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J mouse
Gen. Pharmac., 1976, Vol. 7, pp. 75 to 79, Pergamon Press. Printed In Great Britatn
THE PHARMACODYNAMICS OF METHYL MERCURY IN THE BALB/c MOUSE AND ITS HAIRLESS HOMOLOG, THE HRS/J MOUSE PAUL M. SALVATERRA AND E D W A R D J. MASSARO* Department of Biochemistry, State University of N e w York at Buffalo, Buffalo, N Y 14214, U.S.A.
(Received 9 May 1975) Abstract--1. Tissue/organ pharmacodynamics of methyl mercury were investigated in the BALB/c mouse and its hairless (homozygous) homolog, the HRS/J mouse. 2. The order of maximum tissue/organ Hg uptake was simiIar for both strains. For most tissues/ organs, the rate of Hg elimination was slower and the concentration maxima greater in the BALB/c mouse. 3. Urinary excretion totaled 10~o of the dose for the BALB/c mouse and 6 ~ for the HRS/J mouse while facal Hg excretion totaled 63 ~ for the BALB/c strain and 86 ~o for the HRS/J.
INTRODUCTION Human populations, especially those of industrialized countries, are constantly exposed to environmental contaminants (e.g. Methyl Mercury in Fish, 1971, Lead: Airborne Lead in Perspective, 1972). Some of these such as methyl mercury (MeHg), which is a potent neurotoxic agent, have been responsible for incidents of human poisoning of epidemic proportions (Methyl Mercury in Fish, 1971; Bakir et al., 1973). F o r the most part, however, human exposure to environmental pollutants is on a low-level, long-term basis. Little is known of the effects of such exposure, but certain environmentally available toxic substances such as methyl mercury are cumulative poisons (Methyl Mercury in Fish, 1971). In apparently "normal" hnmans there is considerable individual variability in tissue mercury levels (e.g. Massaro et al., 1974). The mechanisms responsible for maintaining this variability may be genetically based. Presently, our understanding of these mechanisms and their, possible genetic control is primitive (Pharmacogenetics, 1968) . . . . . . It is well known that hair binds heavy metals (Underwood, 1971). Quantitatively, since it i s metabolically inactive and because of its large concentration factor (Salvaterra et al., 1975) and continuous growth, hair may play a major role in the regulation of tissue MeHg levels in hairier species (e.g. rodents, lower primates) vs relatively hairless animals such as man (~stlund, 1969; Ulfvarson, 1970). Individual variability in hair mercury levels in mice (Salvaterra et al., 1975) and in humans (Birke et aL, 1972) may be related to individual differences in susceptibility to MeHg intoxication. * To w h o m allcorrespondence should be addressed.
Thus, (i) hair of different composition may have a greater or lesser capacity to bind (and, therefore, sequester) mercury or, (ii) assuming a finite rate of binding, the more rapid the turnover (hair growth and fallout), the less susceptible the animal to intoxication. To gain insight into the role of hair in M e H g metabolism, we have undertaken a study o f the pharmacokinetics of MeHg in the BALB/c mouse and its hairless homozygons homolog, the HRS/J mouse. MATERIALS AND METHODS
Animals Five week old (averaging 18 g) female BALB/c and HRS/J mice were obtained from the Jackson Laboratories (Bar Harbor, Maine) and housed in groups of five.
Mercury administration MeHgCI (Alpha Inorganics, Beverly, Massachusetts) and Me~°SHgCI (New England Nuclear, Boston, Massachusetts) were dissolved in 0-14 M NaC1 to yield a solution containing 0.25 mg Hg/ml at a specific activity of 20 pCi/nd. A single dose containing 2.5 mg Hg (as MeHg: specific activity 200/zCi)/kg body weight was injected intraperitoneally.
Tissue sampling and collection of excretia Five animals of each strain were sacrificed by cervical dislocation at 0.25, 0.5, 1, 2, 4, 8, 16 and 32 days post MeHg administration. The 32 day animals were housed individually in stainless steel metabolism cages equipped with a wire mesh floor to trap feces. Urine was collected directly into a counting vial (containing 5 ml of 1.0% bovine serum albumin to bind any bolatile Hg compounds eliminated in the urine). Tissue/organ samples were placed in tared airtight polyethylene vials. Tissue sampling proceeded as follows: 75
76
PAUL M. SALVATERRAAND EDWARDJ. MASSARO
blood (approximately 100/zl) was obtained from the heart by syringe: the right lobe of the liver, the spleen, the left kidney, the left gastrocenemius muscle and both lenses were removed: the brain was extirpated and dissected into cerebral cortex and cerebellum: the suprascapular fat pads provided fat tissue: skin and skin plus hair samples were removed from the back just below the neck: hair (BALB/c) was shaved from the skin plus hair samples: "whole body" homogenates were prepared from frozen/thawed carcasses by blending with 2 vol of distilled water at high speed in a Waring blender.
Determinationofmercurylevels The levels °f radi°activity in all tissue/°rgan samples
were quantitated by gamma scintillation spectrometry (Packard Gamma Spectrometer, Model 3315). The amount of Hg present was calculated by comparing the samples with standards prepared from the injection solution. Data for tissue/organ Hg concentrations are expressed as pg Hg/g wet wt. Data for urine and feces are expressed as percentage of the dose.
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RESULTS Figure 1 (a-k) depicts the uptake and elimination of MeHg by the tissues/organs of the two strains of mice studied. It is evident that MeHg is rapidly transported to and absorbed by the tissues/organs (Giblin & Massaro, 1974). Blood, spleen, liver, kidney, fat and skin attain their maxima (~g Hg/g wet wt of tissue) within 12hr after which concentrations decrease (Fig. 1: a-d, f and j). Muscle (Fig. 1: e), cerebellum (Fig. 1: g), cortex (Fig. 1: h), lens (Fig. 1: i), and hair (Fig. 1: k) attain Hg concentration maxima at later times suggesting a more effective barrier between each of these tissues/organs and the circulating Hg. Maximum Hg concentrations are reached in muscle tissue 1 and 2 days postadministration in HRS/J and BALB/c, respectively; in the lens, 8 and 16 days, respectively, are required while 4 days are required for the cerebrum and cerebellum of both strains. The pattern of uptake for hair is one of rapid increase within 12hr followed by rapid decline and another, slower, uptake phase reaching a second maximum on day 8. The order of maximum tissue/organ Hg uptake is similar for both strains although the relative amounts and rates of uptake and elimination vary. For BALB/c mice, the order is: kidney > liver > blood > spleen > muscle > fat > skin plus hair > cortex > cerebellum > lens > hair. For HRS/J mice, it is kidney > liver > spleen > blood > muscle > fat > skin > cortex > cerebellum > lens. The rate (T½) of elimination for Hg administered as MeHg varies as a function of time [appears to be biphasic (Ulfvarson, 1969; Giblin & Massaro, 1973)]. However, it does provide a means of comparing elimination rates if cognizance is taken of the time span for which it has been calculated. In this study, the Hg elimination rates of the various
Blood
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Time, days Fig. I. Uptake and elimination of Hg by tissues/organs of BALB/c and HRS/J mice after a single i.p. injection of 10 mg Hg (as MeHg)/kg body weight. Each point represents the mean + S.E.M. for 5 animals.
77
The pharmacodynamics of methyl mercury in the mouse Table 1. Half-time (Ti)" of elimination of MeHg from various tissues and whole bodies of BALB/c and HR~/J mice I ]BAL~/c
flRSI3
T~ (days)
lZ ~½(daya)
31ood
6.09
(10) c
4.69
SpleeR
6.43
(09)
5.50
(08)
Liver
7,37
(07)
5.81
(09)
(09)
(10)
Kidney
7.41
(06)
5,23
}luscZe
7.93
(05)
5.78
(06)
Fae
7,L6
(08)
5.58
(07)
Cerebellum
9,51
(04)
7.83
(04)
Cortex
10,58
(02)
8.53
(02)
Lens
27.39
(01)
33.59
(01)
(Hair)
10.14
(03)
8,19
(03)
Hair
23.40
Skln +
~hole 9odyb I0,045
5.80
a.
Calculated From ellmiuation phase of data in ~igure I.
b.
Calculated from whole body homogenates (Figure 2).
c.
The number in parentheses represents the rank order of the T~ from the longest T½ (lens) to the shortest (blood),
tissues/organs investigated differ markedly. The T½s (Table 1), calculated from the linear component of log/linear elimination rate curves (Fig. 1), show that blood has the fastest elimination rate while the lens has the slowest. The 7"} for hair from the % Refalned dose-calculated from whole body leo
i
-.
BALB/c mice is of the same order of magnitude as that of the lens. Also, compared to other tissues/organs, the cortex and cerebellum exhibit a relatively slow rate of Hg release. The overall rate of elimination of Hg (administered as MeHg) differs considerable between the BALB/c and HRS/J mouse. Except for the lens, the T½s for the individual tissues/organs and carcass are longer for the BALB/c mouse (Table 1). "Whole body" (carcass) elimination rates (Fig. 2) were obtained from log/linear plots of the percentage of the dose retained vs time for the two strains. The values for the percentage of the dose retained were calculated from the amount of 2°gHg present in "whole body" homogenates (ref. section on Materials and Methods). In addition, the maximum Hg concentrations were greater in all tissues/organs of the BALB/c mouse (except kidney and cerebral cortex) up to 2 days postadministration (Fig. 1: d and h) and for the skin vs skin plus hair at all times up to 16 days. The rate and cumulative urinary excretion of Hg calculated as percentage of the dose are illustrated in Fig. 3. The rate of urinary excretion appears constant during the 32 day period of observation for both strains with the BALB/c mice exhibiting a slightly higher cumulative excretion. The amount of Hg eliminated by this route is small; less than 10% and 6% for BALB/c and HRS/J, respectively. The rate of fecal excretion of 2°3Hg is strikingly different for each strain (Fig. 4). Via this route, there is an initial rapid rate of elimination followed by a gradual decline in rate. The rate of fecal excretion is higher for the HRS/J mice up to 14 days postadministration, after which the rates for the two strains are approximately equal. Cumulatively, the HRS/J mice excreted 86 % of the administered Hg in 32 days while the BALB/c mice excreted 63 ~o. The proportion of total fecal to total urinary excretion 32 days postadministration is 6.3 for BALB/c and 14.3 for HRS/J mice. Urinary exere'Honof2°~-Ig
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Fig. 2. Whole-body retention of Hg in BALB/c and HRS/J mice after a single i.p. injection of 2.5 mg Hg (as MeHg~/kg body weight. The log of the percentage of the dose retained was calculated from the WHg levels present in whole body homogenates.
0
0 I Z
4
8
12 Tim%
16
20
24
28
32
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Fig. 3. Daily and cumulative urinary excretion of Hg in BALB/c and HRS/3 mice after a single i.p. injection of 2.5 mg I-Ig (as MeHg)/kg body weight. Each point represents the mean_+S.E.M. for 5 animals.
78
PAUL M. SALVATERRAAND EDWARD J. MASSARO
Fecal exchange of 2°3Hg 9o
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Time, days Fig. 4. Daily and cumulative fecal excretion of Hg in BALB/c and HRS/J mice after a single i.p. injection of 2.5 mg Hg (as MeHg)/kg body weight. DISCUSSION
The influence of strain difference on the pharmacokinetics of MeHg in animals has not been extensively studied. In mice, it has been concluded (Miller & Csonka, 1968) to be of no consequence. However, in this study, large differences in the uptake, distribution and elimination of MeHg were observed between the BALB/c mouse and its hairless (homozygous) homolog, the HRS/J mouse. It has been suggested (0stlund, 1969; Ulfvarson, 1970; Salvaterra et aL, 1974) that hair may play a very important role in sequestration and excretion of MeHg. As shown in this study, there are profound differences between the strains, not only in total tissue/organ uptake of MeI-Ig; but also in elimination rates. These differences may be the consequence of the presence of hair. The HRS/J mouse more effectively excretes MeHg via the feces. This characteristic may have been selected for to compensate for the inability of this strain to sequester (i.e. detoxify) Hg compounds and those of other metals via the hair. In addition, the HRS/J mouse shows a larger uptake of Hg administered as MeHg by the kidney and, at early times, by the skin, compared to B A L B / c skin plus hair. The cerebral cortex of the HRS/J mouse also takes up Hg at a faster rate than that of the BALB/c mouse. Quantitatively, the most important route of excretion of Hg administered as MeHg is via the feces ((~)stlund, 1969; Ulfvarson, 1970) although significant amounts are detected in urine. The relative importance of these two routes differs considerably between the two strains of mice
studied in this investigation as evidenced by fecal to urinary ratios of Hg of 6.3 and 14"3 for BALB/c and HRS/J, respectively. However, within the strains the ratios change with time indicating an alteration in the importance of these modes of elimination in short and long term excretion. The fecal route exhibits a continually declining rate (0stlund, 1969) while the urinary rate is relatively constant (Fig. 3); but has been reported to increase with time (Ostlund, 1969). The relative rates of elimination via these routes may be related to tissue/organ differences in the rate of biotransformation of MeHg to inorganic Hg ~+ (Norseth, 1972; Norseth & Clarkson, 1970a, b). Hair influences "whole body" retention of Hg due to prolonged retention of bound Hg (Table 1 ; Ostlund, 1969; Ulfvarson, 1970). Quantitatively, sequestration in hair does not account for a great proportion of the Hg excreted following a single administration of MeHg. However, its importance increases upon repeated exposure (Salvaterra et al., 1974). Different tissues/organs exhibit differences in rates of uptake, biotransformation and release of Hg administered as MeHg. The rate of Hg elimination is influenced by the turnover rates of Hgbinding tissue proteins. For example, the longer T½s of Hg in the lens and brain compared to blood and the spleen may be due, primarily, to a slow turnover rate of Hg-binding proteins in the lens and brain. Metallothionein, a metal binding protein found in liver and kidney (Jakubowski et al., 1970; Pulido et aL, 1966), accounts for the ability of these tissues to concentrate Hg. Since MeHg is transported by the blood (Giblin & Massaro, 1974), the barriers to circulatory exchange with the various tissues/organs must also be considered. The blood-brain barrier, for example, is a very selective and effective protective screen (Katzman, 1972). An analogous barrier may protect the lens. However, the slow rate of Hg elimination by the lens and brain may account for the apparent target nature of these tissues in cases of MeHg poisoning (B/ickstrSm, 1969; Takeuchi, 1972). Whether a low rate of turnover of Hgbinding proteins, of biotransformation or some combination of both processes is responsible for this phenomenon is unknown. SUMMARY 1. Female BALB/c and HRS/J mice (5 weeks old; averaging 18 g) were injected i.p. with 2.5 mg Hg [as methyl mercury (MeHg) +Me~°3Hg]/kg b.w. at a specific activity of 200/LCi. 2. Five animals of each strain were sacrificed at 0.25, 0.5, 1, 2, 4, 16 and 32 days postadministration. 3. MeHg uptake and elimination rates were quantitated in the blood, liver, spleen, kidney, gastrocenemius muscle, lens, cerebral cortex,
The pharmacodynamics of methyl mercury in the mouse
79
LA Du B. N. & KALOWW. (Editors) (1968) Pharmacogenetics. Ann. NY. Acad. Sci. 151, 691-1001. Lead: Airborne Lead in Perspective (1972) Committee on Biological Effects of Atmospheric Pollutants. National Academy of Sciences, Washington. 330p. MASSAROE. J., YAFFES. J. & THOMASC. C., Jr. (1974) Mercury levels in human brain, skeletal muscle and body fluids. Life Sci. 14, 1939-1946. Methyl Mercury in Fish. A Toxicologic-Epidemiologic Evaluation of Risks (1971) Report from an expert group. Nord. Hyg. Tidskr., (Suppl. 4) 364p. MILER V. L. & CSomcA E. (1968) Mercury retention in two strains of mice. Toxicol. appl. PharmacoL 13, 207-211. No~E3aa T. (1972) Biotransformation of methyl mercuric salts in the rat with chronic administration of methyl mercuric cysteine. Acta Pharmacol. ToxicoL 31, 138-148. NOI~SETHT. & CLARKSONT. W. (1970a) Blotransformation of methyl mercury salts in the rat studied by specific determination of inorganic mercury. Biochem. Pharmac. 19, 2775-2783. NORSETH T. & CLARKSONT. W. (1970b) Studies on the biotransformation of ~°~Hg-labeled methyl mercury chloride in rats. Arch. Environ. Health 21, 717-727. REFERENCES OSTLUNI)K. (1969) Studies on the metabolism of methyl mercury and dimethyl mercury in mice. Acta BXCKSTROM J. (1969) Distribution studies of mercuric Pharmacol. Toxicol. 27, (Suppl. 1). pesticides in quail and some fresh-water fishes. Acts PULn)O P., K~QI J. H. R. & VALImEB. L. (1966) Isolation Pharmacol. Toxicol. (Suppl. 3) 27. and some properties of human metallothionein. BAKIR F., DAMLUn S. F., AMIN-ZAKIL., ]V[URTADHA Biochemistry 5, 1768-1777. M., KHALIDIA., AL-RAWI N. Y., TmRrrt S., DnAnm H. I., CLARKSONT. W., SMITHJ. C. & DOI~RTY R. A. SALVATERRA P., MASSARQE. J., MORGANTI J. B. & LOWN B. A. (1975) Time dependent tissue/organ (1973) Methlmercu~ poisoning in Iraq. Science, N. Y. uptake and distribution of Z°aHg in mice exposed to 181, 230-241. multiple sublethal doses of methyl mercury. Toxicol. GmLIN F. J. & MASSAROE. J. (1973) Pharmacodynamics appl. Pharmacol. 32, 432-442 of methyl mercury in the rainbow trout (Salmo gairdneri): tissue uptake, distribution and excretion. TAKEUCmT. (1972) Biological reactions and pathological changes in human beings and animals caused by Toxicol. appl. Pharmacol. 24, 81-91. organic mercury contamination. In Environmental GmLIN F. J. & MASSAROE. J. (1974) The mechanism of Mercury Contamination (Edited by HARTUNG R. & methylmercury transport and transfer to the tissues DrNMAN B. D.), pp. 247-289. Ann Arbor Science, of the rainbow trout (Salmo gairdneri). In Trace Ann Arbor. Substances in Environmental Health--VIII (Edited by I-IE~HILL D. D.), pp. 349-355. University of ULFVAnSONU. (1969) The effect of the size of the dose on the distribution and excretion of mercury in rats Missouri Press. after single intravenous injection of various mercury JAKUBOWSKI M., PIOTROWSKI J. & TROJANOWSKAB. compounds. Toxicol. appl. PharmacoL 15, 517-524. (1970) Binding of mercury in the rat: studies using 2°aHgClt and gel filtration. Toxicol. appL Pharmacol. ULWm,sos U. (1970) Transportation of mercury in animals. Stud. Lab. Salut. 6, 1-63. 16, 743-753. KATZMANR. (1972) Basic Neurochemistry, pp. 327-339. UNDERWOOD E. J. (1971) Trace Elements in Human and Animal Nutrition. 543p. Academic Press, New York. Little & Brown, Boston. cerebellum, suprascapular fat pad, skin, hair and carcass by gamma scintillation spectrometry. 4. The order of maximum tissue/organ Hg uptake was similar for both strains. 5. Blood had the fastest Hg elimination rate (shortest T½); lens and BALB]c hair, the slowest. Cortex and cerebellum also exhibit slow rates of release. 6. Except for lens, the T½s for the tissue/organs and carcass were slower for the BALB/c mouse. 7. Except for kidney and cortex, maximum tissue/organ Hg concentrations were greater in the BALB/c mouse up to 2 days postadministration. 8. Urinary H g excretion rate was constant for both strains. The amount of Hg eliminated by this route was 10% for the BALB/c mouse and 6% for the HRS/J. 9. The rate of fecal Hg excretion was much higher for the HRS/J mouse up to 14 days postadministration. In 32 days, it excreted 869/o of the dose while the BALB/c mouse excreted 63 %.
THE PHARMACODYNAMICS OF METHYL MERCURY IN THE BALB/c MOUSE AND ITS HAIRLESS HOMOLOG, THE HRS/J MOUSE PAUL M. SALVATERRA AND E D W A R D J. MASSARO* Department of Biochemistry, State University of N e w York at Buffalo, Buffalo, N Y 14214, U.S.A.
(Received 9 May 1975) Abstract--1. Tissue/organ pharmacodynamics of methyl mercury were investigated in the BALB/c mouse and its hairless (homozygous) homolog, the HRS/J mouse. 2. The order of maximum tissue/organ Hg uptake was simiIar for both strains. For most tissues/ organs, the rate of Hg elimination was slower and the concentration maxima greater in the BALB/c mouse. 3. Urinary excretion totaled 10~o of the dose for the BALB/c mouse and 6 ~ for the HRS/J mouse while facal Hg excretion totaled 63 ~ for the BALB/c strain and 86 ~o for the HRS/J.
INTRODUCTION Human populations, especially those of industrialized countries, are constantly exposed to environmental contaminants (e.g. Methyl Mercury in Fish, 1971, Lead: Airborne Lead in Perspective, 1972). Some of these such as methyl mercury (MeHg), which is a potent neurotoxic agent, have been responsible for incidents of human poisoning of epidemic proportions (Methyl Mercury in Fish, 1971; Bakir et al., 1973). F o r the most part, however, human exposure to environmental pollutants is on a low-level, long-term basis. Little is known of the effects of such exposure, but certain environmentally available toxic substances such as methyl mercury are cumulative poisons (Methyl Mercury in Fish, 1971). In apparently "normal" hnmans there is considerable individual variability in tissue mercury levels (e.g. Massaro et al., 1974). The mechanisms responsible for maintaining this variability may be genetically based. Presently, our understanding of these mechanisms and their, possible genetic control is primitive (Pharmacogenetics, 1968) . . . . . . It is well known that hair binds heavy metals (Underwood, 1971). Quantitatively, since it i s metabolically inactive and because of its large concentration factor (Salvaterra et al., 1975) and continuous growth, hair may play a major role in the regulation of tissue MeHg levels in hairier species (e.g. rodents, lower primates) vs relatively hairless animals such as man (~stlund, 1969; Ulfvarson, 1970). Individual variability in hair mercury levels in mice (Salvaterra et al., 1975) and in humans (Birke et aL, 1972) may be related to individual differences in susceptibility to MeHg intoxication. * To w h o m allcorrespondence should be addressed.
Thus, (i) hair of different composition may have a greater or lesser capacity to bind (and, therefore, sequester) mercury or, (ii) assuming a finite rate of binding, the more rapid the turnover (hair growth and fallout), the less susceptible the animal to intoxication. To gain insight into the role of hair in M e H g metabolism, we have undertaken a study o f the pharmacokinetics of MeHg in the BALB/c mouse and its hairless homozygons homolog, the HRS/J mouse. MATERIALS AND METHODS
Animals Five week old (averaging 18 g) female BALB/c and HRS/J mice were obtained from the Jackson Laboratories (Bar Harbor, Maine) and housed in groups of five.
Mercury administration MeHgCI (Alpha Inorganics, Beverly, Massachusetts) and Me~°SHgCI (New England Nuclear, Boston, Massachusetts) were dissolved in 0-14 M NaC1 to yield a solution containing 0.25 mg Hg/ml at a specific activity of 20 pCi/nd. A single dose containing 2.5 mg Hg (as MeHg: specific activity 200/zCi)/kg body weight was injected intraperitoneally.
Tissue sampling and collection of excretia Five animals of each strain were sacrificed by cervical dislocation at 0.25, 0.5, 1, 2, 4, 8, 16 and 32 days post MeHg administration. The 32 day animals were housed individually in stainless steel metabolism cages equipped with a wire mesh floor to trap feces. Urine was collected directly into a counting vial (containing 5 ml of 1.0% bovine serum albumin to bind any bolatile Hg compounds eliminated in the urine). Tissue/organ samples were placed in tared airtight polyethylene vials. Tissue sampling proceeded as follows: 75
76
PAUL M. SALVATERRAAND EDWARDJ. MASSARO
blood (approximately 100/zl) was obtained from the heart by syringe: the right lobe of the liver, the spleen, the left kidney, the left gastrocenemius muscle and both lenses were removed: the brain was extirpated and dissected into cerebral cortex and cerebellum: the suprascapular fat pads provided fat tissue: skin and skin plus hair samples were removed from the back just below the neck: hair (BALB/c) was shaved from the skin plus hair samples: "whole body" homogenates were prepared from frozen/thawed carcasses by blending with 2 vol of distilled water at high speed in a Waring blender.
Determinationofmercurylevels The levels °f radi°activity in all tissue/°rgan samples
were quantitated by gamma scintillation spectrometry (Packard Gamma Spectrometer, Model 3315). The amount of Hg present was calculated by comparing the samples with standards prepared from the injection solution. Data for tissue/organ Hg concentrations are expressed as pg Hg/g wet wt. Data for urine and feces are expressed as percentage of the dose.
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RESULTS Figure 1 (a-k) depicts the uptake and elimination of MeHg by the tissues/organs of the two strains of mice studied. It is evident that MeHg is rapidly transported to and absorbed by the tissues/organs (Giblin & Massaro, 1974). Blood, spleen, liver, kidney, fat and skin attain their maxima (~g Hg/g wet wt of tissue) within 12hr after which concentrations decrease (Fig. 1: a-d, f and j). Muscle (Fig. 1: e), cerebellum (Fig. 1: g), cortex (Fig. 1: h), lens (Fig. 1: i), and hair (Fig. 1: k) attain Hg concentration maxima at later times suggesting a more effective barrier between each of these tissues/organs and the circulating Hg. Maximum Hg concentrations are reached in muscle tissue 1 and 2 days postadministration in HRS/J and BALB/c, respectively; in the lens, 8 and 16 days, respectively, are required while 4 days are required for the cerebrum and cerebellum of both strains. The pattern of uptake for hair is one of rapid increase within 12hr followed by rapid decline and another, slower, uptake phase reaching a second maximum on day 8. The order of maximum tissue/organ Hg uptake is similar for both strains although the relative amounts and rates of uptake and elimination vary. For BALB/c mice, the order is: kidney > liver > blood > spleen > muscle > fat > skin plus hair > cortex > cerebellum > lens > hair. For HRS/J mice, it is kidney > liver > spleen > blood > muscle > fat > skin > cortex > cerebellum > lens. The rate (T½) of elimination for Hg administered as MeHg varies as a function of time [appears to be biphasic (Ulfvarson, 1969; Giblin & Massaro, 1973)]. However, it does provide a means of comparing elimination rates if cognizance is taken of the time span for which it has been calculated. In this study, the Hg elimination rates of the various
Blood
"Cortex ~
tJ 32 0124 8 Time, days
~.
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(h)
32
Skin and hair,skin
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(i)
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i~
3a
Time, days Fig. I. Uptake and elimination of Hg by tissues/organs of BALB/c and HRS/J mice after a single i.p. injection of 10 mg Hg (as MeHg)/kg body weight. Each point represents the mean + S.E.M. for 5 animals.
77
The pharmacodynamics of methyl mercury in the mouse Table 1. Half-time (Ti)" of elimination of MeHg from various tissues and whole bodies of BALB/c and HR~/J mice I ]BAL~/c
flRSI3
T~ (days)
lZ ~½(daya)
31ood
6.09
(10) c
4.69
SpleeR
6.43
(09)
5.50
(08)
Liver
7,37
(07)
5.81
(09)
(09)
(10)
Kidney
7.41
(06)
5,23
}luscZe
7.93
(05)
5.78
(06)
Fae
7,L6
(08)
5.58
(07)
Cerebellum
9,51
(04)
7.83
(04)
Cortex
10,58
(02)
8.53
(02)
Lens
27.39
(01)
33.59
(01)
(Hair)
10.14
(03)
8,19
(03)
Hair
23.40
Skln +
~hole 9odyb I0,045
5.80
a.
Calculated From ellmiuation phase of data in ~igure I.
b.
Calculated from whole body homogenates (Figure 2).
c.
The number in parentheses represents the rank order of the T~ from the longest T½ (lens) to the shortest (blood),
tissues/organs investigated differ markedly. The T½s (Table 1), calculated from the linear component of log/linear elimination rate curves (Fig. 1), show that blood has the fastest elimination rate while the lens has the slowest. The 7"} for hair from the % Refalned dose-calculated from whole body leo
i
-.
BALB/c mice is of the same order of magnitude as that of the lens. Also, compared to other tissues/organs, the cortex and cerebellum exhibit a relatively slow rate of Hg release. The overall rate of elimination of Hg (administered as MeHg) differs considerable between the BALB/c and HRS/J mouse. Except for the lens, the T½s for the individual tissues/organs and carcass are longer for the BALB/c mouse (Table 1). "Whole body" (carcass) elimination rates (Fig. 2) were obtained from log/linear plots of the percentage of the dose retained vs time for the two strains. The values for the percentage of the dose retained were calculated from the amount of 2°gHg present in "whole body" homogenates (ref. section on Materials and Methods). In addition, the maximum Hg concentrations were greater in all tissues/organs of the BALB/c mouse (except kidney and cerebral cortex) up to 2 days postadministration (Fig. 1: d and h) and for the skin vs skin plus hair at all times up to 16 days. The rate and cumulative urinary excretion of Hg calculated as percentage of the dose are illustrated in Fig. 3. The rate of urinary excretion appears constant during the 32 day period of observation for both strains with the BALB/c mice exhibiting a slightly higher cumulative excretion. The amount of Hg eliminated by this route is small; less than 10% and 6% for BALB/c and HRS/J, respectively. The rate of fecal excretion of 2°3Hg is strikingly different for each strain (Fig. 4). Via this route, there is an initial rapid rate of elimination followed by a gradual decline in rate. The rate of fecal excretion is higher for the HRS/J mice up to 14 days postadministration, after which the rates for the two strains are approximately equal. Cumulatively, the HRS/J mice excreted 86 % of the administered Hg in 32 days while the BALB/c mice excreted 63 ~o. The proportion of total fecal to total urinary excretion 32 days postadministration is 6.3 for BALB/c and 14.3 for HRS/J mice. Urinary exere'Honof2°~-Ig
,o
"""''""']
1 5 - (o)
~; ,o
o'-: .
o HRS/J ~
.
'
l
.~ ~,..I---I"I
o_
(
(hi
,~ 012
4
8
16
Time,
- BALB/C
5
o HRS/J
32
doys
Fig. 2. Whole-body retention of Hg in BALB/c and HRS/J mice after a single i.p. injection of 2.5 mg Hg (as MeHg~/kg body weight. The log of the percentage of the dose retained was calculated from the WHg levels present in whole body homogenates.
0
0 I Z
4
8
12 Tim%
16
20
24
28
32
days
Fig. 3. Daily and cumulative urinary excretion of Hg in BALB/c and HRS/3 mice after a single i.p. injection of 2.5 mg I-Ig (as MeHg)/kg body weight. Each point represents the mean_+S.E.M. for 5 animals.
78
PAUL M. SALVATERRAAND EDWARD J. MASSARO
Fecal exchange of 2°3Hg 9o
{_#.~-{"{
I a!
7o
{~.-~"{"{'2" }/~"
>~ 5o
~
/Z
~/
..~..!i
i t .~i~
~i-''~'~'" '" ....
.ii:" fO
~t/.,'t1'
0
- i - - i - - I-- i- - I - - i - - I - - i - - I - - I - - i - - I - -
>.
15
~)/°",.o....o
6
,5
o'" ,t ...... i l l ~ _ --i-- i--i--i--i--i--i--i--i--i--i--iz~ 0 I 4 8 12 16 2 0 24
I--1-- [--I-
28
32
Time, days Fig. 4. Daily and cumulative fecal excretion of Hg in BALB/c and HRS/J mice after a single i.p. injection of 2.5 mg Hg (as MeHg)/kg body weight. DISCUSSION
The influence of strain difference on the pharmacokinetics of MeHg in animals has not been extensively studied. In mice, it has been concluded (Miller & Csonka, 1968) to be of no consequence. However, in this study, large differences in the uptake, distribution and elimination of MeHg were observed between the BALB/c mouse and its hairless (homozygous) homolog, the HRS/J mouse. It has been suggested (0stlund, 1969; Ulfvarson, 1970; Salvaterra et aL, 1974) that hair may play a very important role in sequestration and excretion of MeHg. As shown in this study, there are profound differences between the strains, not only in total tissue/organ uptake of MeI-Ig; but also in elimination rates. These differences may be the consequence of the presence of hair. The HRS/J mouse more effectively excretes MeHg via the feces. This characteristic may have been selected for to compensate for the inability of this strain to sequester (i.e. detoxify) Hg compounds and those of other metals via the hair. In addition, the HRS/J mouse shows a larger uptake of Hg administered as MeHg by the kidney and, at early times, by the skin, compared to B A L B / c skin plus hair. The cerebral cortex of the HRS/J mouse also takes up Hg at a faster rate than that of the BALB/c mouse. Quantitatively, the most important route of excretion of Hg administered as MeHg is via the feces ((~)stlund, 1969; Ulfvarson, 1970) although significant amounts are detected in urine. The relative importance of these two routes differs considerably between the two strains of mice
studied in this investigation as evidenced by fecal to urinary ratios of Hg of 6.3 and 14"3 for BALB/c and HRS/J, respectively. However, within the strains the ratios change with time indicating an alteration in the importance of these modes of elimination in short and long term excretion. The fecal route exhibits a continually declining rate (0stlund, 1969) while the urinary rate is relatively constant (Fig. 3); but has been reported to increase with time (Ostlund, 1969). The relative rates of elimination via these routes may be related to tissue/organ differences in the rate of biotransformation of MeHg to inorganic Hg ~+ (Norseth, 1972; Norseth & Clarkson, 1970a, b). Hair influences "whole body" retention of Hg due to prolonged retention of bound Hg (Table 1 ; Ostlund, 1969; Ulfvarson, 1970). Quantitatively, sequestration in hair does not account for a great proportion of the Hg excreted following a single administration of MeHg. However, its importance increases upon repeated exposure (Salvaterra et al., 1974). Different tissues/organs exhibit differences in rates of uptake, biotransformation and release of Hg administered as MeHg. The rate of Hg elimination is influenced by the turnover rates of Hgbinding tissue proteins. For example, the longer T½s of Hg in the lens and brain compared to blood and the spleen may be due, primarily, to a slow turnover rate of Hg-binding proteins in the lens and brain. Metallothionein, a metal binding protein found in liver and kidney (Jakubowski et al., 1970; Pulido et aL, 1966), accounts for the ability of these tissues to concentrate Hg. Since MeHg is transported by the blood (Giblin & Massaro, 1974), the barriers to circulatory exchange with the various tissues/organs must also be considered. The blood-brain barrier, for example, is a very selective and effective protective screen (Katzman, 1972). An analogous barrier may protect the lens. However, the slow rate of Hg elimination by the lens and brain may account for the apparent target nature of these tissues in cases of MeHg poisoning (B/ickstrSm, 1969; Takeuchi, 1972). Whether a low rate of turnover of Hgbinding proteins, of biotransformation or some combination of both processes is responsible for this phenomenon is unknown. SUMMARY 1. Female BALB/c and HRS/J mice (5 weeks old; averaging 18 g) were injected i.p. with 2.5 mg Hg [as methyl mercury (MeHg) +Me~°3Hg]/kg b.w. at a specific activity of 200/LCi. 2. Five animals of each strain were sacrificed at 0.25, 0.5, 1, 2, 4, 16 and 32 days postadministration. 3. MeHg uptake and elimination rates were quantitated in the blood, liver, spleen, kidney, gastrocenemius muscle, lens, cerebral cortex,
The pharmacodynamics of methyl mercury in the mouse
79
LA Du B. N. & KALOWW. (Editors) (1968) Pharmacogenetics. Ann. NY. Acad. Sci. 151, 691-1001. Lead: Airborne Lead in Perspective (1972) Committee on Biological Effects of Atmospheric Pollutants. National Academy of Sciences, Washington. 330p. MASSAROE. J., YAFFES. J. & THOMASC. C., Jr. (1974) Mercury levels in human brain, skeletal muscle and body fluids. Life Sci. 14, 1939-1946. Methyl Mercury in Fish. A Toxicologic-Epidemiologic Evaluation of Risks (1971) Report from an expert group. Nord. Hyg. Tidskr., (Suppl. 4) 364p. MILER V. L. & CSomcA E. (1968) Mercury retention in two strains of mice. Toxicol. appl. PharmacoL 13, 207-211. No~E3aa T. (1972) Biotransformation of methyl mercuric salts in the rat with chronic administration of methyl mercuric cysteine. Acta Pharmacol. ToxicoL 31, 138-148. NOI~SETHT. & CLARKSONT. W. (1970a) Blotransformation of methyl mercury salts in the rat studied by specific determination of inorganic mercury. Biochem. Pharmac. 19, 2775-2783. NORSETH T. & CLARKSONT. W. (1970b) Studies on the biotransformation of ~°~Hg-labeled methyl mercury chloride in rats. Arch. Environ. Health 21, 717-727. REFERENCES OSTLUNI)K. (1969) Studies on the metabolism of methyl mercury and dimethyl mercury in mice. Acta BXCKSTROM J. (1969) Distribution studies of mercuric Pharmacol. Toxicol. 27, (Suppl. 1). pesticides in quail and some fresh-water fishes. Acts PULn)O P., K~QI J. H. R. & VALImEB. L. (1966) Isolation Pharmacol. Toxicol. (Suppl. 3) 27. and some properties of human metallothionein. BAKIR F., DAMLUn S. F., AMIN-ZAKIL., ]V[URTADHA Biochemistry 5, 1768-1777. M., KHALIDIA., AL-RAWI N. Y., TmRrrt S., DnAnm H. I., CLARKSONT. W., SMITHJ. C. & DOI~RTY R. A. SALVATERRA P., MASSARQE. J., MORGANTI J. B. & LOWN B. A. (1975) Time dependent tissue/organ (1973) Methlmercu~ poisoning in Iraq. Science, N. Y. uptake and distribution of Z°aHg in mice exposed to 181, 230-241. multiple sublethal doses of methyl mercury. Toxicol. GmLIN F. J. & MASSAROE. J. (1973) Pharmacodynamics appl. Pharmacol. 32, 432-442 of methyl mercury in the rainbow trout (Salmo gairdneri): tissue uptake, distribution and excretion. TAKEUCmT. (1972) Biological reactions and pathological changes in human beings and animals caused by Toxicol. appl. Pharmacol. 24, 81-91. organic mercury contamination. In Environmental GmLIN F. J. & MASSAROE. J. (1974) The mechanism of Mercury Contamination (Edited by HARTUNG R. & methylmercury transport and transfer to the tissues DrNMAN B. D.), pp. 247-289. Ann Arbor Science, of the rainbow trout (Salmo gairdneri). In Trace Ann Arbor. Substances in Environmental Health--VIII (Edited by I-IE~HILL D. D.), pp. 349-355. University of ULFVAnSONU. (1969) The effect of the size of the dose on the distribution and excretion of mercury in rats Missouri Press. after single intravenous injection of various mercury JAKUBOWSKI M., PIOTROWSKI J. & TROJANOWSKAB. compounds. Toxicol. appl. PharmacoL 15, 517-524. (1970) Binding of mercury in the rat: studies using 2°aHgClt and gel filtration. Toxicol. appL Pharmacol. ULWm,sos U. (1970) Transportation of mercury in animals. Stud. Lab. Salut. 6, 1-63. 16, 743-753. KATZMANR. (1972) Basic Neurochemistry, pp. 327-339. UNDERWOOD E. J. (1971) Trace Elements in Human and Animal Nutrition. 543p. Academic Press, New York. Little & Brown, Boston. cerebellum, suprascapular fat pad, skin, hair and carcass by gamma scintillation spectrometry. 4. The order of maximum tissue/organ Hg uptake was similar for both strains. 5. Blood had the fastest Hg elimination rate (shortest T½); lens and BALB]c hair, the slowest. Cortex and cerebellum also exhibit slow rates of release. 6. Except for lens, the T½s for the tissue/organs and carcass were slower for the BALB/c mouse. 7. Except for kidney and cortex, maximum tissue/organ Hg concentrations were greater in the BALB/c mouse up to 2 days postadministration. 8. Urinary H g excretion rate was constant for both strains. The amount of Hg eliminated by this route was 10% for the BALB/c mouse and 6% for the HRS/J. 9. The rate of fecal Hg excretion was much higher for the HRS/J mouse up to 14 days postadministration. In 32 days, it excreted 869/o of the dose while the BALB/c mouse excreted 63 %.