernelov, 1969). However, very little effort has been expended on establishing the overall mechanism of toxic action (Berlin et al., 1965; Chang and Hartmann, 1972a, b; Cremer, 1962; Diamond and Sleight, 1972; Klein et al., 1972; Kuwahara, 1970; Patterson and Usher, 1971; Salvaterra et al., 1973; Yoshino et al., 1966a, b). 1 To whom all correspondence should be addressed. Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214. 432 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction Printed in Great Britain
in any form reserved.
METHYLMERCURY
‘IISSUE/ORGAN
DlSTRIBUTION
433
Tissue/organ distribution and pharmacodynamic studies have been performed using a variety of species-eg., rats (Norseth, 1969, 1970; Swensson et al., 1959; Ulfvarson, 1969), cats (Kitamura, 1968), dogs (Swensson et al., 1959), guinea pigs (Iverson et ul., 1973), fish (Giblin and Massaro, 1973), and mice (ijstlund, 1969; Suzuki et al., 1963 ; Suzuki, 1969). Most of these studies monitored the fate only of single doses. Also tissue/organ uptake and release rates have been followed only for short time periods (Berlin and Ullberg, 1963; &tlund, 1969; Suzuki et al., 1963; Swensson and Ulfvarson, 1968). Moreover, where data are available, the Hg content of only a limited number of tissues/organs has been assessed. In this study, we administered repeated doses (up to 10) of MeHg and determined concentrations in 10 tissues/organs at intervals of 3 and 6 days following each dose. The dose level selected for multiple administration was approximately 20% of the single dose 7-day LD50. The time period between doses was 3 days since major symptoms of MeHgintoxication are related to CNS dysfunction (Expert Group Report, 1971, Takeuchi, 1972) and 3 days are required to achieve maximal Hg concentrations in the brain of the mouse after a single dose of MeHg (Salvaterra et al., 1973). METHODS Subjects Mice. Two hundred male Swiss-Webster mice (Carworth Breeding Laboratories, New York) which had a mean weight of 37 g at the beginning of the experiment and 42 g at the end served as subjects. They were housed individually in stainless steel cages with wire mesh bottoms and had free access to food and water. Materials MethyZ mercury.
MeHgOH (Alfa Inorganics, Beverly, Mass.) was dissolved in 0.14 M NaCl to yield a solution containing approximately 0.25 mg Hg/ml as MeHgCl.’ This was combined with 203Hg-labeled MeHgCl (New England Nuclear, Boston, Mass.) in 0.01 M Na&O, (4.97 mCi/ml) to produce solutions containing 0.25 mg Hg/ml with specific activities of approximately 3 and 10 &i/ml. The lo-&i/ml solution was employed in the initial five series of intraperitoneal (ip) injections and the 3-@/ml solution in the last five series, Procedure LD.50 determination.
25 mg Hg (as MeHg)/kg,
Groups of eight mice were injected ip with 5, 10, 15, 20, or respectively, of stable MeHgCl in NaCl solution. The LD50
z To check the stability of the isotope solutions and the identity of the anion associated with the MeHg, thin-layer chromatography was employed. Fresh samples and samples of I-mo-old injection solution, MeHgOH in H20, HgC12 in 0.14 M NaCl, MeHgCl in 0.14 M NaClandmixtureswerechromatographed on commercially prepared (Analtech, Inc., Newark, Delaware) cellulose and silica gel G thin layer plates (250 pm thick, 1 x 3.5 or 3.5 x 6 in.); 5 or 1Oq.d samples were applied with a Hamilton microliter syringe (Hamilton Co., Whittier, Calif.) and the plates developed at room temperature, in 3 M NH.+OH. After drying at room temperature, Hg-containing spots (a yellow spot on a green/brown background) were visualized by spraying with a 0.4% (w/v) benzene solution of dithiazone (diphenylthiocarbazone, Merck). The plates were then scored in l-cm sections, scraped into test tubes, and radioactivity determined by gamma counting. The results indicate complete conversion of the OHto the Cl- in 0.14 M NaCl and also indicated a radiochemical purity of >95% after storage of the solution for 1 mo.
434
SALVATERRA
ET AL.
and slope function, along with 95 % confidence limits, were determined according to Litchfield and Wilcoxon (1948). Dosing of animals. This study was designed so that the total quantity of MeHg administered would be fatal to all animals if administered in a single dose. One hundred animals received 2.5 mg Hg/kg (1.0 ml of injection solution/100 g body wt) ip, and an equal number of controls received 1.Oml of 0.14 M NaCl/lOO g. Tissue ‘03Hg concentrations were monitored 3 and 6 days after each dose by gamma spectrometry (de i@a). Three days after the first dose, ten animals were selected randomly from each group. Five animals from the MeHg-treated group were sacrificed immediately for determination of tissue concentrations of ‘03Hg and the remaining five were sacrificed 3 days later. The remaining experimental animals were reinjected with MeHg and the controls with saline. This experimental design ultimately generated two series (sacrificed at 3 and 6 days, respectively) consisting of ten groups of five mice each receiving a total of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5, and 25.0 mg Hg/kg (as MeHgCl), respectively. The last group of 6-day postinjection animals contained only four experimentals and four controls since one animal of each group died before the completion of the experiment. Collection of tissue samples. The animals were sacrificed by cervical dislocation. Tissues were rapidly dissected, blotted dry and placed in tared, air-tight polyethylene vials, and weighed. Hair and fat samples were obtained from carcasses stored at -20°C for approximately 3 wk. A preliminary investigation had shown no alteration in hair and fat 203Hg contents of carcasses stored as long as 6 wk. Tissue and organ sampling proceeded as follows : The pericardial cavity was opened and whole blood was drawn from the heart by syringe (an aliquot of approx. 100 ~1 was monitored for radioactivity). A portion of the right lobe of the liver was removed (no significant differences were found in 203Hg content among the lobes or various areas of individual lobes of this organ). The spleen was dissected free of connective tissue and removed in toto. The left kidney was exposed and the posterior half removed (no significant differences in 203Hg concentrations were found between the anterior and posterior halves of this organ). The right hind leg was skinned and the gastrocnemius muscle removed. Skin was removed from the head and the whole brain was extirpated and dissected into cerebellum and cerebral cortex. Normally, both lenses were assayed together (no significant difference in 203Hg concentrations between the left and right lens was observed). Hair (approx. 10 mg) was shaved from the middle portion of the back just below the neck with a scalpel and small pieces of contaminating skin were removed by freehand dissection under a magnifying lens. Subcutaneous fat was scraped from the skin beneath the hair sampling site. Of all the data, those for hair were the most variable. An explanation of this phenomenon was sought. To assessthe contribution of possible surface contamination to this variability, hair samples were washed with detergent, rinsed with distilled water, oven dried, and counted. No difference between washed and unwashed hair was found, nor was 203Hg detected in hair of control animals. Hair from the hind limb of some animals also was sampled and the results obtained agreed with those for hair taken from the back. For determination of “whole body” concentrations of 203Hg, the frozen carcasses were fragmented and homogenized with an equal amount of water (w/v) at high speed
METHYLMERCURY
TISSUE/ORGAN
435
DISTRIBUTION
in a Waring Blendor until a smooth homogenate was obtained. An aliquot corresponding to approximately 2 g of original tissue weight was used for counting. Determination
of radioactivity
and calculation
of tissue/organ
Hg concentrations.
Tissue radioactivity was measured in a Packard gamma scintillation spectrometer (model 5319). In general, enough counts were collected for each sample to hold the counting error to less than 1%. Counting efficiency averaged 28.1 “//o. The total amount of mercury in each tissue was calculated by comparison of experimental samples with triplicate standards prepared each injection day from 10-/d aliquots of the injection solution which were placed in 0.3 ml of 10% bovine serum albumin (BSA) and sealed in glass ampules. No detectable loss of 203Hg from the MgHg-BSA standards or the injection solution occurred during the course of the experiment. Terminology. The ability of each tissue to concentrate Hg relative to the dose can be expressed by the term concentration factor (CF) (Giblin and Massaro, 1973). CF is calculated by dividing the tissue Hg concentration (pg Hg/g wet wt) by the total accumulated dose (mg Hg/kg). A number greater than 1 indicates the ability of that tissue/organ to accumulate mercury above the cumulative dose. RESULTS
The LD50 and 95 % confidence limits for 7 days (or 3 mo) was estimated to be 13.5 (9.3-19.6) mg Hg (as MeHg)/kg. The dose-response curve was rather steep with a slope function of 1.6 (1.3-2.0). Table 1 shows the survival rate of the mice for each dose TABLE SURVIVAL
RATE
Time after dose (days) 1 2 3 4 5 6 7
3mo
1
OF MICE AT INCREASING INJECTED INTRAPERITONEALLY
LEVELS
OF MeHg
5
Dose level (mg Hg/kg) ______10 15 20
25
gb 8 8 8 8 8 8 8
8 8 8 7 7 7 6 6
2 2 2 2 2 2 2 2
8 6 3 2 2 2 2 2
5 3 2 1 0 0 0 0
a As MeHgCl.
* Number of animals (out of eight) surviving at this time and dose level.
level. All animals which survived the first day, but eventually died, exhibited weight loss and certain CNS signs of MeHg intoxication (e.g., loss of muscular control and piloerection). However, no tremors were observed in any group.
436
SALVATERRA ET AL.
Maximal Hg concentrations occurred in most tissues/organs 3 days after administration of doses 5 or 6 (a total of 12.5-15.0 mg Hg/kg) and then decreased, though not statistically significantly (e.g., the tissue Hg concentrations had reached a plateau). This dose-response pattern is illustrated in Fig. 1 for the 3-day sampling period. It was similar for the 6-day period. For hair and fat (8 doses = 20 mg Hg/kg) and lens (9 doses = 22.5 mg Hg/kg) the plateau was shifted to the right (e.g., these tissues
1-1-1-1-1-1-1-1-1-1 LiverQluscle-,Lenso
3
6
/-,-1-,-f 9 12 15 18 21 24 27 30
Doys
FIG. 1. Total accumulated tissue/organ concentrations of mercury (Hg) 3 days after the terminal dose of a series of intraperitoneal doses of methyl mercury (MeHg). Each dose consisted of 2.5 mg Hg (as MeHg)/kg and was administered every 3 days. Each point represents the mean + SE for five animals.
Cumulative
Dose (mq Hq/kq)
2. Whole body concentrations of Hg 3 days after the terminal dose of a series of doses of MeHg administered ip. Each dose consisted of 2.5 mg Hg (as MeHg)/kg. The Hg concentrations were calculated from measurements of 203Hg in homogenates of five pooled carcasses. FIG.
required a higher dose and/or a longer time to reach a plateau). The “whole body” concentration of Hg was maximal after 7 doses (17.5 mg Hg/kg) as shown in Fig. 2. Since five animals were pooled before homogenization, no measure of individual variability is available. Six aliquots of homogenate were counted and gave standard errors of 2 % or less.
METHYLMERCURY
TISSUE/ORGAN
437
DISTRIBUTION
In Fig. 3 the CF for each tissue 3 and 6 days after each dose is plotted vs the total accumulated dose. The only tissues exhibiting CF values greater than 1 at any time were: kidney (max CF >9.6), liver (max CF >1.5), and hair (max CF >5.5). Hair differed from the other tissues with respect to the number of doses required to reach the maximum CF. For all tissues/organs except hair, the maximum CF was reached 3 days after the initial dose. For hair, the value of the maximum CF was variable and was reached only after 7 or 8 doses (17.5-20.0 mg Hg/kg; Fig. 3). All CF values except that of hair decreased as total dose increased. Moreover, it should be noted that, in relation to Hg uptake, hair does not appear to exhibit saturation kinetics (Fig. 1). Apparently, most tissues were not able to concentrate Hg at the same rate at which total dose was increased. This is not explained by the expected decrease
CF ?
CF
4-
B Spleen
I Kidney
Cerebellum
0
50
(00
150
200
250
Cumulative Dose ( mg Hg/kg I FIG. 3. Concentration factors (CF) for zo3Hg for tissues/organs of the mouse 3 and 6 days after the terminal dose of a series of doses of MeHg administered ip. Each dose consisted of 2.5 mg Hg (as MeHg)/kg. Each point represents the mean for five animals + SE.
438
SALVATERRA ET
AL.
in CF (as the total dose was increased) due to weight gain during the course of the experiment. That is, the (partial) correction for weight gain obtained by multiplying the CF by the ratio, average weight of each dose group over initial average weight of each group, makes no significant difference. Two other features of Fig. 3 should be noted. Initially, the rates of decrease of the CF values of certain tissues (kidney, blood, spleen, muscle, and liver) are relatively large while those of other tissues (lens, cerebellum, cortex, and fat) are considerably smaller. Also the 3- and 6-day data are of similar magnitude and, in some cases (lens, cerebellum, cortex, and fat), the curves intersect indicating occasional continued uptake through the 6-day sampling interval. For each tissue, the ratio, Hg concentration 6 days after each dose divided by Hg concentration 3 days after each dose, did not vary in any apparent systematic manner
FIG. 4. Averageof the ratio, Hg(tissue/organ) concentration6 daysafter eachdosedividedby Hg concentration3 daysafter eachdose,of a series of dosesof 203Hg-labeled MeHg (2.5 mgHg/kg) administered ip. BarsrepresentSE.
with dose. Therefore, the ratios of the individual tissues were averaged for all ten dose levels and are presented in order of decreasing magnitude in Fig. 4. The only tissue exhibiting a significantly higher concentration of Hg 6 days after each dose compared to 3 days was hair. Fat, lens, cortex, and cerebellum retained 75-100x of the 3-day values at 6 days while the remaining tissues retained only 70 % or less. The rapid tissue distribution of ip injected MeHg in animals indicates that it must be transported primarily by the blood (White and Rothstein, 1973). In order to assess any dose-dependent changes in the partitioning of Hg between the blood and the tissues, the blood Hg/tissue Hg ratios were calculated and plotted vs total dose in Fig. 5. For clarity, only the 6-day data are presented (the 3-day values are similar). A relatively constant ratio is observed for the liver, kidney, spleen, and muscle indicating a constant distribution of Hg between blood and these tissues. However, in the case of lens, cortex, and cerebellum the ratio decreases reflecting a greater proportion of Hg
METHYLMERCURY
IO 0 I-
TISSUE/ORGAN
439
DISTRIBUTION
\
O’
Fat Hair
.-J-~;~~
5
10
15
zo-
i5-
CumulativeDose(mg Hg /kg 1 FIG. 5. Blood-tissue ratios of 203Hg 6 days after the terminal dose of a series of 2.5 mg Hg (as MeHg)/kg doses administered ip. Each point represents the average of five animals.
in these tissues compared to blood as the total dose increased. Hair and fat show an initial increase in the ratio followed by a decrease. This initial increase reflects the fact that there is a much slower rate of uptake of Hg by these tissues compared to blood at the time of the first few sampling periods (Fig. 1). DISCUSSION
Saturation kinetics for MeHg uptake were exhibited by all tissues with the possible exception of hair. This was somewhat unexpected since saturating tissue concentrations have not been identified as such in the literature for MeHg administered in repeated doses (Friberg, 1959; Gage, 1964; Suzuki, 1969; Suzuki et al., 1963; Swensson et al., 1959; Ulfvarson, 1962). Possible explanations for this could include: (a) an insufficient time period between doses for the MeHg to reach a state of dynamic equilibrium among the tissues; (b) use of very large doses which caused extensive tissue necrosis; or (c) very small doses which did not. permit accumulation of sufficient tissue concentrations to show saturation kinetics; (d) insufficient number and/or distribution of sampling intervals. In this study nearly all tissues achieved maximal Hg uptake during the 3-day interval between doses (Fig. 4), indicating the establishment of dynamic equilibria. The only tissue which did not consistently attain a maximum Hg concentration between sampling intervals (3 and 6 days after each dose) was hair (Fig. 4). Since hair is metabolically inactive and concentrates Hg significantly, it may function as an important excretion route of this compound (i)stlund, 1969; Ulfvarson, 1970). Quantitatively [e.g., its large CF (Fig. 3) and continuous growth], hair may play a major role in the regulation of tissue MeHg concentrations in hairier species (e.g., mice, rats, and lower primates) vs relatively hairless animals such as man. The continuous uptake of MeHg by hair and its high capacity for this compound may explain, in part, why mice are able to tolerate a relatively large dose of MeHg administered in small repeated doses. It should be noted that none of the animals in this study developed any CNS signs characteristic of MeHg intoxication.
440
SALVATERRA ETA.
The individual variability in hair Hg concentrations seen in this study (Fig. 1) and in humans (Birke et al., 1972) may be related to individual differences in susceptibility to MeHg intoxication. Thus, hair of different composition may have a greater or lesser capacity to bind Hg, or assuming a finite rate of binding, the more rapid the turnover (growth and fallout) the less susceptible the animals to intoxication. Saturation kinetics for tissue MeHg concentrations could be explained by an increase in elimination rate, a decrease in absorption, or both. In this study, an apparent increase in elimination rate could be seen for the Hg eliminated via the hair. Other major excretion routes of MeHg such as the urine and feces (Friberg, 1959; Gage, 1964; &tlund, 1969) were not investigated in this study. Thus, no definite conclusions can be drawn concerning the effects of dose and time on the rate of Hg excretion via these routes. However, in other studies (unpublished observations) we have observed that fecal excretion plays an important role in MeHg elimination after a single dose. In contrast, only a minor quantity of Hg is excreted via the urine. A decrease in the rate of absorption of the MeHg may not be ruled out, however, since a thickening of the peritoneum was observed in some experimental as well as a few control animals and probably represents a side effect of the ip route of administration. No grossly visible peritonitis or loss of subcutaneous fat was observed as has been reported for rats receiving weekly ip doses of 10 mg Hg/kg (Diamond and Sleight, 1972). Thickening of the spleen capsule also was observed in most animals receiving six or more doses of MeHg (not in controls). The constant blood/tissue Hg ratios for kidney, liver, spleen, and muscle (Fig. 5A) indicate a simple distribution equilibrium. On the other hand, a decrease in this value as seen for the lens, cerebellum, and cortex, indicates a more complex partitioning mechanism. Thus, in contrast to previous suggestions (Expert Group Report, 1971), this observation indicates that blood may not be the ideal tissue for estimating Hg content of brain after repeated exposure. Moreover, the greater ability of the lens, cerebellum, and cortex than the blood to bind Hg (Fig. 4) on repeated exposure (Fig. 5B) may explain the apparent target nature of these tissues in MeHg poisoning (Backstriim, 1969; Suzuki, 1969). Metallothionein (Jakubowski et al., 1970; Pulido et al., 1966) may be responsible for the ability of the kidney and, to some extent, the liver, to concentrate Hg (Fig. 1, 3). This study provides data on the dynamics of MeHg in a model system after repeated exposure to sublethal doses. These data may be of value in the understanding of the biochemical and behavioral changes observed after repeated exposure to MeHg.
REFERENCES studies of mercuric pesticides in quail and some freshwater fishes. Acta Pharmacol.Toxicol. 27 (Suppl. 3), l-103. BERGLUND, F. AND BERLIN, M. (1969). Risk of methylmercury cumulation in man and mammals and the relation between body burden of methylmercury and toxic effects. In ChemicalFallout (M. W. Miller and G. G. Berg, eds.), pp. 258-273. Thomas, Springfield. BERLIN, M., JERKSELL, L. G. AND NORDBERG, G. (1965). Accelerated uptake of mercury by brain caused by 2,3-dimercaptopropanol (BAL) after injection into the mouse of a methylmercuric compound. Acta Pharmacol.Toxicol. 23, 312-320. BACKSTR~M, J. (1969). Distribution
METHYLMERCURY TISSUE/ORGAN DISTRIBUTION BERLIN,
M.
AND
ULBERG,
441
S. (1963). Accumulation and retention of mercury in the mouse.
Arch. Environ. Health 6, 610-616.
G., JOHNELS, G., PLANTIN,L.-O., SJ~STRAND, B., SKERFVING, S. ANDWESTERMARK, T. (1972). Studieson humansexposedto methylmercury through fish consumption. Arch. Environ. Health 25, 77-91. CHANG, L. W. AND HARTMANN, H. A. (1972a).Ultrastructural studieson the nervoussystem after mercuryintoxication. I. Pathologicalchangesin the nervecell bodies.Acta Neuroparhol. 20, 122-138. CHANG, L. W. AND HARTMANN, H. A. (197213).Blood-brain barrier dysfunction in experimental mercury intoxication. Acta Neuropathol. 21, 179-184. CREMER, J. E. (1962).The action of triethyl tin, triethyl lead,ethyl mercury and other inhibitors on the metabolismof brain and kidney slicesin vitro using substrateslabeled with 14C. BIRKE,
J. Neurochem.
9,289-298.
S. D. (1972).Acute and subchronicmethyl mercury toxicosis in the rat. Toxicol. Appl. Pharmacol. 23,197-207. EXPERT GROUP REPORT (1971).Methyl mercury in fish: A toxicologic-epidemiologic evaluation of risks. Nord. Hyg. Tidskr. Suppl. 4, 364 pages. FRIBERG, L. (1959). Studieson the metabolismof mercuric chloride and methyl mercury dicyandiamide.AMA Arch. Industr. Health 20,42-49. FRIBERG, L. AND VOSTAL, J. (eds.)(1970).Mercury in the environment: An Epidemiological and Toxicological Appraisal. ChemicalRubber Co. Press,Cleveland,Ohio. GAGE, J. L. (1964). Distribution and excretion of methyl and phenyl mercury salt. Brit. J. Industr. Med. 21, 197-202. GIBUN, F. J. AND MASSARO, E. J. (1973).Pharmacodynamicsof methyl mercury in the rainbow trout (Salmo gairdneri) : Tissueuptake, distribution andexcretion. Toxicol. Appl. Pharmacol. 24, 81-91. IVERSON, F., DOWNIE, R. H., PAUL, C. ANDTRENHOLM, H. L. (1973).Methyl mercury: Acute toxicity tissuedistribution and decay profiles in the guineapig. Toxicol. Appl. Pharmacol. DIAMOND,
S. S. AND SLEIGHT,
z&4,545-554.
JAKUEZ~WSKI, J., PIOTROWSKI, J. ANDTROJANOWSKA, B. (1970).Binding of mercury in the rat : Studiesusing203HgC12 and gel filtration. Toxicol. Appl. Pharmacol. 16, 743-753. JERNELBV, A. (1969).Conversionof mercury compounds.In Chemical Fallout (M. W. Miller and G. G. Berg, eds.),pp. 68-74. Thomas,Springfield. KITAMURA,S. (1968).Determination of mercury content in bodiesof inhabitants,cats,fishes, and shellsin Minamata district and in the mud of Minamata Bay. In Minamata Disease (M. Kutsuna, ed.), pp. 257-266.Kumamato University, Japan. KLEIN, R., HERMAN,S. P., BRUBAKER, P. E., LUCIER,G. W. ANDKRIGMAN, M. R. (1972). A model of acute methylmercury intoxication in rats. Arch. Pathol. 93, 408-418. KUWAHARA, S. (1970).Effect of mercurialson the function of the brain. J. Kumamoto Med. sot. 44,214221. LITCHFIELD, J. T., JR. AND WILCOXON, F. (1948).A simplifiedmethodof evaluatingdose-effect experiments.J. Pharmacol. Exp. Ther. 96,99-l 13. NORSETH, T. (1969). Studies on the Biotransformation of Methylmercury Salts in the Rat. Ph.D. thesis,the University of Rochester,Rochester,New York. NORSETH, T. (1970). Studies on the biotransformation of ‘03Hg-labelledmethyl mercury chloride in rats. Arch. Environ. Health 21, 717-727. ~STLUND, K. (1969). Studieson the metabolismof methylmercury and dimethylmercury in mice. Acta Pharmacol. Toxicol. 27 (Suppl. l), 1-132. PATTERSON, R. A. AND USHER, D. R. (1971).Acute toxicity of methylmercury on glycolytic intermediatesand adeninenucleotidesof rat brain. Life Sci. 10 (Part II), 121-128. PULIDO, R., KAGI, J. H. R. AND VALLEE, B. L. (1966).Isolation and somepropertiesof human metallothionein.Biochemistry 5, 1768-1777. SALVATERRA, P., LOWN, B., MORGANTI, J. AND MASSARO, E. J. (1973).Alterations in neurochemicaland behavioral parametersin the mouseinducedby low dosesof methyl mercury. Acta Pharmacol. Toxicol. 33, 177-l 90.
442
SALVATERRA
ET AL.
SUZUKI, T. (1969). Neurological symptoms from concentration of mercury in the brain. In Chemical Fallout (M. W. Miller and G. G. Berg, eds.), pp. 245-257. Thomas, Springfield. SUZUKI, T., MIYAMA, T. AND KATSUNUMA, H. (1963).Comparativestudy of bodily distribution of mercury in miceafter subcutaneousadministrationof methyl, ethyl and n-propyl mercury acetates.Jap. J. Exp. Med. 33, 177-282. SWENSSON, A., LUNDGREN, K.-D. AND LINDSTR~M, 0. (1959). Retention of various mercury compoundsafter subacuteadministration. Arch. Industr. Health 20, 467-472. SWENSSON, A. AND ULFVARSON, U. (1968).Distribution and excretion of mercury compounds in rats over a long period after a singleinjection. Acta Pharmacol.Toxicol. 26, 273-283. TAKEUCHI, T. (1972). Biological reactions and pathological changesof human beingsand animalsunder the condition of organic mercury contamination. In EnvironmentalMercury Contamination(R. Hartung and B. D. Dinman, eds.), pp. 247-289. AM Arbor Science, AM Arbor. ULFVARSON, U. (1969).The effect of the size of the doseon the distribution and excretion of mercury in rats after intravenousinjection of various mercury compounds.Toxicol. Appl. Pharmacol.15, 517-524. ULFVARSON, U. (1970).Transportation of mercury in animals.StudiaLaborisSalutis6, l-63. ULFVARSON, U. (1962). Distribution and excretion of somemercury compoundsafter long term exposure.Znt. Arch. Gewerbepath.19,412-422. WEST@ G. (1969).Methylmercury compoundsin animalfoods. In ChemicalFallout (M. W. Miller and G. G. Berg, eds.),pp. 75-93. Thomas,Springfield. WHITE, J. F. AND ROTHSTEIN, A. (1973).The interaction of methylmercury with erythrocytes. Toxicol. Appl. Pharmacol.26, 310-384. YOSHINO, Y., MOZAI, T. AND NAKAO, K. (1966a).Distribution of mercury in the brain and its subcellularunits in experimentalorganic mercury poisonings.J. Neurochem.13, 397-406. YOSHINO, Y., MOZAI, T. AND NAKAO, K. (1966b).Biochemicalchangesin the brain in rats poisonedwith alkylmercury compound, with specialreferenceto the inhibition of protein synthesisin brain cortex slices.J. Neurochem.13, 1223-1230.
