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Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Disposition and retention of mercuric chloride in mice after oral and parenteral administration a
Jesper Bo Nielsen & Ole Andersen
b
a
Department of Environmental Medicine , Odense University , J. B. Winslöwsvej 19, Odense C, DK‐5000, Denmark b
Department of Environmental Medicine , Odense University , Odense, Denmark Published online: 20 Oct 2009.
To cite this article: Jesper Bo Nielsen & Ole Andersen (1990) Disposition and retention of mercuric chloride in mice after oral and parenteral administration, Journal of Toxicology and Environmental Health: Current Issues, 30:3, 167-180, DOI: 10.1080/15287399009531420 To link to this article: http://dx.doi.org/10.1080/15287399009531420
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
DISPOSITION AND RETENTION OF MERCURIC CHLORIDE IN MICE AFTER ORAL AND PARENTERAL ADMINISTRATION Jesper Bo Nielsen, Ole Andersen
Downloaded by [University of Cambridge] at 07:09 06 September 2015
Department of Environmental Medicine, Odense University, Odense, Denmark
The present study compares effects of dose size on whole-body retention and relative organ distribution of 203HgCl2, after oral and intraperitoneal administration to female mice of two strains (inbred CBA/Bom and outbred Bom:NMRI). Using whole-body retention data of oral and intraperitoneal administration, an estimated "true absorption" of a single oral dose of inorganic mercury was calculated to be about 20% at two different dose levels. At the highest oral dose, a delay in fecal elimination of nonabsorbed mercury was observed, indicating a decreased peristaltic rate. The relative hepatic deposition was larger after oral than after intraperitoneal administration, presumably due to a first-pass effect, and a correspondingly lower relative renal deposition was seen. Increasing doses at both exposure routes resulted in increasing relative deposition in liver, stomach, intestines, and spleen but decreasing relative deposition in lungs and kidneys. Bom:NMRI mice deposited a larger fraction of the whole-body burden in the kidneys and a smaller fraction in the livers than did CBA/Bom mice. Comparison to a previous study with male mice (Nielsen and Andersen, 1989) demonstrates that male and female mice deposit similar fractions of their body burden in the liver, while male mice deposit significantly larger amounts of mercury in the kidneys and smaller amounts in the carcass than do female mice. Thus, the toxicokinetics of inorganic mercury in mice depend on dose size, administration route, and sex; the mouse strain is of less importance than the other factors investigated. The absorption of inorganic mercury was estimated to be about 20%, that is, twice as high as earlier estimates.
INTRODUCTION The acute and chronic toxicity of inorganic mercury in humans has been studied during several centuries (Paracelsus, 1567; Ramazzini, 1700). The critical organs after acute oral exposure of humans are the mucous membranes of the gastrointestinal tract and the kidneys (Berlin, 1986; Ellenhorn and Barceleux, 1987). Human data on the toxicokinetics of mercuric mercury have primarily been focused on rate of elimination and body retention calculated on the basis of whole-body countings and fecal or urinary excretion (Rahola This study was supported by grant 12-8108 from the Danish Medical Research Council to Jesper Bo Nielsen. Requests for reprints should be sent to Jesper Bo Nielsen, Department of Environmental Medicine, Odense University, J. B. Winslöwsvej 19, DK-5000 Odense C, Denmark. 167 Journal of Toxicology and Environmental Health, 30:167-180, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
Downloaded by [University of Cambridge] at 07:09 06 September 2015
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et al., 1973; Miettinen, 1973). Calculation of the intestinal absorption on the basis of these investigations is seriously undermined by the fact that part of the intestinally absorbed mercury will be reexcreted to the gastrointestinal tract during the experimental period. Accordingly, the authors did not try to establish the intestinal absorption, but rather the wholebody retention from their data. Thus a clear distinction must be made between body retention and the true intestinal absorption. Investigations of the toxicokinetics of soluble mercury(ll) salts after administration via various routes indicate that differences in the absorption, relative organ distribution, and rate of elimination occur (Miyama et al., 1968; Nielsen and Andersen, 1989). However, most experimental studies of the toxicokinetics of inorganic mercury have used parenteral exposure (Rothstein and Hayes, 1960; Berlin et al., 1966; Cember and Donagi, 1963; Magos and Webb, 1976; Kristensen and Hansen, 1980). As several different soluble salts of inorganic mercury(ll) and also various animal species and strains were used, a general conclusion is not easily drawn on the basis of these studies. However, the rate of intestinal absorption of mercury from various soluble compounds would be expected to be similar, as these salts easily dissociate when dissolved in the gastrointestinal contents. Further, since the mercury(ll) ion in vivo is entirely bound to proteins or other thiolcontaining ligands, the systemic transport and the excretion of absorbed mercury would be expected to be the same regardless of the type of soluble salt administered. Due to difficulties in comparing results obtained in different laboratories using different study designs and animal models, the present study was performed to allow a direct comparison between the toxicokinetics of orally and iritraperitoneally administered inorganic mercury using the same experimental model. Because of possible strain differences in the toxicokinetics of inorganic mercury, two strains of mice were used. MATERIALS AND METHODS Seven- to 8-wk-old female mice were kept on beechwood bedding in a well-controlled environment (50 ± 5% relative humidity, 20 air changes/h, temperature 21 ± 1°C, light/dark periods 12/12 h with 0.5 h twilight) with free access to standard mouse pellets (Brogaarden, Chr. Petersen, Ringsted, Denmark) and water. Eighty CBA/Bom mice were randomly assigned to 8 experimental groups of 10 animals. Each animal was marked and weighed and given one dose of HgCI2 (Merck, analytical grade) in water either by stomach tube (1, 5, 25, or 100 ^mol/kg body weight) or by intraperitoneal injection (0.05, 0.25,1.25, or 5 /*mol/kg body
Downloaded by [University of Cambridge] at 07:09 06 September 2015
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weight). Thus, the ip doses were chosen at 5% of the oral doses. Eighty Bom:NMRI mice were treated in the same way. The dose range was chosen on the background of earlier investigations in mice to cover a range between nontoxic doses and doses reaching levels where slight and reversible kidney damage could be expected. The HgCI2 solutions were labeled with 203Hg (203HgCI2 in HCI; 1.5 mCi/mg Hg; Amersham, U.K.) to allow determination of the whole-body retention and organ distribution of mercury. Within 15 min after dosing, each animal was imobilized by placing it into a plastic vial and counted in a whole-body counter (Nal well crystal, diameter 50 mm, 125 mm deep). The amount of radioactivity added to the HgCI2 solutions was adjusted so each animal initially counted between 500,000 and 1,000,000 cpm. The backgrounds in the whole-body counter and the organ counter were below 400 and 30 cpm, respectively. A lower limit for the use of counting results (detection limit) was set at the average background level plus three standard deviations. Five backgrounds and an HgCI2 standard of known intensity were counted at the beginning of the counting session for each experimental group. The window was set to include the 279-keV peak of the isotope. Calculated from the manufacturer's description of specific activity, this setting yielded a counting efficiency of 45% for the standard. Whole-body counting was repeated 1, 2, 3, 4, 7,10, and 14 d after dosing, and weighing was repeated 4 times during the experimental period. On d 14, all surviving animals were killed by cervical dislocation. Brain, lungs, heart, uteri (ovaries and uterine horns), spleen, kidneys, liver, stomach, and the intestinal tract were counted in a Searle 1195R gamma counter. The remaining carcass was counted in the wholebody counter. For each animal, the whole-body retention of the dose was expressed as percent of its initial count (%WBR). The time needed for elimination of 70% of the oral dose (7"07) quantifies delayed initial elimination and is used as a measure of toxic effects on the peristaltic process. Earlier studies of the toxicokinetics of orally administered cadmium have demonstrated that the time for elimination of the major part of a nonabsorbed toxic oral dose of a metal is a quantitative measure of the toxicity toward the peristaltic process (Andersen et a!., 1988). The T07 was estimated from the whole-body counting curves. After adjustment for differences in counting efficiency between the whole-body counter and the counter used for organ counting, each animal's organ counts were expressed as percent of the animal's WBR on d 14. This proportion is a measure of the fraction of the residual body burden present in the organ at d 14, that is, the relative distribution of mercury in different compartments. The results from the different groups were compared using the nonparametric Mann-Whitney U-test with a significance limit of .05.
). B. NIELSEN AND O. ANDERSEN
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0.1
FIGURE 1. Whole-body counting curves (semilogarithmic diagram) showing effect of dose of HgCI2 on whole-body retention of mercury after a single oral dose to female Bom:NMRI mice: A, 1 ^mol/kg (n - 10); O, 5 ^mol/kg (n - 9); V, 25 ^mol/kg (n - 10); D, 100 /jmol/kg (n - 9).
203
RESULTS The slopes of the whole-body counting curves (Figs. 1-4) showing the fractional retention of the dose indicates the rate of (fecal and urinary) elimination of mercury. Mice given 100 /tmol/kg of HgCI2 orally had a significantly decreased elimination of mercury within the first 3-4 d after dosing compared to mice given 1, 5, or 25 ^mol/kg (Figs. 1 and 2). The reduced elimination rate is illustrated (Table 1) by an increased T07 in the groups given 100 /imol/kg HgCI2 orally, as compared to the groups given the lower doses (both mice strains). A decreased initial elimination rate after the highest dose of mercuric mercury was not observed after intraperitoneal administration; in contrast, the initial elimination rate of intraperitoneally administered mercury was highest at the highest dose (Figs. 3 and 4). The rate of excretion of systemic mercury as estimated from the slope of the last part of the whole-body counting curves was found to be independent of the dose within the dose range used in these experiments, except that Bom:NMRI mice eliminated the absorbed part of the highest oral dose more rapidly (Fig. 1). The excretion rate as estimated
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from the whole-body counting curves was similar in the two mice strains after oral or intraperitoneal administration of HgCI2. Both mouse strains excreted intraperitoneally administered HgCI2 more slowly than orally administered HgCI2. Within the experimental period of 2 wk a constant elimination rate was not achieved. Accordingly, rate constants have not been calculated from the whole-body counting curves. The relative organ distribution after exposure to HgCI2 by the oral or the intraperitoneal route is shown in Tables 2 and 3. The relative deposition in liver, intestines, and spleen increased with the dose size in both Bom:NMRI and CBA/Bom mice (Tables 2 and 3). The relative deposition in these organs was doubled from the lowest dose in both mice strains, except in the livers of CBA/Bom mice where the increase was only marginal. The relative deposition in kidneys, lungs, and carcass decreased with increasing dose. Within the dose range used, the relative deposition in kidneys and lungs decreased considerably more in Bom:NMRI mice than in CBA/Bom mice. The relative deposition of orally administered mercury in stomach, uteri, and brain did not depend on the dose. Depo-
IOO-II
FIGURE 2. Whole-body counting curves (semilogarithmic diagram) showing effect of dose of HgCI2 on whole-body retention of mercury after a single oral dose to female CBA/Bom mice: A , 1 #mol/kg (n - 10); O, 5 jimol/kg (n - 10); V, 25 /xmol/kg (n - 10); D, 100 ^mol/kg (n - 10).
203
). B. NIELSEN AND O. ANDERSEN
172
100-i
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10-
en
1-
0.1 • 7
10
Days FIGURE 3. Whole-body counting curves (semilogarithmic diagram) showing effect of dose of HgCI2 on whole-body retention of mercury after a single intraperitoneal dose to female Bom:NMRI mice: A, 0.05 fimol/kg (n - 10); O, 0.25 ^mol/kg (n - 10); V, 1.25 pmol/kg (n - 8); D, 5.00 ftmol/kg (n - 8). 203
sition of orally administered mercury in the heart was below the detection, limit at all doses. The ratio of the relative deposition of orally administered HgCI2 in liver to kidneys of Bom:NMRl mice changed from 0.4 to 1.3 when the dose was increased from 1 /imol/kg to 100 /*mol/kg, but the combined relative deposition in liver and kidneys was constant between 56 and 58% at all dose levels (Table 2). The combined deposition in liver and kidneys was similar in CBA/Bom mice after oral administration of HgCI2 (54-60%). However the liver/kidney ratio was not changed to the same extent (Table 3), although the relative deposition in the liver reached the same level as in the kidneys. The relative deposition of intraperitoneally administered mercury in liver, stomach, and spleen increased somewhat with the dose, but the increase in the relative deposition in intestines and uteri was considerably higher (Tables 2 and 3). The relative deposition of mercury in kidneys, lungs, and brain decreased with increasing dose, and the relative
MERCURIC CHLORIDE TOXICOKINETICS
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100 -i
10o
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ID
1-
0.1 7 Days
10
FIGURE 4. Whole-body counting curves (semilogarithmic diagram) showing effect of dose of HgCI2 on whole-body retention of mercury after a single intraperitoneal dose to female CBA/Bom mice: A, 0.05 /imol/kg (n - 9); O, 0.25 /^mol/kg (n - 10); V, 1.25 ^mol/kg (n - 10); D, 5.00 /itnol/kg (n - 10). 203
deposition in heart and carcass remained constant within the dose range used. The increase in relative deposition in the liver and the decrease in relative kidney deposition at increasing intraperitoneal HgCl2 doses led to an increase in the ratio between the mercury burden in liver and kidneys from 0.2 to 0.6. However, the kidneys remained the major depot for mercury besides the carcass. TABLE 1. Time (in h) from dosage to elimination of 70% of the dose (To 7) of 203HgCI2. Oral dose (n mo I/kg)
Bom:NMRI
CBA/Bom
1 5 25 100
18 18 19 41
19 19 18 36
J. B. NIELSEN AND O. ANDERSEN
174
TABLE 2. Effect of dose on relative organ distribution of 203HgCI2 in female Bom:NMRI mice. The table shows percentage of residual body burden, median (25 and 75 percentiles), in various organs and carcass from animals sacrified at d 14.
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Oral dose (/*mol HgCI2/kg)
Liver Kidneys Stomach Intestine Uteri Heart Spleen Lungs Brain Carcass
1
5
10
9
10
9
15.70 (14.34-18.79)ab 39.91 (35.83-43.09)ab 0.85 (0.70-0.90) 4.49 (4.08-4.78) abc 0.56 (0.34-1.04) 0.15 (0.12-0.21)* 0.30 (0.26-0.39)ab 0.82 (0.66-1.03)b 1.36(1.11-1.84) 34.85 (30.67-37.45)3
17.10 (15.50-20.97)ab 39.24 (34.94-43.68)a 0.83(0.54-1.41) 5.65 (5.00-6.55)3 0.60 (0.49-0.86)6 0.12 (0.05-0.21)* 0.38 (0.10-0.60) 0.59 (0.49-0.83)6 1.47(1.19-2.15) 34.64 (28.77-36.35)
22.71 (20.04-28.81)3 31.38 (29.01-37.19)3 0.73 (0.61-0.98) 6.36 (5.85-7.04)a 0.37 (0.23-0.54) 0.20 (0.07-0.34)* 0.49 (0.40-0.65) 0.45 (0.32-0.58) 1.17(0.86-1.52) 33.31 (26.93-38.11)
33.48 (26.76-37.76) 26.99 (18.35-30.40) 0.91 (0.87-1.17) 8.21 (6.56-10.12) 0.56 (0.34-0.80) 0.02 (0.00-0.25)* 0.60 (0.50-0.76) 0.34 (0.18-0.62)* 1.36(1.15-1.55) 29.48 (26.48-30.94)
a
Significantly different Significantly different Significantly different *Below detection limit b
25 No. of animals
100
(p < .05) from group given 100 jjmol HgCI 2 /kg. (p < .05) from group given 25 /^mol HgCI2/kg. (p < .05) from group given 5 ^mol HgCI2/kg. (background level + 3 SD). ip Dose (fimol HgC!2/kg)
Liver Kidneys Stomach Intestine Uteri Heart Spleen Lungs Brain Carcass
0.05
0.25
1.25 No. of animals
10
10
8
8
10.69 (8.48-12.40)ab 46.53 (43.93-58.35)a 0.53 (0.43-0.60)a-b 4.18 (3.27-4.41) abc 0.29 (0.20-0.76)ab 0.16 (0.13-0.18) 0.37 (0.30-0.44)ab 0.68 (0.60-0.77)ab 1.30 (1.10-1.45)ab 32.54 (27.32-36.94)
9.83 (8.95-10.63)ab 50.71 (45.73-55.03)ab 0.47 (0.37-0.56)ab 5.22 (4.63-5.97)ab 0.56 (0.30-0.89)ab 0.18 (0.13-0.21) 0.38 (0.35-0.45)ab 0.61 (0.50-0.70)a 1.08(1.02-1.29)a 31.25 (27.17-35.09)
14.02 (12.99-14.95)a 44.24 (42.89-45.20)a 0.91 (0.86-1.01)a 7.36 (6.52-8.30)a 1.83 (0.64-2.71) 0.17 (0.13-0.24) 0.53 (0.46-0.59) 0.57 (0.47-0.66)3 0.98(0.73-1.11) 29.77 (29.05-30.80)
19.98 (15.84-23.65) 33.79(25.24-41.11) 1.13(1.11-1.55) 9.75 (8.34-12.55) 1.41 (0.90-2.36) 0.05 (0.00-0.13)* 0.73 (0.49-1.22) 0.34 (0.20-0.45) 0.81 (0.73-0.96) 31.53 (27.65-36.26)
Significantly different (p < .05) from group given 5.00 /imol HgCI2/kg. b Significantly different (p < .05) from group given 1.25 /tmol HgCI2/kg. Significantly different (p < .05) from group given 0.25 /
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Disposition and retention of mercuric chloride in mice after oral and parenteral administration a
Jesper Bo Nielsen & Ole Andersen
b
a
Department of Environmental Medicine , Odense University , J. B. Winslöwsvej 19, Odense C, DK‐5000, Denmark b
Department of Environmental Medicine , Odense University , Odense, Denmark Published online: 20 Oct 2009.
To cite this article: Jesper Bo Nielsen & Ole Andersen (1990) Disposition and retention of mercuric chloride in mice after oral and parenteral administration, Journal of Toxicology and Environmental Health: Current Issues, 30:3, 167-180, DOI: 10.1080/15287399009531420 To link to this article: http://dx.doi.org/10.1080/15287399009531420
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/ terms-and-conditions
DISPOSITION AND RETENTION OF MERCURIC CHLORIDE IN MICE AFTER ORAL AND PARENTERAL ADMINISTRATION Jesper Bo Nielsen, Ole Andersen
Downloaded by [University of Cambridge] at 07:09 06 September 2015
Department of Environmental Medicine, Odense University, Odense, Denmark
The present study compares effects of dose size on whole-body retention and relative organ distribution of 203HgCl2, after oral and intraperitoneal administration to female mice of two strains (inbred CBA/Bom and outbred Bom:NMRI). Using whole-body retention data of oral and intraperitoneal administration, an estimated "true absorption" of a single oral dose of inorganic mercury was calculated to be about 20% at two different dose levels. At the highest oral dose, a delay in fecal elimination of nonabsorbed mercury was observed, indicating a decreased peristaltic rate. The relative hepatic deposition was larger after oral than after intraperitoneal administration, presumably due to a first-pass effect, and a correspondingly lower relative renal deposition was seen. Increasing doses at both exposure routes resulted in increasing relative deposition in liver, stomach, intestines, and spleen but decreasing relative deposition in lungs and kidneys. Bom:NMRI mice deposited a larger fraction of the whole-body burden in the kidneys and a smaller fraction in the livers than did CBA/Bom mice. Comparison to a previous study with male mice (Nielsen and Andersen, 1989) demonstrates that male and female mice deposit similar fractions of their body burden in the liver, while male mice deposit significantly larger amounts of mercury in the kidneys and smaller amounts in the carcass than do female mice. Thus, the toxicokinetics of inorganic mercury in mice depend on dose size, administration route, and sex; the mouse strain is of less importance than the other factors investigated. The absorption of inorganic mercury was estimated to be about 20%, that is, twice as high as earlier estimates.
INTRODUCTION The acute and chronic toxicity of inorganic mercury in humans has been studied during several centuries (Paracelsus, 1567; Ramazzini, 1700). The critical organs after acute oral exposure of humans are the mucous membranes of the gastrointestinal tract and the kidneys (Berlin, 1986; Ellenhorn and Barceleux, 1987). Human data on the toxicokinetics of mercuric mercury have primarily been focused on rate of elimination and body retention calculated on the basis of whole-body countings and fecal or urinary excretion (Rahola This study was supported by grant 12-8108 from the Danish Medical Research Council to Jesper Bo Nielsen. Requests for reprints should be sent to Jesper Bo Nielsen, Department of Environmental Medicine, Odense University, J. B. Winslöwsvej 19, DK-5000 Odense C, Denmark. 167 Journal of Toxicology and Environmental Health, 30:167-180, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
Downloaded by [University of Cambridge] at 07:09 06 September 2015
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et al., 1973; Miettinen, 1973). Calculation of the intestinal absorption on the basis of these investigations is seriously undermined by the fact that part of the intestinally absorbed mercury will be reexcreted to the gastrointestinal tract during the experimental period. Accordingly, the authors did not try to establish the intestinal absorption, but rather the wholebody retention from their data. Thus a clear distinction must be made between body retention and the true intestinal absorption. Investigations of the toxicokinetics of soluble mercury(ll) salts after administration via various routes indicate that differences in the absorption, relative organ distribution, and rate of elimination occur (Miyama et al., 1968; Nielsen and Andersen, 1989). However, most experimental studies of the toxicokinetics of inorganic mercury have used parenteral exposure (Rothstein and Hayes, 1960; Berlin et al., 1966; Cember and Donagi, 1963; Magos and Webb, 1976; Kristensen and Hansen, 1980). As several different soluble salts of inorganic mercury(ll) and also various animal species and strains were used, a general conclusion is not easily drawn on the basis of these studies. However, the rate of intestinal absorption of mercury from various soluble compounds would be expected to be similar, as these salts easily dissociate when dissolved in the gastrointestinal contents. Further, since the mercury(ll) ion in vivo is entirely bound to proteins or other thiolcontaining ligands, the systemic transport and the excretion of absorbed mercury would be expected to be the same regardless of the type of soluble salt administered. Due to difficulties in comparing results obtained in different laboratories using different study designs and animal models, the present study was performed to allow a direct comparison between the toxicokinetics of orally and iritraperitoneally administered inorganic mercury using the same experimental model. Because of possible strain differences in the toxicokinetics of inorganic mercury, two strains of mice were used. MATERIALS AND METHODS Seven- to 8-wk-old female mice were kept on beechwood bedding in a well-controlled environment (50 ± 5% relative humidity, 20 air changes/h, temperature 21 ± 1°C, light/dark periods 12/12 h with 0.5 h twilight) with free access to standard mouse pellets (Brogaarden, Chr. Petersen, Ringsted, Denmark) and water. Eighty CBA/Bom mice were randomly assigned to 8 experimental groups of 10 animals. Each animal was marked and weighed and given one dose of HgCI2 (Merck, analytical grade) in water either by stomach tube (1, 5, 25, or 100 ^mol/kg body weight) or by intraperitoneal injection (0.05, 0.25,1.25, or 5 /*mol/kg body
Downloaded by [University of Cambridge] at 07:09 06 September 2015
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weight). Thus, the ip doses were chosen at 5% of the oral doses. Eighty Bom:NMRI mice were treated in the same way. The dose range was chosen on the background of earlier investigations in mice to cover a range between nontoxic doses and doses reaching levels where slight and reversible kidney damage could be expected. The HgCI2 solutions were labeled with 203Hg (203HgCI2 in HCI; 1.5 mCi/mg Hg; Amersham, U.K.) to allow determination of the whole-body retention and organ distribution of mercury. Within 15 min after dosing, each animal was imobilized by placing it into a plastic vial and counted in a whole-body counter (Nal well crystal, diameter 50 mm, 125 mm deep). The amount of radioactivity added to the HgCI2 solutions was adjusted so each animal initially counted between 500,000 and 1,000,000 cpm. The backgrounds in the whole-body counter and the organ counter were below 400 and 30 cpm, respectively. A lower limit for the use of counting results (detection limit) was set at the average background level plus three standard deviations. Five backgrounds and an HgCI2 standard of known intensity were counted at the beginning of the counting session for each experimental group. The window was set to include the 279-keV peak of the isotope. Calculated from the manufacturer's description of specific activity, this setting yielded a counting efficiency of 45% for the standard. Whole-body counting was repeated 1, 2, 3, 4, 7,10, and 14 d after dosing, and weighing was repeated 4 times during the experimental period. On d 14, all surviving animals were killed by cervical dislocation. Brain, lungs, heart, uteri (ovaries and uterine horns), spleen, kidneys, liver, stomach, and the intestinal tract were counted in a Searle 1195R gamma counter. The remaining carcass was counted in the wholebody counter. For each animal, the whole-body retention of the dose was expressed as percent of its initial count (%WBR). The time needed for elimination of 70% of the oral dose (7"07) quantifies delayed initial elimination and is used as a measure of toxic effects on the peristaltic process. Earlier studies of the toxicokinetics of orally administered cadmium have demonstrated that the time for elimination of the major part of a nonabsorbed toxic oral dose of a metal is a quantitative measure of the toxicity toward the peristaltic process (Andersen et a!., 1988). The T07 was estimated from the whole-body counting curves. After adjustment for differences in counting efficiency between the whole-body counter and the counter used for organ counting, each animal's organ counts were expressed as percent of the animal's WBR on d 14. This proportion is a measure of the fraction of the residual body burden present in the organ at d 14, that is, the relative distribution of mercury in different compartments. The results from the different groups were compared using the nonparametric Mann-Whitney U-test with a significance limit of .05.
). B. NIELSEN AND O. ANDERSEN
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0.1
FIGURE 1. Whole-body counting curves (semilogarithmic diagram) showing effect of dose of HgCI2 on whole-body retention of mercury after a single oral dose to female Bom:NMRI mice: A, 1 ^mol/kg (n - 10); O, 5 ^mol/kg (n - 9); V, 25 ^mol/kg (n - 10); D, 100 /jmol/kg (n - 9).
203
RESULTS The slopes of the whole-body counting curves (Figs. 1-4) showing the fractional retention of the dose indicates the rate of (fecal and urinary) elimination of mercury. Mice given 100 /tmol/kg of HgCI2 orally had a significantly decreased elimination of mercury within the first 3-4 d after dosing compared to mice given 1, 5, or 25 ^mol/kg (Figs. 1 and 2). The reduced elimination rate is illustrated (Table 1) by an increased T07 in the groups given 100 /imol/kg HgCI2 orally, as compared to the groups given the lower doses (both mice strains). A decreased initial elimination rate after the highest dose of mercuric mercury was not observed after intraperitoneal administration; in contrast, the initial elimination rate of intraperitoneally administered mercury was highest at the highest dose (Figs. 3 and 4). The rate of excretion of systemic mercury as estimated from the slope of the last part of the whole-body counting curves was found to be independent of the dose within the dose range used in these experiments, except that Bom:NMRI mice eliminated the absorbed part of the highest oral dose more rapidly (Fig. 1). The excretion rate as estimated
Downloaded by [University of Cambridge] at 07:09 06 September 2015
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from the whole-body counting curves was similar in the two mice strains after oral or intraperitoneal administration of HgCI2. Both mouse strains excreted intraperitoneally administered HgCI2 more slowly than orally administered HgCI2. Within the experimental period of 2 wk a constant elimination rate was not achieved. Accordingly, rate constants have not been calculated from the whole-body counting curves. The relative organ distribution after exposure to HgCI2 by the oral or the intraperitoneal route is shown in Tables 2 and 3. The relative deposition in liver, intestines, and spleen increased with the dose size in both Bom:NMRI and CBA/Bom mice (Tables 2 and 3). The relative deposition in these organs was doubled from the lowest dose in both mice strains, except in the livers of CBA/Bom mice where the increase was only marginal. The relative deposition in kidneys, lungs, and carcass decreased with increasing dose. Within the dose range used, the relative deposition in kidneys and lungs decreased considerably more in Bom:NMRI mice than in CBA/Bom mice. The relative deposition of orally administered mercury in stomach, uteri, and brain did not depend on the dose. Depo-
IOO-II
FIGURE 2. Whole-body counting curves (semilogarithmic diagram) showing effect of dose of HgCI2 on whole-body retention of mercury after a single oral dose to female CBA/Bom mice: A , 1 #mol/kg (n - 10); O, 5 jimol/kg (n - 10); V, 25 /xmol/kg (n - 10); D, 100 ^mol/kg (n - 10).
203
). B. NIELSEN AND O. ANDERSEN
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Days FIGURE 3. Whole-body counting curves (semilogarithmic diagram) showing effect of dose of HgCI2 on whole-body retention of mercury after a single intraperitoneal dose to female Bom:NMRI mice: A, 0.05 fimol/kg (n - 10); O, 0.25 ^mol/kg (n - 10); V, 1.25 pmol/kg (n - 8); D, 5.00 ftmol/kg (n - 8). 203
sition of orally administered mercury in the heart was below the detection, limit at all doses. The ratio of the relative deposition of orally administered HgCI2 in liver to kidneys of Bom:NMRl mice changed from 0.4 to 1.3 when the dose was increased from 1 /imol/kg to 100 /*mol/kg, but the combined relative deposition in liver and kidneys was constant between 56 and 58% at all dose levels (Table 2). The combined deposition in liver and kidneys was similar in CBA/Bom mice after oral administration of HgCI2 (54-60%). However the liver/kidney ratio was not changed to the same extent (Table 3), although the relative deposition in the liver reached the same level as in the kidneys. The relative deposition of intraperitoneally administered mercury in liver, stomach, and spleen increased somewhat with the dose, but the increase in the relative deposition in intestines and uteri was considerably higher (Tables 2 and 3). The relative deposition of mercury in kidneys, lungs, and brain decreased with increasing dose, and the relative
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FIGURE 4. Whole-body counting curves (semilogarithmic diagram) showing effect of dose of HgCI2 on whole-body retention of mercury after a single intraperitoneal dose to female CBA/Bom mice: A, 0.05 /imol/kg (n - 9); O, 0.25 /^mol/kg (n - 10); V, 1.25 ^mol/kg (n - 10); D, 5.00 /itnol/kg (n - 10). 203
deposition in heart and carcass remained constant within the dose range used. The increase in relative deposition in the liver and the decrease in relative kidney deposition at increasing intraperitoneal HgCl2 doses led to an increase in the ratio between the mercury burden in liver and kidneys from 0.2 to 0.6. However, the kidneys remained the major depot for mercury besides the carcass. TABLE 1. Time (in h) from dosage to elimination of 70% of the dose (To 7) of 203HgCI2. Oral dose (n mo I/kg)
Bom:NMRI
CBA/Bom
1 5 25 100
18 18 19 41
19 19 18 36
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TABLE 2. Effect of dose on relative organ distribution of 203HgCI2 in female Bom:NMRI mice. The table shows percentage of residual body burden, median (25 and 75 percentiles), in various organs and carcass from animals sacrified at d 14.
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Oral dose (/*mol HgCI2/kg)
Liver Kidneys Stomach Intestine Uteri Heart Spleen Lungs Brain Carcass
1
5
10
9
10
9
15.70 (14.34-18.79)ab 39.91 (35.83-43.09)ab 0.85 (0.70-0.90) 4.49 (4.08-4.78) abc 0.56 (0.34-1.04) 0.15 (0.12-0.21)* 0.30 (0.26-0.39)ab 0.82 (0.66-1.03)b 1.36(1.11-1.84) 34.85 (30.67-37.45)3
17.10 (15.50-20.97)ab 39.24 (34.94-43.68)a 0.83(0.54-1.41) 5.65 (5.00-6.55)3 0.60 (0.49-0.86)6 0.12 (0.05-0.21)* 0.38 (0.10-0.60) 0.59 (0.49-0.83)6 1.47(1.19-2.15) 34.64 (28.77-36.35)
22.71 (20.04-28.81)3 31.38 (29.01-37.19)3 0.73 (0.61-0.98) 6.36 (5.85-7.04)a 0.37 (0.23-0.54) 0.20 (0.07-0.34)* 0.49 (0.40-0.65) 0.45 (0.32-0.58) 1.17(0.86-1.52) 33.31 (26.93-38.11)
33.48 (26.76-37.76) 26.99 (18.35-30.40) 0.91 (0.87-1.17) 8.21 (6.56-10.12) 0.56 (0.34-0.80) 0.02 (0.00-0.25)* 0.60 (0.50-0.76) 0.34 (0.18-0.62)* 1.36(1.15-1.55) 29.48 (26.48-30.94)
a
Significantly different Significantly different Significantly different *Below detection limit b
25 No. of animals
100
(p < .05) from group given 100 jjmol HgCI 2 /kg. (p < .05) from group given 25 /^mol HgCI2/kg. (p < .05) from group given 5 ^mol HgCI2/kg. (background level + 3 SD). ip Dose (fimol HgC!2/kg)
Liver Kidneys Stomach Intestine Uteri Heart Spleen Lungs Brain Carcass
0.05
0.25
1.25 No. of animals
10
10
8
8
10.69 (8.48-12.40)ab 46.53 (43.93-58.35)a 0.53 (0.43-0.60)a-b 4.18 (3.27-4.41) abc 0.29 (0.20-0.76)ab 0.16 (0.13-0.18) 0.37 (0.30-0.44)ab 0.68 (0.60-0.77)ab 1.30 (1.10-1.45)ab 32.54 (27.32-36.94)
9.83 (8.95-10.63)ab 50.71 (45.73-55.03)ab 0.47 (0.37-0.56)ab 5.22 (4.63-5.97)ab 0.56 (0.30-0.89)ab 0.18 (0.13-0.21) 0.38 (0.35-0.45)ab 0.61 (0.50-0.70)a 1.08(1.02-1.29)a 31.25 (27.17-35.09)
14.02 (12.99-14.95)a 44.24 (42.89-45.20)a 0.91 (0.86-1.01)a 7.36 (6.52-8.30)a 1.83 (0.64-2.71) 0.17 (0.13-0.24) 0.53 (0.46-0.59) 0.57 (0.47-0.66)3 0.98(0.73-1.11) 29.77 (29.05-30.80)
19.98 (15.84-23.65) 33.79(25.24-41.11) 1.13(1.11-1.55) 9.75 (8.34-12.55) 1.41 (0.90-2.36) 0.05 (0.00-0.13)* 0.73 (0.49-1.22) 0.34 (0.20-0.45) 0.81 (0.73-0.96) 31.53 (27.65-36.26)
Significantly different (p < .05) from group given 5.00 /imol HgCI2/kg. b Significantly different (p < .05) from group given 1.25 /tmol HgCI2/kg. Significantly different (p < .05) from group given 0.25 /
