9 1986 by The Iqumana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/86/1001-0047503.40
The Fate of Cd, Cu, Ca, Zn, and Fe in Rat During the Recovery Period Following Cessation of Repeated Exposure to Cd ELIZABETH KOMSTA-SZUMSKA *'1 AND MARGARET CZUBA2
'Environmental Toxicology Section, A/berta Environmental Centre, Bag 4000, Vegreville, Alberta, Canada TOB 4LO; and 2 Ecotoxicology Group, National Research Council of Canada, Ottawa, Canada KIA OR6 Received May 20, 1985; Accepted December 9, 1985
ABSTRACT Cadmium was administered subcutaneously to male Wistar rats, 0.1 mL/rat in 0.9% saline 3 times a wk for 4 wk at 3 mg Cd/kg. Saline was administered to control animals in an equivalent manner, without Cd. After the end of the dosing period, the distribution and excrefion of Cd, Cu, Ca, Zn, and Fe were observed in some organs and excreta for 35 d (1, 7, 14, 21, 28, and 35 d). Cadmium dosing caused significant disturbances in the metabolism of Zn, Cu, Fe, and Ca, especially during the recovery period. Growth in Cd-dosed animals did not accelerate, even after 5 wk of recovery. There was evidence of mobilization of some elements among organs. Accumulation of Cd occurred in liver, kidney, and spleen during dosing, and during the recovery period it was retained in kidney and testes (for 2 wk) and cleared steadily in liver and RBC (for 5 wk), but increased in spleen (first 3 wk). The pattern of Cd excretion was closely associated with the binding of Cd with metallothioneins in kidney and liver for the first 21 and 7 d, respectively. This was associated with the excretion of Cd-metallothioneins (Cd-MT) in urine from d 1 to 21 during recovery. Cadmium caused higher Ca accumulations in testes and liver, which were probably associated with the lesions observed in these organs. Significant increases of Cu (in kidney d 7) and Fe (in liver) were *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
47
vol. ]0, 1986
Komsta-Szumska and Czuba
48
observed during recovery. Furthermore, significant reductions of Cu and Fe were found in plasma, spleen, and RBC (after 5 wk) and kidney, spleen, and testes (on d 7), and blood (after 5 wk). Index Entries: Cd toxicity; recovery period; trace element mobilization; metallothionein binding of Cd and Cu.
INTRODUCTION Cadmium, a toxicologically important metal, accumulates in liver and kidneys following long- and short-term exposures (1-3), and the concentrations in these organs depend upon both the dose and route of administration (4,5). Cadmium may also alter the metabolism of essential trace elements by effecting normal tissue distribution of Zn, Cu, and Fe (6-9). In competition for ligands in the biological system, Cd may affect the deposition and homeostasis of these elements and, thus, the manifestation of Cd toxicity (7,10). Previously, it has been accepted that Cdinduced anemia was not dependent on the route of administration (11-13), but, rather, that the competition of Cd and Fe in the gastrointestinal mucosa contributes closely to the development of anemia (14). Administration of Cd has resulted in dose-related increases in the urinary excretion of Cd, Zn, Cu, and Fe (15,16). It has also been proposed that the elevated excretion of Cd-metallothionein (Cd-MT) in the urine may be a specific index of Cd exposure (17,18). The purpose of the present study was to examine the metabolic fate of Cd after the cessation of repeated injections and its influence on organ concentrations of Zn, Cu, Ca, and Fe. In addition, measurements were made on the urinary and fecal excretion of cadmium, zinc, and copper, as well as the investigation of metal-binding profiles of kidney, liver, and urinary MT at different times after the last dose of Cd.
MATERIALS AND METHODS Animals Male rats (6-wk-old) of Wistar strain were obtained from the local breeder (National Research Council of Canada) with an initial body weight of 170 _+ 10 g. Animals were divided into 2 groups: (1) control---12 rats, (2) Cd-treated---36 rats. All animals were housed in plastic cages with free access to both food and water. The animals were quarantined for 1 wk and then used for experimental purposes. The standard diet contained, among others, the following elements: Zn (20 ppm), Cu (15 ppm), Ca (20,000 ppm), and Fe (300 ppm). Cadmium levels were not detectable.
Biological Trace Element Research
Vol. 10, 1986
Fate of Trace Elements During RecoveryPeriod
49
Injections of Cd and Saline Cadmium chloride was injected subcutaneously (sc) at a dose of 3 mg Cd/kg body weight as a 0.1 mL solution/rat, 3 times a wk (Mon., Wed., and Fri.) for 4 wk (12 doses). The saline (0.9% NaC1) (or control) group was injected with the same volume and in the same manner, but without Cd. Six animals from the Cd-dosed group and two from the control group were killed on d 1, 7, 14, 21, 28, and 35 after the last dose of Cd treatment. Animals were weighed once a week.
Collection of Urine and Feces During the Cd treatment (after 4, 8, and 12 doses), the animals were moved to metabolic cages (both groups) and had their urine and feces collected for 24 h. After the cessation of Cd administration, the collection of excreta was also repeated at the end of each week (for up to 5 wk).
Sample Preparation The animals were killed by vascular perfusion with physiological saline under Nembutal anesthesia. Heparinized blood was collected by heart puncture. Plasma and red blood cells (RBC) were separated by centrifuging whole blood at 300 rpm for 15 min at room temperature. The kidneys, liver, spleen, and testes were removed and weighed (wet tissue).
Determination of ~etal Concentrations One gram portions of the liver, kidney, and testes from each rat were digested with 6 mL of acid mixture (HClO4: HNO3 = 1 : 5 v/v) and then diluted to 10 mL with double-distilled water. A 0.2--0.3 g portion of the spleen, RBC, and plasma in each rat was also digested in the same manner. The total concentrations of Cd, Cu, Zn, Fe, and Ca were determined by Atomic Absorption Spectrophotometry (AAS), Jarrel-Ash, Model 850, with background correction. Urine, collected for 24 h, was centrifuged at 300g for 10 min, and 1.0 mL portions of the supernatant were digested with I mL of acid mixture. Feces, collected for 24 h, approximately 0.3 g portions, were digested with 2.0 mL of acid mixture. The total levels of Cd, Cu, and Zn were deterrnined by A3 q
Separation of Kidney, Liver, and (Jdnary Supernatant Proteins by Gel Filtration Kidney and liver (2.0 g of tissue) were homogenized in 10 mM TrisHC1 buffer at pH 8.6 (5 mL). Kidney and liver cytosols were obtained by
Biological Trace Element Research
VoL I O, 1986
50
Komsta-Szumska and Czuba
differential centrifugation of homogenates at 27,000g for 20 min. The supernatants were subsequently centrifuged at 105,000g for 90 min. The organ cytosols and urine supernatants (8 mL) were fractionated by column chromatography on Sephadex G-75 (2.5 x 60 cm column), using 10 mM Tris-HC1 buffer at pH 8.6 as eluant. Five-milliliter fractions were collected. The elution volume was calibrated with Dextran blue (2 x 106), Cytochrome c (12,400) and potassium chromate (200). The gel filtration fractions were analyzed by AAS for Cd and Zn without digestion.
Statistical Analyses Statistical analyses of metal concentrations were calculated as means + SD of six samples and significant differences were obtained by Student's t-test.
RESULTS
Body Weight Average body weights recorded during the course of the study are shown in Fig. 1. The body weights of the control (saline injected) animals increased rapidly during the first 3 wk, then slowly for the rest of the 9-wk period. Cadmium-treated animals had a slower weight gain both during (first 4 wk) and after (wk 5-9) Cd dosing. The body weight of the Cd-treated animals at the end of 9 wk was 71% of the control animals.
Tissue Distribution of Cd Twenty-four hours after the last dose of cadmium chloride, the highest accumulations of Cd were found in the liver, kidney, and spleen (Fig. 2). During the first 2 wk of recovery, the Cd concentration in the kidney did not decrease significantly, but subsequently, fell rapidly. Five weeks after the cessation of Cd treatment, the Cd level in the kidney was about one-fourth of that found on d 1. The hepatic Cd concentration decreased rapidly (wk 1-3) then slowly (from wk 3-5). The concentration of Cd in the spleen continued to increase for 3 wk after the final injection of Cd, then fell sharply, and by wk 5 remained at a slighly lower level compared with the value found on d 1 after the last dose of Cd. The Cd level in the RBC had declined by d 7 after the cessation of Cd treatment (compared with d 1); however, by d 35 it was almost the same as that on d 1. The Cd concentration in plasma was about 5-6 times lower than in RBC. Cadmium amounts in the plasma had increased slightly by wk I and after that showed a plateau between d 14 and 35 of the recovery period. The Cd concentration in the testes had risen significantly by d 7
Biological Trace Element Research
Vol. 1O, 1986
Fate of Trace Elements During RecoveryPeriod
51
GROWTH CURVES 4 0 0 --
jo Control group
I~
oI o
o~oI
30O 1~176 1o
/ 9
.~Ip 9
~''
~o
~,~0 ~
0~0
Cadmium group
IOO--
I
I
I
I
I
I
I
I
I
I
2
3
4 5 6 WEEKS
7
8
9
Fig. 1. Body weight gain of rats during 4 wk of Cd injectionns and 5 wk after cessation of Cd administration (9 Controls received saline only (O). and 14, then rapidly decreased with no further changes after 2 wk (Fig.
2). Effect on (lrinary Excretion of Cd, Zn, and Cu Repeated injections of CdCI2 caused an increased excretion of Cd in urine after the 8th dose, and excretion sharply increased after the 12th dose (Fig. 3). The urinary excretion level in controls is given in the figure caption. Urinary Cd excretion continued to increase even after the cessation of Cd treatment (until d 14), after which it fell sharply between d 21 and 35. The amount of Zn excreted in the urine increased after the fourth dose of Cd and remained at significantly elevated levels until d 21, compared with the saline group (even 3 wk after the cessation of Cd dosing). Urinary excretion of Cu was also enhanced by injections of Cd. The highest level of urinary Cu compared with the saline group was observed after the last dose of Cd (the 12th). Excretion of Cu during the recovery period was almost unchanged (Fig. 3).
Biological Trace Element Research
Vol. I O, 1986
Komsta-Szumska and Czuba
52
KIDNEY
160.0
LIVER
160.0 9
~~Z,~,~
120.0
SPLEEN
160.0 12o.o 8o.0
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=-
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7
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11'40"
~ 40"0
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,~
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after
...--"'~
"~ the
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~,
last dose of
2'8
3'5
Cd
~'
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3'5
Administration
Fig. 2. Cadmium concentration (p,g/g wet tissue) in organs of experimental rats, at different times after cessation of Cd administration. The values are expressed as means + SD of six animals.
Effect on Fecal Excretion of Cd, Zn, and Cu The a m o u n t of Cd excreted in feces increased sharply with injections of Cd (Fig. 3). Excretion levels in the control are given in the figure caption. During the recovery period, Cd excretion fell sharply (between d 1 and 7) to a concentration below that of the control (by d 14). However, 35 d after the cessation of Cd treatment, Cd excretion in feces d r o p p e d to the level found in the control group (i.e., 23 ~g/24 h). There was a significantly enhanced excretion of Zn in feces after Cd injections (by about 400 ~g/g). This level was maintained during the first week after the last dose. The level dropped (by 200 ~g/g) during the remainder of the recovery period, but never reached control levels (500.0 + 50.0 ~g/d/rat). Fecal excretion of Cu was only slightly elevated after the last dose of Cd and u n c h a n g e d (compared with the control) during the recovery period (Fig. 3).
Trace-Element Distribution in Tissue During Recovery Pe~od Zinc
Twenty-four hours after the last dose of Cd treatment, Zn levels were increased at least 2 or 3 times in all organs except RBC (Table 1). The Biological Trace Element Research
Vol. 1O, 1986
Fate of Trace Elements During Recovery Period
53
days after lost dose of Cd [
Cd-lnjection (dose No)
0
o
80.0
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The Fate of Cd, Cu, Ca, Zn, and Fe in Rat During the Recovery Period Following Cessation of Repeated Exposure to Cd ELIZABETH KOMSTA-SZUMSKA *'1 AND MARGARET CZUBA2
'Environmental Toxicology Section, A/berta Environmental Centre, Bag 4000, Vegreville, Alberta, Canada TOB 4LO; and 2 Ecotoxicology Group, National Research Council of Canada, Ottawa, Canada KIA OR6 Received May 20, 1985; Accepted December 9, 1985
ABSTRACT Cadmium was administered subcutaneously to male Wistar rats, 0.1 mL/rat in 0.9% saline 3 times a wk for 4 wk at 3 mg Cd/kg. Saline was administered to control animals in an equivalent manner, without Cd. After the end of the dosing period, the distribution and excrefion of Cd, Cu, Ca, Zn, and Fe were observed in some organs and excreta for 35 d (1, 7, 14, 21, 28, and 35 d). Cadmium dosing caused significant disturbances in the metabolism of Zn, Cu, Fe, and Ca, especially during the recovery period. Growth in Cd-dosed animals did not accelerate, even after 5 wk of recovery. There was evidence of mobilization of some elements among organs. Accumulation of Cd occurred in liver, kidney, and spleen during dosing, and during the recovery period it was retained in kidney and testes (for 2 wk) and cleared steadily in liver and RBC (for 5 wk), but increased in spleen (first 3 wk). The pattern of Cd excretion was closely associated with the binding of Cd with metallothioneins in kidney and liver for the first 21 and 7 d, respectively. This was associated with the excretion of Cd-metallothioneins (Cd-MT) in urine from d 1 to 21 during recovery. Cadmium caused higher Ca accumulations in testes and liver, which were probably associated with the lesions observed in these organs. Significant increases of Cu (in kidney d 7) and Fe (in liver) were *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
47
vol. ]0, 1986
Komsta-Szumska and Czuba
48
observed during recovery. Furthermore, significant reductions of Cu and Fe were found in plasma, spleen, and RBC (after 5 wk) and kidney, spleen, and testes (on d 7), and blood (after 5 wk). Index Entries: Cd toxicity; recovery period; trace element mobilization; metallothionein binding of Cd and Cu.
INTRODUCTION Cadmium, a toxicologically important metal, accumulates in liver and kidneys following long- and short-term exposures (1-3), and the concentrations in these organs depend upon both the dose and route of administration (4,5). Cadmium may also alter the metabolism of essential trace elements by effecting normal tissue distribution of Zn, Cu, and Fe (6-9). In competition for ligands in the biological system, Cd may affect the deposition and homeostasis of these elements and, thus, the manifestation of Cd toxicity (7,10). Previously, it has been accepted that Cdinduced anemia was not dependent on the route of administration (11-13), but, rather, that the competition of Cd and Fe in the gastrointestinal mucosa contributes closely to the development of anemia (14). Administration of Cd has resulted in dose-related increases in the urinary excretion of Cd, Zn, Cu, and Fe (15,16). It has also been proposed that the elevated excretion of Cd-metallothionein (Cd-MT) in the urine may be a specific index of Cd exposure (17,18). The purpose of the present study was to examine the metabolic fate of Cd after the cessation of repeated injections and its influence on organ concentrations of Zn, Cu, Ca, and Fe. In addition, measurements were made on the urinary and fecal excretion of cadmium, zinc, and copper, as well as the investigation of metal-binding profiles of kidney, liver, and urinary MT at different times after the last dose of Cd.
MATERIALS AND METHODS Animals Male rats (6-wk-old) of Wistar strain were obtained from the local breeder (National Research Council of Canada) with an initial body weight of 170 _+ 10 g. Animals were divided into 2 groups: (1) control---12 rats, (2) Cd-treated---36 rats. All animals were housed in plastic cages with free access to both food and water. The animals were quarantined for 1 wk and then used for experimental purposes. The standard diet contained, among others, the following elements: Zn (20 ppm), Cu (15 ppm), Ca (20,000 ppm), and Fe (300 ppm). Cadmium levels were not detectable.
Biological Trace Element Research
Vol. 10, 1986
Fate of Trace Elements During RecoveryPeriod
49
Injections of Cd and Saline Cadmium chloride was injected subcutaneously (sc) at a dose of 3 mg Cd/kg body weight as a 0.1 mL solution/rat, 3 times a wk (Mon., Wed., and Fri.) for 4 wk (12 doses). The saline (0.9% NaC1) (or control) group was injected with the same volume and in the same manner, but without Cd. Six animals from the Cd-dosed group and two from the control group were killed on d 1, 7, 14, 21, 28, and 35 after the last dose of Cd treatment. Animals were weighed once a week.
Collection of Urine and Feces During the Cd treatment (after 4, 8, and 12 doses), the animals were moved to metabolic cages (both groups) and had their urine and feces collected for 24 h. After the cessation of Cd administration, the collection of excreta was also repeated at the end of each week (for up to 5 wk).
Sample Preparation The animals were killed by vascular perfusion with physiological saline under Nembutal anesthesia. Heparinized blood was collected by heart puncture. Plasma and red blood cells (RBC) were separated by centrifuging whole blood at 300 rpm for 15 min at room temperature. The kidneys, liver, spleen, and testes were removed and weighed (wet tissue).
Determination of ~etal Concentrations One gram portions of the liver, kidney, and testes from each rat were digested with 6 mL of acid mixture (HClO4: HNO3 = 1 : 5 v/v) and then diluted to 10 mL with double-distilled water. A 0.2--0.3 g portion of the spleen, RBC, and plasma in each rat was also digested in the same manner. The total concentrations of Cd, Cu, Zn, Fe, and Ca were determined by Atomic Absorption Spectrophotometry (AAS), Jarrel-Ash, Model 850, with background correction. Urine, collected for 24 h, was centrifuged at 300g for 10 min, and 1.0 mL portions of the supernatant were digested with I mL of acid mixture. Feces, collected for 24 h, approximately 0.3 g portions, were digested with 2.0 mL of acid mixture. The total levels of Cd, Cu, and Zn were deterrnined by A3 q
Separation of Kidney, Liver, and (Jdnary Supernatant Proteins by Gel Filtration Kidney and liver (2.0 g of tissue) were homogenized in 10 mM TrisHC1 buffer at pH 8.6 (5 mL). Kidney and liver cytosols were obtained by
Biological Trace Element Research
VoL I O, 1986
50
Komsta-Szumska and Czuba
differential centrifugation of homogenates at 27,000g for 20 min. The supernatants were subsequently centrifuged at 105,000g for 90 min. The organ cytosols and urine supernatants (8 mL) were fractionated by column chromatography on Sephadex G-75 (2.5 x 60 cm column), using 10 mM Tris-HC1 buffer at pH 8.6 as eluant. Five-milliliter fractions were collected. The elution volume was calibrated with Dextran blue (2 x 106), Cytochrome c (12,400) and potassium chromate (200). The gel filtration fractions were analyzed by AAS for Cd and Zn without digestion.
Statistical Analyses Statistical analyses of metal concentrations were calculated as means + SD of six samples and significant differences were obtained by Student's t-test.
RESULTS
Body Weight Average body weights recorded during the course of the study are shown in Fig. 1. The body weights of the control (saline injected) animals increased rapidly during the first 3 wk, then slowly for the rest of the 9-wk period. Cadmium-treated animals had a slower weight gain both during (first 4 wk) and after (wk 5-9) Cd dosing. The body weight of the Cd-treated animals at the end of 9 wk was 71% of the control animals.
Tissue Distribution of Cd Twenty-four hours after the last dose of cadmium chloride, the highest accumulations of Cd were found in the liver, kidney, and spleen (Fig. 2). During the first 2 wk of recovery, the Cd concentration in the kidney did not decrease significantly, but subsequently, fell rapidly. Five weeks after the cessation of Cd treatment, the Cd level in the kidney was about one-fourth of that found on d 1. The hepatic Cd concentration decreased rapidly (wk 1-3) then slowly (from wk 3-5). The concentration of Cd in the spleen continued to increase for 3 wk after the final injection of Cd, then fell sharply, and by wk 5 remained at a slighly lower level compared with the value found on d 1 after the last dose of Cd. The Cd level in the RBC had declined by d 7 after the cessation of Cd treatment (compared with d 1); however, by d 35 it was almost the same as that on d 1. The Cd concentration in plasma was about 5-6 times lower than in RBC. Cadmium amounts in the plasma had increased slightly by wk I and after that showed a plateau between d 14 and 35 of the recovery period. The Cd concentration in the testes had risen significantly by d 7
Biological Trace Element Research
Vol. 1O, 1986
Fate of Trace Elements During RecoveryPeriod
51
GROWTH CURVES 4 0 0 --
jo Control group
I~
oI o
o~oI
30O 1~176 1o
/ 9
.~Ip 9
~''
~o
~,~0 ~
0~0
Cadmium group
IOO--
I
I
I
I
I
I
I
I
I
I
2
3
4 5 6 WEEKS
7
8
9
Fig. 1. Body weight gain of rats during 4 wk of Cd injectionns and 5 wk after cessation of Cd administration (9 Controls received saline only (O). and 14, then rapidly decreased with no further changes after 2 wk (Fig.
2). Effect on (lrinary Excretion of Cd, Zn, and Cu Repeated injections of CdCI2 caused an increased excretion of Cd in urine after the 8th dose, and excretion sharply increased after the 12th dose (Fig. 3). The urinary excretion level in controls is given in the figure caption. Urinary Cd excretion continued to increase even after the cessation of Cd treatment (until d 14), after which it fell sharply between d 21 and 35. The amount of Zn excreted in the urine increased after the fourth dose of Cd and remained at significantly elevated levels until d 21, compared with the saline group (even 3 wk after the cessation of Cd dosing). Urinary excretion of Cu was also enhanced by injections of Cd. The highest level of urinary Cu compared with the saline group was observed after the last dose of Cd (the 12th). Excretion of Cu during the recovery period was almost unchanged (Fig. 3).
Biological Trace Element Research
Vol. I O, 1986
Komsta-Szumska and Czuba
52
KIDNEY
160.0
LIVER
160.0 9
~~Z,~,~
120.0
SPLEEN
160.0 12o.o 8o.0
80.(;
=-
..=
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7
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TESTIS
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~ 40"0
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after
...--"'~
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,',
~,
last dose of
2'8
3'5
Cd
~'
,',
i,
2'e
3'5
Administration
Fig. 2. Cadmium concentration (p,g/g wet tissue) in organs of experimental rats, at different times after cessation of Cd administration. The values are expressed as means + SD of six animals.
Effect on Fecal Excretion of Cd, Zn, and Cu The a m o u n t of Cd excreted in feces increased sharply with injections of Cd (Fig. 3). Excretion levels in the control are given in the figure caption. During the recovery period, Cd excretion fell sharply (between d 1 and 7) to a concentration below that of the control (by d 14). However, 35 d after the cessation of Cd treatment, Cd excretion in feces d r o p p e d to the level found in the control group (i.e., 23 ~g/24 h). There was a significantly enhanced excretion of Zn in feces after Cd injections (by about 400 ~g/g). This level was maintained during the first week after the last dose. The level dropped (by 200 ~g/g) during the remainder of the recovery period, but never reached control levels (500.0 + 50.0 ~g/d/rat). Fecal excretion of Cu was only slightly elevated after the last dose of Cd and u n c h a n g e d (compared with the control) during the recovery period (Fig. 3).
Trace-Element Distribution in Tissue During Recovery Pe~od Zinc
Twenty-four hours after the last dose of Cd treatment, Zn levels were increased at least 2 or 3 times in all organs except RBC (Table 1). The Biological Trace Element Research
Vol. 1O, 1986
Fate of Trace Elements During Recovery Period
53
days after lost dose of Cd [
Cd-lnjection (dose No)
0
o
80.0
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