TOXICOLOGY

Food

AND

APPLIED

36, 19-30 (1976)

PHARMACOLOGY

Intake, Body Weight, following Chronic HANS R. BERTHOUD,’

and Brain Methylmercury

Histopathology Treatment’

in Mice

ROBERTH. GARMAN, AND BERNARD WEISS

Environmental Health Sciences Center, Department of Radiation Biology and Biophysics, and Division of Laboratory Animal Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York I4642 Received

May

21, 1975;

accepted

October

31, 1975

Food Intake, Body Weight, and Brain Histopathology in Mice following Chronic Methylmercury Treatment. BERTHOUD, H. R., GARMAN, R. H., AND WEISS, B. (1976). Toxicol. Appl. Pharmacol. 36, 19-30. Forty-eight male CD-l mice were given daily intragastric dosesof 0, 0.25, 1.0, and 4.0 mg of Hg/kg as methylmercury for up to 60 days. The intake of a liquid diet and body weight were recorded daily. Onset of hypophagia, body weight loss, and weaknessin the hind legs of more than half the animalsappearedafter 1, 7, and 14 days, respectively, in the 4-mg/kg group and after 23,23, and 44 days in the I-mg/kg group. The lowestdose level did not changeintake and body weight. Evidenceof neurondegeneration waspresentwithin the corpusstriatum, the cerebralcortex, the thalamus, and the hypothalamus. Autoradiography showedaccumulation of *03Hgin the cytoplasm of periventricular glial cells for all dosegroups. Loss of appetite and body weight are commonly reported signsof intoxication by toxic agents. They are rarely quantified, however, perhaps becausethey seemso common and nonspecific. This is an unfortunate oversight for two reasons.First, defects in food and energy regulation may provide early indications of an incipient overt intoxication. Second, a fuller understanding of the basis for these defects may help disclose the mechanismsby which the toxic substanceacts. The present report representsan attempt to study methylmercury poisoning from such a standpoint. Methylmercury is primarily a central nervous system poison. Intoxication typically is accompanied by paresthesias(numbness and tingling) of the lips, tongue, and fingertips, followed by the onset of ataxia and incoordination, dysarthria, and peripheral visual disturbances (Hunter er al., 1940; Bakir et al., 1973; Brown, 1954; Takeuchi, 1972; Prick et al., 1967; Maghazaji, 1974). Metallic mercury vapor poisoning has also been associatedwith paresthesias(Vroom and Greer, 1972). Decreasedappetite (Smith, 1972; Maghazaji, 1974)and a metallic taste (Brown, 1954) are additional sequelaeof mercury intoxication in humans,but may be the consequences, i Supported in part by grants, No. ES-01247 from the National Institute of Environmental Health Science, No. NSF-Gl-300978 from the National Science Foundation (RANN program), No. MH11752 from the National Institute of Mental Health, No. NS-08048 from the National Institute of Neurological Diseases and Stroke, and in part by a contract with the U.S. Energy Research and Development Administration at the University of Rochester Biomedical and Environmental Research Project and has been assigned Report No. UR-3490-757. z Fellow of the Swiss National Foundation for Scientific Research, Grant No. 841.177.73. Copyright 0 1976 by Academic Press, Inc. 19 All rights of reproduction in any form reserved. Printed

in Great

Britain

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AYD

WEISS

particularly if they appear during the later stages of poisoning, of difficulties in chewing and swallowing, diarrhea, or ulceration of the oral mucosa (Takeuchi, 1972: Prick et al., 1967; Vroom and Greer. 1972). Experimental studies in animals have also reported weight loss to be an early sign of methylmercury toxicity (Suzuki and Miyama, 197 1; Miyakawa et al., 1970; Diamond and Sleight, 1972: Stillings et al., 1974; Klein et a/., 1973; Ideka et a/., 1973), along with motor deficits, but the progression of weight loss in relation to food intake has not been extensively monitored. Spyker ( 1975), who made such measurements in mice. reported that prenatal exposures decreased food and water intake in maturity and, at the same time, led to obesity in some and inanition in others. Schroeder and Mitchener (I 975) found significantly increased body weights in mice given I ppm of methylmercury in the drinking water during their life span and significantly decreased body weights after 10 weeks with 5 ppm of methylmercury. Given the many ways by which methylmercury might act to produce disorders of food regulation, it seemed important to examine further, to quantify, and to seek some clues to the basis for the phenomenon.

METHODS

Animals. The animals were 48 CD-l mice (Crl :CD-I (ICR)BR).3 They were 50 days old at the beginning of the experiment and housed, six per cage, in plastic cages with wood shavings for bedding. Their only food was a liquid diet consisting of evaporated milk enriched with 0.3 ml/liter of a polyvitamin preparation;4 this diet had a caloric density of 1.35 Cal/g. This formula was fed ad libitum, as the only fluid source, for 18 hr daily. Water only was available between I 100 and 1700 hr. The room temperature was maintained at 22 + 2°C and the lights were on from 0600 to 1800 hr. After 64 days on the liquid diet, all surviving animals were given free access to Purina mouse chow and water. Zntubations. Four different dose levels of methylmercuric chloride (MeHg) (0, 0.25, 1.O, and 4.0 mg of HE/kg) were given daily to four groups of 12 mice each by intragastric intubation. Stock solutions were prepared by dissolving methylmercuric chloride in 0.005 M Na,CO, and adding radioactive metllyl-203Hg. The dosing solutions were freshly prepared every day by adding L-cysteine solution to the stock solution so that the administered solution contained twice as much L-cysteine as methylmercury on a molar basis. This was done to reduce the irritant effects of MeHg on the gastrointestinal tract. The specific activities of the intubation solutions were about 0.3, I .3, and 5.6 @i/mg of Hg for the high, intermediate, and low dose, respectively. The intubation volume for all groups was 1 ml/100 g body weight. Procedure. Between 0900 and 1100 hr all mice were weighed and dosed. The amount of liquid diet consumed by the six mice in each cage during the previous 18 hr was determined by measuring the decrease in weight of the preweighed bottles. On Days 11, 22, 31, and 41 (first MeHg administration = Day 0), the liquid diet was sweetened by adding 60 mg/lOO ml of sodium saccharin; on days 13, 24, 33, and 43 the diet was 3 Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts. ’ VI-SYNERAL, USV Pharmaceutical Corp., Tuckahoe, New York.

FOOD INTAKE AFTER METHYLMERCURY

21

made bitter by adding 20 mg/lOO ml of quinine sulfate. These challengeswere undertaken becausehypothalamic lesions have been reported to exaggerate the responseto these substances(Teitelbaum, 1975). Motor coordination tests. Between Days 15 and 21 the ability to remain on a grooved rotating plastic rod of 30-mm diameter was measured for the controls and the two lower dose groups. The original rotarod device had been modified so that it could be made to accelerate. The starting speedfor this study was set at 4 rpm and the acceleration to 5 rpm/min. The first two of the five sessionswere considered training sessions, and animals that fell off before 1 min had elapsedwere put back on the rod. Between Days 36 and 38 the ability to hang onto a horizontal wire mesh plane was measuredfor the controls, the two low dosegroups, and the survivors of the high dose group. The animals were first put on the upper side of the plane; the plane was then slowly turned upside down. Duration of time on the inverted plane (i.e., prior to falling off) wasmeasuredwith a stopwatch. The wire meshconsistedof wires 1 mm in diameter spaced5 mm apart. The first of the three sessionswasconsideredto be a training session. Brain mercury concentrations. For the brain Hg determinations, autoradiography, and histology, the mice were injected with a lethal dose of sodium pentobarbital and perfused through the heart with 0.9 % NaCl followed by 10% neutral buffered formalin. For weighing and counting, the wet brains were transferred into preweighed plastic vials. All counts were made in a liquid scintillation counter with an efficiency of 36 % (Packard Model 466).5 The mean counts of aliquots of the dosing solutions used on Days 1-3 served as standards. Autoradiography and histology. Frontal or sagittal slicesof the perfused brains were processed by a paraffin technique, and 6-pm sections of the resulting blocks were placed on glassmicroslides. Paraffin was eliminated from the sections for autoradiography. They were then hydrated and transported to the darkroom where they were dipped into liquid nuclear track emulsion6 held at 40°C. The slides were placed in light-tight boxes containing a desiccant and placed in a refrigerator (4°C) for 2-3 weeks. After exposure for this period, the emulsion-coated slideswere developed and stained with hematoxylin and eosin. RESULTS Food Intake and Body Weight Body weight and food intake curves are shown in Fig. 1. The mean daily intake of the liquid diet before the start of treatment was about 12.5 g/mouse, except for the 1-mg/kg group, in which it was 13.5 g/mouse. During the 2 months of the experiment, the control animals showed a slight but steady decreasein daily consumption to about 80 % of the baselinelevel, while increasing their body weight by 20 %. The 0.25-mg/kg group showed some irregularities in diet consumption and body weight between Days 12 and 36, but toward the end of the 60-day period, theseanimals consumed the same amount of diet as the controls and their mean body weight slightly surpassed that of the controls. The food intake and body weight of the I-mg/kg group did not differ from that of the controls until Day 22. At this point, mean intake and mean body weight began a steady decreasethat was maintained until Day 39, when two animals 5 Packard Instrument Company, Inc., LaGrange, Illinois. 6 NTB-2, Eastman Kodak Co., Rochester, New York.

22

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WEISS

died. At Day 48, the four most severely affected of the surviving animals were sacriticed. For the six remaining mice of this group, mercury administration was discontinued. Nevertheless, they developed hypophagia during the following2 weeks. (Discontinuities in Fig. 1 represent changes in treatment or in number of subjects.) The mean food consumption of the animals treated with the high dose (4 mg:kg) started to decrease after the first methylmercury administration, and by Day 16 their

FIG. 1. Mean liquid diet intake and mean body weight changes as affected by methylmercury. Each point of the intake curves is the mean of 3-7 successive days, except for the days with 0.06% saccharin (dotted vertical lines) and the days with 0.02% quinine (dashed vertical lines). The filled symbols represent the part of the experiment with MeHg treatment, while the open symbols indicate ceased treatment. The number of mice in each group was 12, unless otherwise indicated. Thus, on day 39, two mice in the l.O-mg of Hg/kg group died, leaving II = 10. The next series of symbols for this group indicates the weights of these 10 animals. The next series of symbols for this group shows n = 6 due to the sacrifice of four mice, and, since these symbols are unfdled, that no methylmercury was administered during this time. The next series, with II = 4, shows the weights of the final survivors. Similar interpretations apply to the other groups. From Day 64 to 120 all animals had free access to dry chow and water. “Rota-rod” and “wire-mesh” indicate the time when motor coordination tests were carried out.

mean intake was 50% of the control value. Their body weight started to decreaseafter 7 days, and by Day 16 their mean body weight had dropped to 10% below their initial weight. At this time, the eight mice showing the greatest decreasein body weight were sacrificed. The four survivors initially exhibited normal intake and body weights near those of the controls. After 10 days their intake and body weight began a prolonged decline that continued until the end of the 2-month period. During the next 2 months, however, with normal food and water available, their body weights recovered to control values. Diarrhea was observed transiently in someanimals during the period of adaptation to the liquid diet regimen but was never seenafter the start of the Hg dosing.

FOOD INTAKE AFTER METHYLMERCURY

23

In some of the cages,frequent fighting was observed, necessitating the isolation of two of the most aggressivemice at the end of the first 2 months. Someof the irregularities in the weight curves may be due to this phenomenon. EfSectsof Taste Quality All groups, except the high dosegroup (which already had been severely affected by Day II), increased intake on saccharin days and decreasedintake on quinine days. The poisoned groups tended to be more reactive to the taste quality. Their saccharin enhancement and quinine depression of intake were larger compared to the controls. Separate analysis of variance (Siegel, 1956) of the intake difference between test and preceding and following control days for the four test periods, however, revealed no significant differences among the four groups. Motor Coordination Rotarod performance data are shown in Fig. 2. The decreasedperformance of the 1-mg/kg animals was statistically significant in spite of the high incidence of animals from all groups accidentally falling off at low speeds(H = 4.10, p < 0.001, KruskalWallis test; Kirk, 1968). 16 -

0

-

0 ROTATION

10

20 SPEED

30

40 (RPM

50

60 I

FIG. 2. Histogram of rotarod performance. The rotation speed on the abscissa indicates the rotation speed of the rotating rod at which an animal fell off. “Frequency” refers to the number of trials (3 x 12 mice) on which a mouse fell off at a specified speed as the speed of rotation progressively increased. The figures are based on the combined data of three successive daily sessions. A KruskalWallis analysis of variance (Kirk, 1968) revealed a significant difference between the controls and the 1 .O-mg of Hg/kg group.

The results of the wire mesh test are summarized in Table 1. Although half of the animals of the 1-mg/kg group were severely hypophagic and had already suffered a substantial body weight lossby the time thesetestswere given, all except two mice with paralyzed hind legs still were able to hang for at least 4 set under the wire meshplane. The mean fall times indicate, however, that the low dose (0.25 mg/kg) animals also showed a slight deficit compared to the controls. Brain Hg Concentrations, Histopathologic Changes,and Autoradiography Brain Hg concentrations are given in Table 2. Histopathologic changes associated with methylmercury intoxication consisted of acute eosinophilic neuron degeneration

24

BERTHOUD,

GARMAN

TABLE

AND

WEISS

I

ON THE ABILITY OF MICE TO HANG FROM AN INVERTED AS MEASURED BY DURATION OF TIME PRIOR TO FALLING

EFFECT OF METHYLMERCURY WIRE MESH PLANE

Controls Proportion of mice which fell off before 40 set Mean fall times of the mice which fell off (set)

0.25 mg Hgjkg

I .O mg Hg/kg

HOKIZ( )& I AI. OFF

4.0 mg Hg:kg”

3112

4’17 ! L

6112

I :4

24.0

12.2

1 1.5b

10.5

a Surviving animals, only tested after discontinuance of the MeHg. * Included are two mice with hind leg paralysis and zero-duration times. TABLE RELATIONSHIP

Daily dose (mg Wk4 0.25 1.0 4.0

BETWEEN

DAILY BRAIN

Duration of administration (days) 61 45 14

2

DOSE GIVEN, DURATION OF ADMINISTRATION, MERCURY CONCENTRATION IN MICE’

AND

RESULTING

Time until sacrifice (days)

Total dose (mg Hdk)

Mean brain Hg concentration Cm Wg)

If

64 48 18

15.25 45.0 56.0

2.0 (0.68-4.50)b 16.5 (6.9-21.8) 32.1 (19.2-52.0)

6 4 7

a The animals selected for the measurement of brain mercury concentration were the most severely affected ones, with the exception of the I.O-mg/kg group. In this group, two mice died on day 40, and these were not included in the mean. ’ Values in parentheses are ranges.

with an associatedmicrogliocytosis. Affected neurons had pyknotic or karyorrhectic nuclei and shrunken, brightly eosinophilic cytoplasm. Areas of neuron loss were often denoted by the presenceof nuclear fragments (“nuclear dust”). Microglial cells were in evidence in the vicinity of the degenerating neurons but were also present in areas where no neuron degeneration could be detected. The distribution of the MeHg-induced degeneration was similar in all three groups, but lesions were minimal in extent in the low dose group. In these mice, slightly increasednumbers of microglial cells werepresent within the hypothalamus and thalamus, and eosinophilic neurons were occasionally seenwithin the cerebral cortex, but these degenerating neurons were difficult to find. The mice in the high dose group had the most extensive degree of neuron loss. but quantitative differencesbetween theseanimalsand those in the intermediate dose group were not considered to be significant. In these mice, the most significant neuronal loss was within the corpus striatum, the putamen being most severely affected (Fig. 3). In the most markedly affected cases, the putamen appeared to be essentially depopulated of neurons with only microglial cells, gemistocytic astrocytes, and nuclear dust remaining.

FOOD INTAKE AFTER METHYLMERCURY

25

FI G. 3. (A) Putamen from an animal receiving the low dose, which is histologically normal. (B) Putamen from an animal receiciog the high dose with acute eosino2hilic neuron degeneration (solid arr .ows). Mic roglial cells (open arrows) surround one of the degenerating neurons (H&E; 600x).

26

BERTHOUD,

FI’ c. 4. (A) Lateral

norn lal. (B) Lateral nken 600x :).

neurons

hypothalamic hypothalamic (solid arrows),

GARMAN

AND

WEISS

area from an animal receiving the low dose, which is histologi tally area from an animal receiving the high dose with neuronal loss, and occasional microglial cells (open arrows): f, fornix (H ME;

FOOD

INTAKE

AFTER

METHYLMERCURY

27

Other subcortical areas which were similarly affected, although to a lesser degree than the putamen, were: the thalamus, hypothalamus and amygdala. The pattern of neuronal loss within the thalamus was diffuse, although the lateral nuclear groupings, particularly in the region of the thalamic radiations, were most markedly affected. Within the hypothalamus, eosinophilic neuronal change was difficult to recognize, but increased numbers of microglial cells were consistently present and were considered to indicate the presence of neuronal degeneration, Eosinophilic neurons were identified, however, within the lateral hypothalamic area (Fig. 4) and mammillary nuclei, and the latter were severely affected in some cases. Subcortical areas which appeared to escape damage included the hippocampus, pineal body, substantia nigra, pontine nucleus, and most of the large neuronal groupings within the brainstem. The medial habenular nuclei were histologically normal, while the lateral habenular nuclei often had microglial cell infiltrates and occasional degenerating neurons. Microglial cell infiltrates were similarly present within the cerebellar nuclei as well as around the fourth ventricle, but the cerebellar cortex was always histologically normal.

FIG. 5. Autoradiograph of MeHg-treated mouse. Black grains indicate the presence of Hg within the periventricular glial cells (solid arrows); V, third ventricle (H&E; 800x).

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BERTHOUD,

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The mice which received the intermediate and high dose had scattered eosinophilic neurons within their cerebral cortices, but regional cortical differences could not be detected, and the overall cortical architecture of these brains was well preserved. Based on the glial reaction, the cortical change appeared to be more acute than that within the most affected subcortical regions. The autoradiographic distribution of ro3Hg was essentially the same in all of the mice. Slightly higher than background levels of radioactivity were present diffusely over the brain sections, but this label was poorly localized. The only cells which were consistently labeled were the periventricular glial cells (Fig. 5). These cells were particularly numerous around the third ventricle but were also present around the lateral and fourth ventricles. Label was occasionally seen over other glial cells, also, particularly within the white matter. In all cases, this label was present over the cytoplasmic processes of these cells. DISCUSSION

The present experiment shows clearly that hypophagia and body weight loss are among the early signs of chronic and subacute methylmercury poisoning in mice. A daily doseas low as I mg of Hgjkg causedmeanfood intake and body weight to decline after 20 days, although motor coordination at that time, asmeasuredwith the rotarod, was only slightly impaired. Suzuki and Miyama (1971) after feeding mice with a methylmercury-containing diet, found the earliest motor deficit to be an inability of the mice to hold their headsin a horizontal position while being suspendedby their tails. The brain Hg concentrations of the affected mice were about 10 pg/g, which is comparable to that of the intermediate doseanimals in the present study at the time of onset of hypophagia. Assuming a daily intake of about 5 g of food, containing 31.5 ppm of methylmercury, Suzuki’s mice consumed about 90 mg of Hg/kg to reach a brain concentration of 16 pg/g after 20 days. The comparable intermediate dose group in this experiment ingesteda total of only 44 mg of Hg/kg to reach this brain concentration. Mice of the samestrain, age, and sex, which were maintained on normal mousechow, did not show detectable changes in body weight and gross behavior after receiving intragastric dosesof 3 mg of Hg/kg of methylmercury on 5 out of every 6 days during 6 weeks of treatment (unpublished observation, H. R. Berthoud). Methylmercury toxicity, thus, is lower in mice fed mousechow than in those fed milk. The finding that the survivors of the intermediate and high dose groups fully recovered body weight during the second2-month period of this experiment, while having ad libitum accessto mouse chow and water, and the relatively high toxicity of 5 ppm of methylmercury in the drinking water of mice fed a controlled diet (Schroeder and Mitchener, 1975) point to the presence of a protective substance in normal mouse chow. Selenium, cysteine, and fish protein have recently been reported to decrease methylmercury toxicity in rats (Stillings et al., 1974) pointing to the need to analyze the contribution of thesesubstances. Histopathologic studies of alkylmercurial encephalopathies have been performed on numerous species,most particularly the rat, but only a few detailed studies are available on the mouse (Takeuchi et al., 1962; Mukai, 1972). Our histopathologic findings differ from those reported by Mukai (1972), who treated mice with tritiated ethyl mercuri-S-cysteine, only with regard to the absenceof cerebellar lesions in our

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INTAKE

AFTER

METHYLMERCURY

29

animals. Mukai also reported the presence of prominent autoradiographic label over hypertrophied subependymal glia. Cassano et al. (1969), using mice treated with 203Hgvapor, also reported high concentrations of label within ependymal cells and subependymal glia. Whole body autoradiography of mice given ‘03HgCI, intravenously (Berlin and Ullberg, 1963)and exposed to *03Hgvapor (Cassanoet al., 1969)have also demonstrated labeling around the ventricles. As suggested by these investigators, mercury may enter the brains to a significant extent via the cerebrospinal fluid. The localization of label primarily over the cytoplasm of glial cells rather than neurons has also been reported in monkeys exposed to methyl ao3Hg(Garman et al., 1975) and indicates that an important site of action of the organic mercurials may be within these glial cells. It is important to note that autoradiographic label associatedwith mercurial compounds may be due to direct action of nonradioactive Hg with the nuclear emulsion (Silberberg et al., 1969). In fact, the bulk of the autoradiographic label present over tissuesections from monkeys receiving methyl- ‘03Hg has been found to be due to this direct effect (R. H. Garman, unpublished information). Nevertheless, we feel that the autoradiographic procedure, as performed in this study, is a valid histochemical technique for the cellAlar localization of Hg. The results of this experiment do not allow us to posit a precise site of action of methylmercury in its disruption of body weight regulation. Although brain lesionsare present in areas involved in the regulation of food intake (Grossman, 1975) and body weight loss was reported after direct intracerebral injections of methylmercury (Richardson and Murphy, 1974), a peripheral contribution cannot be excluded. Recent data indicate, for instance, that the liver and the kidney are alsohighly vulnerable to methylmercury intoxication (Emerick and Holm, 1972; Klein et al., 1973).Measurement of endocrine and neuroendocrine function in the methylmercury-poisoned animals, coupled with detailed histologic examinations, as well as studieson the effects of intracerebral injections of methylmercury into specific areas,should provide a more complete understanding of how this poison affects body energy regulation. REFERENCES BAKIR, F., DAMLUJI, S. F., AMIN-ZAKI, L., MURTADHA, M., KHALIDI, A., AL-RAWI, N. Y., TIKRITI, S., DHAHIR, H. I., CLARKSON, T. W., SMITH, J. C. AND DOHERTY, R. A. (1973). Methylmercury poisoningin Iraq. Science 181,230-241. BERLIN, M. AND ULLBERG, S. (1963). Accumulation and retention of mercury in the mouse. I. Arch. Environ. Health 6, 589-601. BROWN, I. A. (1954). Chronic mercurialism. Arch. Neural. Psychiat. (Chicago) 72, 674681. CASSANO, G. B., VIOLA, P. L., GHETTI, B. AND AMADUCCI, L. (1969). The distribution of inhaled mercury (HgZo3) vapors in the brain of rats and mice. J. Neuropathol. Exp. Neurol. 28,308-320. DIAMOND, S. S. AND SLEIGHT, S. D. (1972). Acute and subchronicmethylmercury toxicosis in the rat. Toxicol. Appl. Pharmacol. 23, 197-207. EMERICK, R. J. AND HOLM, A. M. (1972). Toxicity and tissue distribution of mercury in rats fed various mercurial compounds. Nufr. Rep. Znt. 6, 125-131. GARMAN, R., WEISS, B. AND EVANS, H. (1975). Alkylmercurial encephalopathy in the monkey (Saimiri sciureus and Macaca arctoides). Acta Neuropathol. (Berlin) 32, 61-74. GROSSMAN, S. P. (1975). Role of the hypothalamus in the regulation of food and water intake. Psychol. Rev. 82,200-224.

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D., BOMFORD, R. R. AND RUSSELL. D. S. (1940). Poisoning by methyl mercury compounds. Quart. J. Med. 9, 193-213. IKEDA, Y., TOBE, M., KOBAYASHI, K., SUZUKI, S., KAWASAKI, Y. AND YONEMARU, H. (1973). Long-term toxicity study of methylmercuricchloride in monkeys(first report). Toxicology 1, 361-375. KIRK, R. E. (1968).Experimental Design Procedures for the Behavioral Sciences. BrooksCole, Belmont, Calif. KLEIN, R., HERMAN, S. P., BULLOCK, B. C. AND TALLEY, F. A. (1973).Methyl mercury intoxication in rat kidneys. Arch. Pathol. 96, 83-90. MAGHAZAJI, H. 1. (1974).Psychiatricaspectsof methyl mercury poisoning.J. Neural. NeuroHUNTER,

surg. Psychiat. 37, 954-958.

T., DESHIMARU, M., SUMIYOSHI, S., TERAOKA, A., UDO,N., HATTORI, E. AND S. (1970). Experimental organic mercury poisoning-pathologic changesin peripheralnerves.Acta Neuropathol. 15,45-55. MUKAI, N. (1972). An experimental study of alkylmercurial encephalopathy.Acta Neuropathol. (Berlin) 22, 102-109. PRICK. J. J. G.. SONNEN, A. E. H. AND SLOOFF, J. L. (1967). Organic mercury poisoningI. Proc. Kon. Ned. Akad. Wetensch. (Ser. C) 70, 152-186. RICHARDSON, R. J. AND MURPHY, S. D. (1974). Neurotoxicity produced by intracranial administrationof methylmercury in rats. Toxicol. Appl. Pharmacol. 29, 289-300. SCHROEDER, H. A. AND MITCHENER, M. (1975).Life-term effectsof mercury, methyl mercury, and nine other trace metalson mice.J. Nutr. 105,452458. SIEGEL, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. SILBERBERG, I., PRUTKIN, L. AND LEIDER, M. (1969).Electron microscopicstudiesof transepidermalabsorptionof mercury. Arch. Environ. Health 19, 7-14. SMITH, R. G. (1972).Dose-response relationshipassociatedwith known mercury absorption at low dose levels of inorganic mercury. In Environmental Mercury Contamination (R. Hartung and B. D. Dinman, Eds.), pp. 207-222. Ann Arbor SciencePublications, Ann Arbor, Mich. SPYKER, J. (1975).Behavioral teratology and toxicology. In Behavioral Toxicology (B. Weiss and V. Laties, Eds.), Chap. 12, pp. 31l-350. Plenum,New York. STILLINGS, B. R., LAGALLY, H., BAIJERSFELD, P. AND SOARES, J. (1974).Effects of cysteine, selenium.and fish protein on the toxicity and metabolismof methylmercuryin rats. Toxicol. MIYAKAWA. TATETSU,

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T. AND MIYAMA, T. (1971). Neurological symptomsand mercury concentration in the brain of micefed with methylmercury salts.Znd. Health 9, 51-58. TAKEUCHI, T. (1972). Biological reactions and pathological changesin human beingsand animalscausedby organic mercury contamination. In Environmental Mercury Contamination (R. Hartung and B. D. Dinman, Eds.), pp. 207-222. Ann Arbor SciencePublishers, Ann Arbor, Mich. TAKEUCHI, T., MORIKAWA, N., MATSUMOTO, H. AND SHIRAISHI, Y. (1962). A pathological study of Minamata diseasein Japan. Acta Neuropathol. (Berlin) 2,4&57. TEITELBAUM, P. (1975). Sensory control of hypothalamic hyperphagia. J. Comp. Physiol. Psychol. 48, 156-163. VROOM, F. Q. ANDGREER,M. (1972).Mercury vapor intoxication. Brain 95, 305-318. SUZUKI,