Toxicology, 61 (1990) 73-83 Elsevier Scientific Publishers Ireland Ltd.
Intracellular cadmium mobilization sequelae Shirley G. Jones, Myron A. Holscher, Pramod K. Singh and M a r k M. Jones Departments of Pathology and Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37235 (U.S.A.) (Received June 7th, 1989; accepted October 13th, 1989)
Summary The consequences of the mobilization of aged intracellular cadmium from its in vivo deposits in mice by chelating agents were examined. The chelating agents used were BAL, sodium N-benzyl-Dglucamine dithiocarbamate (NAB), Diisopropyl meso-2,3-dimercaptosuccinate(Di-PDMS) and sodium N-(4-methoxybenzyl)-D-glucamine dithiocarbamate(4-Me0), all previously shown capable of causing statistically significant decreases in either renal or hepatic cadmium burdens in rodents. They were given at a level of 400 # m o l / k g (i.p.) daily for 10 days to mice previously loaded with a total of 10 mg CdCI 2 • 2.5 H 20 /kg. Under these conditions a significant decrease in the renal cadmium level occurred following treatment with BAL, NaB, and 4-MeO; hepatic cadmium levels decreased significantly following treatment with NaB and 4-MeO. Pathological examination of the kidneys, liver, and testes in these animals showed that chelate mobilization of the cadmium produced no noticeable changes in the histopathology of these organs in comparison with that observed for the animals which had been given only cadmium and had undergone no chelate treatment. The results suggest that the mobilization of such aged cadmium from in vivo deposits need not result in any deleterious changes in the kidneys, liver or testes.
Key words: Cadmium mobilization; Chelating agents; Pathology; cadmium mobilization
Introduction One of the principal reasons for the reluctance to use chelate therapy in cases of chronic cadmium intoxication has been the fear that the transport of chelated cadmium through the kidney will lead to further damage in an already compromised organ. In one of the earliest studies of the use of chelating agents in animals with experimental cadmium intoxication, Tepperman [1] reported that treatment with BAL resulted in an increase in the renal cadmium content when this compound was administered shortly after the administration of cadmium chloride to experimental animals. Subsequent animal studies of Friberg and his coworkers showed that additional damage is in fact, found in rats and rabbits treated with NaeCaEDTA or BAL subsequent to the administration of cadmium Address all correspondence to: Mark M. Jones, Box 1583, Station B, Vanderbilt University, Nashville, TN 37235, U.S.A. 0300-483X/90/$03.50 (~) 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
73
salts [2,3]. These studies were carried out with short intervals between the administration of cadmium and that of the chelating agents. The response of cadmium to the injected chelating agents is generally recognized as being very dependent upon the interval elapsing between the administration of the cadmium and that of the chelating agent [4-9]. After a relatively short period of time (a few hours) the cadmium which remains in an animal is concentrated almost exclusively in intracellular sites at which most of it is firmly bound in the form of a complex, metallothionein [4-9], from which its rate of removal by typical chelating agents is extraordinarily slow. At 24 h after injection, plasma cadmium levels have dropped to less than 1% of initial values [10]. In the studies described here, the interval between cadmium injection and chelate treatment was 72 h, so the vast majority of the cadmium may be expected to be in intracellular sites [4-10]. In a previous study [11], we have shown that the behavior of aged cadmium deposits may differ significantly from freshly injected cadmium in terms of the pathological sequelae subsequent to chelate treatment. This study was undertaken to determine if such additional damage is an inevitable result of the chelate mobilization of cadmium from its aged deposits regardless of the type of chelating agent involved or the rate of such mobilization. In recent years a number of novel chelating agents have been reported which, under various conditions, are capable of mobilizing very appreciable fractions of cadmium from its aged deposits in the livers and kidneys of rodents. Whether these have the same effects when cadmium is mobilized, as was reported earlier for the use of BAL and NazCaEDTA, is an important point in determining the feasibility of a chelate treatment of chronic cadmium intoxication. The dosage level of cadmium was selected so that renal cadmium levels would be comparable or somewhat greater than those attained earlier by Dalhamn and Friberg [2] and by Friberg [3] in the demonstration of the enhanced nephrotoxicity due to chelate treatment of such cadmium exposed animals. The chelating agents used here were selected because they enhance primarily the biliary (and hence fecal) excretion of aged cadmium from its aged renal and hepatic deposits. Analogous compounds which enhance the urinary excretion of cadmium from such aged deposits seem not to have been reported. Materials and methods
The compounds used here were obtained as follows: C d C 1 2 • 2.5 H 2 0 , A.C.S. reagent grade from Fisher Sci. Co., Fairlawn, NJ; BAL in ampules (in peanut oil), USP grade from Hynson, Westcott and Dunning, Baltimore, MD. The sodium N-benzyl-D-glucamine dithiocarbamate monohydrate (NAB) was prepared using the method of Kojima et al. [12], while diisopropyl meso-2,3-dimercaptosuccinate(Di-PDMS) was prepared as described previously [13], as was the sodium N-(4-methoxybenzyl)-D-glucamine dithiocarbamate monohydrate (4MeO) [14]. Eighty male, CrI:CD-1 ® (ICR)BR outbred VAF/Plus Swiss mice weighing 31 -+ 3 g were obtained from Charles River Laboratories, Inc., Raleigh, NC, and kept in an A A A L A C approved animal care facility which provided free access to
74
food and water. After a 1 week acclimation period, 40 of the mice were loaded with cadmium using a series of i.p. injections amounting to 1, 3, 3, and 3 mg of CdC12 • 2.5 H 2 0 / k g in 0.5 ml of 0.9% saline on consecutive days for a total of 10 mg CdC12 • 2.5 H20/kg. The remaining 40 mice were given 0.5 ml of 0.9% saline on each of the 4 consecutive days. After a 3-day interval, the 40 cadmium-loaded mice were randomly assigned to 5 groups of eight mice. Each animal in each of the four groups was given an ip injection of one of the chelating agents at a level of 400/xmol/kg in 0.1 ml five times a week (Monday through Friday) for 2 weeks. This dosage, 400/zmol/kg, was selected because it has previously been used with these chelating agents [12-14]. The chelating agents used were: BAL (in peanut oil); diisopropyl meso-2,3-dimercaptosuccinate (in peanut oil), sodium N-(4methoxybenzyl)-D-glucamine dithiocarbamate( in saline), and sodium N-(benzyl)D-glucamine dithiocarbamate(in saline). The fifth group was given saline only. The 40 mice which had been injected with saline were further divided into 5 groups of eight animals each. Each of the four groups received one of the chelating agents as described above. The fifth group, the normal control, again was given 0.9% saline i.p. each time that the chelate control animals were given chelating agent. Two days after the last injection all the mice were sacrificed by cervical dislocation and dissected. Weighed samples of liver, kidney, brain and testes were digested in pure nitric acid on a heating block (80°C), taken to dryness, redissolved in 1% nitric acid solution made with pyrogen free deionized water, and analyzed for cadmium using a Perkin Elmer Model 403 atomic absorption spectrometer. The liver, kidney and testes samples of the cadmium loaded animals were analyzed using the flame mode. All brains, and all the organs from those mice not loaded with cadmium, were analyzed in the flameless mode. Samples of liver, kidneys and testes for pathological examination were taken from each animal at the time of dissection and immediately placed in 10% buffered formalin. The samples were then embedded, processed and stained (the kidneys were stained with PAS and the liver and testes with H & E) after which the slides were examined with light microscopy.
Results
The cadmium levels found in the liver, kidneys, brain, and testes of the mice at the end of the experiment are given in Table I. The amounts of cadmium removed from the organs examined is seen to vary greatly, with the dithiocarbamates removing more than the vicinal dithiols, and sodium N-(4-methoxybenzyl)D-glucamine dithiocarbamate removing a significantly larger amount from both the liver and the kidneys than the unsubstituted benzyl compound itself. The reduction in renal cadmium levels ranged from 8% for Di-PDMS to 53% for 4-MeO. For hepatic cadmium levels, reductions ranged from 0% for BAL to 39% for 4-MeO. The reductions in renal and hepatic cadmium levels obtained with 4-MeO were significantly greater than those obtained with NaB (Table I). These results are in agreement with previously published studies [14].
75
TABLE I O R G A N CADMIUM LEVELS AT THE CONCLUSION OF CHELATE TREATMENT" Treatment
N
Liver (ppm)
Kidney (ppm)
Brian (ppb)
Testes (ppm)
Cd Cd Cd Cd Cd
8 7 8 8 8
45.8 ~ 10.8 46.0 -+ 6.4 34.8 -+ 7.6 b 27.8 -+ 5.4 '~f 39.7 + 6.2 ~
18.7 _+3.3 10.2 + 3./) ~ 17.2 ± 2.0 8.8 -+ 2.6 c~ 16.0+-1.3 t'~
77.8 ± 55.3 175 -+ 108 h 95.4 ± 33.4 55.2 + 30.5 65.1+66.0
1.37 ± 0.39 1.20 ± 0.34 1.34 ± 0.29 1.39 ± 0.21 1.2720.15
17.0_+ 9.3 c ' h ~ 49 -+29 56 _+32 14 -+15 ~ 21 +21'
11.9-+10.8 ch 0.4+ 0.5 6 . 0 -+ 9.5 t3 -+ 7 ~ 5.7-+ 5.4 j
16.8_+18.7 c'' 1.2+- 1.7 3.1-+ 4.1 9.8± 6.0 40 +-70 J
only + BAL + Di-PDMS + 4-MeO + NaB
A n i m a l s n o t loaded with c a d m i u m :
Normals(allppb) Sal+BAL(ppb) SaI+Di-PDMS(ppb) Sal+4-MeO(ppb) Sal+NaB(ppb)
8 8 8 8 8
l/).1_+ 5.8 ~'h'k 1113 -+80 26 +16 g 13 -+13 h 13 -+ 12h
aAll data are mean values ± standard deviations. All mice labeled Cd (top groups) were given injections of CdCI 2 • 2.5 H20 of 1, 3, 3, and 3 mg/kg over a period of 4 days with one injection per day. After an interval of 3 days they were given a daily injection of the indicated chelating agent at a level of 400/xmol/kg/day, five times a week for 2 weeks with all injections being given on weekdays. Two days after the end of the chelate treatment the animals were sacrificed and dissected to obtain organ samples for cadmium analyses and histopathological examination. bSignificantly different from Cd only: 0.025 < P
Intracellular cadmium mobilization sequelae Shirley G. Jones, Myron A. Holscher, Pramod K. Singh and M a r k M. Jones Departments of Pathology and Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37235 (U.S.A.) (Received June 7th, 1989; accepted October 13th, 1989)
Summary The consequences of the mobilization of aged intracellular cadmium from its in vivo deposits in mice by chelating agents were examined. The chelating agents used were BAL, sodium N-benzyl-Dglucamine dithiocarbamate (NAB), Diisopropyl meso-2,3-dimercaptosuccinate(Di-PDMS) and sodium N-(4-methoxybenzyl)-D-glucamine dithiocarbamate(4-Me0), all previously shown capable of causing statistically significant decreases in either renal or hepatic cadmium burdens in rodents. They were given at a level of 400 # m o l / k g (i.p.) daily for 10 days to mice previously loaded with a total of 10 mg CdCI 2 • 2.5 H 20 /kg. Under these conditions a significant decrease in the renal cadmium level occurred following treatment with BAL, NaB, and 4-MeO; hepatic cadmium levels decreased significantly following treatment with NaB and 4-MeO. Pathological examination of the kidneys, liver, and testes in these animals showed that chelate mobilization of the cadmium produced no noticeable changes in the histopathology of these organs in comparison with that observed for the animals which had been given only cadmium and had undergone no chelate treatment. The results suggest that the mobilization of such aged cadmium from in vivo deposits need not result in any deleterious changes in the kidneys, liver or testes.
Key words: Cadmium mobilization; Chelating agents; Pathology; cadmium mobilization
Introduction One of the principal reasons for the reluctance to use chelate therapy in cases of chronic cadmium intoxication has been the fear that the transport of chelated cadmium through the kidney will lead to further damage in an already compromised organ. In one of the earliest studies of the use of chelating agents in animals with experimental cadmium intoxication, Tepperman [1] reported that treatment with BAL resulted in an increase in the renal cadmium content when this compound was administered shortly after the administration of cadmium chloride to experimental animals. Subsequent animal studies of Friberg and his coworkers showed that additional damage is in fact, found in rats and rabbits treated with NaeCaEDTA or BAL subsequent to the administration of cadmium Address all correspondence to: Mark M. Jones, Box 1583, Station B, Vanderbilt University, Nashville, TN 37235, U.S.A. 0300-483X/90/$03.50 (~) 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
73
salts [2,3]. These studies were carried out with short intervals between the administration of cadmium and that of the chelating agents. The response of cadmium to the injected chelating agents is generally recognized as being very dependent upon the interval elapsing between the administration of the cadmium and that of the chelating agent [4-9]. After a relatively short period of time (a few hours) the cadmium which remains in an animal is concentrated almost exclusively in intracellular sites at which most of it is firmly bound in the form of a complex, metallothionein [4-9], from which its rate of removal by typical chelating agents is extraordinarily slow. At 24 h after injection, plasma cadmium levels have dropped to less than 1% of initial values [10]. In the studies described here, the interval between cadmium injection and chelate treatment was 72 h, so the vast majority of the cadmium may be expected to be in intracellular sites [4-10]. In a previous study [11], we have shown that the behavior of aged cadmium deposits may differ significantly from freshly injected cadmium in terms of the pathological sequelae subsequent to chelate treatment. This study was undertaken to determine if such additional damage is an inevitable result of the chelate mobilization of cadmium from its aged deposits regardless of the type of chelating agent involved or the rate of such mobilization. In recent years a number of novel chelating agents have been reported which, under various conditions, are capable of mobilizing very appreciable fractions of cadmium from its aged deposits in the livers and kidneys of rodents. Whether these have the same effects when cadmium is mobilized, as was reported earlier for the use of BAL and NazCaEDTA, is an important point in determining the feasibility of a chelate treatment of chronic cadmium intoxication. The dosage level of cadmium was selected so that renal cadmium levels would be comparable or somewhat greater than those attained earlier by Dalhamn and Friberg [2] and by Friberg [3] in the demonstration of the enhanced nephrotoxicity due to chelate treatment of such cadmium exposed animals. The chelating agents used here were selected because they enhance primarily the biliary (and hence fecal) excretion of aged cadmium from its aged renal and hepatic deposits. Analogous compounds which enhance the urinary excretion of cadmium from such aged deposits seem not to have been reported. Materials and methods
The compounds used here were obtained as follows: C d C 1 2 • 2.5 H 2 0 , A.C.S. reagent grade from Fisher Sci. Co., Fairlawn, NJ; BAL in ampules (in peanut oil), USP grade from Hynson, Westcott and Dunning, Baltimore, MD. The sodium N-benzyl-D-glucamine dithiocarbamate monohydrate (NAB) was prepared using the method of Kojima et al. [12], while diisopropyl meso-2,3-dimercaptosuccinate(Di-PDMS) was prepared as described previously [13], as was the sodium N-(4-methoxybenzyl)-D-glucamine dithiocarbamate monohydrate (4MeO) [14]. Eighty male, CrI:CD-1 ® (ICR)BR outbred VAF/Plus Swiss mice weighing 31 -+ 3 g were obtained from Charles River Laboratories, Inc., Raleigh, NC, and kept in an A A A L A C approved animal care facility which provided free access to
74
food and water. After a 1 week acclimation period, 40 of the mice were loaded with cadmium using a series of i.p. injections amounting to 1, 3, 3, and 3 mg of CdC12 • 2.5 H 2 0 / k g in 0.5 ml of 0.9% saline on consecutive days for a total of 10 mg CdC12 • 2.5 H20/kg. The remaining 40 mice were given 0.5 ml of 0.9% saline on each of the 4 consecutive days. After a 3-day interval, the 40 cadmium-loaded mice were randomly assigned to 5 groups of eight mice. Each animal in each of the four groups was given an ip injection of one of the chelating agents at a level of 400/xmol/kg in 0.1 ml five times a week (Monday through Friday) for 2 weeks. This dosage, 400/zmol/kg, was selected because it has previously been used with these chelating agents [12-14]. The chelating agents used were: BAL (in peanut oil); diisopropyl meso-2,3-dimercaptosuccinate (in peanut oil), sodium N-(4methoxybenzyl)-D-glucamine dithiocarbamate( in saline), and sodium N-(benzyl)D-glucamine dithiocarbamate(in saline). The fifth group was given saline only. The 40 mice which had been injected with saline were further divided into 5 groups of eight animals each. Each of the four groups received one of the chelating agents as described above. The fifth group, the normal control, again was given 0.9% saline i.p. each time that the chelate control animals were given chelating agent. Two days after the last injection all the mice were sacrificed by cervical dislocation and dissected. Weighed samples of liver, kidney, brain and testes were digested in pure nitric acid on a heating block (80°C), taken to dryness, redissolved in 1% nitric acid solution made with pyrogen free deionized water, and analyzed for cadmium using a Perkin Elmer Model 403 atomic absorption spectrometer. The liver, kidney and testes samples of the cadmium loaded animals were analyzed using the flame mode. All brains, and all the organs from those mice not loaded with cadmium, were analyzed in the flameless mode. Samples of liver, kidneys and testes for pathological examination were taken from each animal at the time of dissection and immediately placed in 10% buffered formalin. The samples were then embedded, processed and stained (the kidneys were stained with PAS and the liver and testes with H & E) after which the slides were examined with light microscopy.
Results
The cadmium levels found in the liver, kidneys, brain, and testes of the mice at the end of the experiment are given in Table I. The amounts of cadmium removed from the organs examined is seen to vary greatly, with the dithiocarbamates removing more than the vicinal dithiols, and sodium N-(4-methoxybenzyl)D-glucamine dithiocarbamate removing a significantly larger amount from both the liver and the kidneys than the unsubstituted benzyl compound itself. The reduction in renal cadmium levels ranged from 8% for Di-PDMS to 53% for 4-MeO. For hepatic cadmium levels, reductions ranged from 0% for BAL to 39% for 4-MeO. The reductions in renal and hepatic cadmium levels obtained with 4-MeO were significantly greater than those obtained with NaB (Table I). These results are in agreement with previously published studies [14].
75
TABLE I O R G A N CADMIUM LEVELS AT THE CONCLUSION OF CHELATE TREATMENT" Treatment
N
Liver (ppm)
Kidney (ppm)
Brian (ppb)
Testes (ppm)
Cd Cd Cd Cd Cd
8 7 8 8 8
45.8 ~ 10.8 46.0 -+ 6.4 34.8 -+ 7.6 b 27.8 -+ 5.4 '~f 39.7 + 6.2 ~
18.7 _+3.3 10.2 + 3./) ~ 17.2 ± 2.0 8.8 -+ 2.6 c~ 16.0+-1.3 t'~
77.8 ± 55.3 175 -+ 108 h 95.4 ± 33.4 55.2 + 30.5 65.1+66.0
1.37 ± 0.39 1.20 ± 0.34 1.34 ± 0.29 1.39 ± 0.21 1.2720.15
17.0_+ 9.3 c ' h ~ 49 -+29 56 _+32 14 -+15 ~ 21 +21'
11.9-+10.8 ch 0.4+ 0.5 6 . 0 -+ 9.5 t3 -+ 7 ~ 5.7-+ 5.4 j
16.8_+18.7 c'' 1.2+- 1.7 3.1-+ 4.1 9.8± 6.0 40 +-70 J
only + BAL + Di-PDMS + 4-MeO + NaB
A n i m a l s n o t loaded with c a d m i u m :
Normals(allppb) Sal+BAL(ppb) SaI+Di-PDMS(ppb) Sal+4-MeO(ppb) Sal+NaB(ppb)
8 8 8 8 8
l/).1_+ 5.8 ~'h'k 1113 -+80 26 +16 g 13 -+13 h 13 -+ 12h
aAll data are mean values ± standard deviations. All mice labeled Cd (top groups) were given injections of CdCI 2 • 2.5 H20 of 1, 3, 3, and 3 mg/kg over a period of 4 days with one injection per day. After an interval of 3 days they were given a daily injection of the indicated chelating agent at a level of 400/xmol/kg/day, five times a week for 2 weeks with all injections being given on weekdays. Two days after the end of the chelate treatment the animals were sacrificed and dissected to obtain organ samples for cadmium analyses and histopathological examination. bSignificantly different from Cd only: 0.025 < P
