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The effect of sodium diethyldithiocarbamate treatment on copper and zinc concentrations in rat brain
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
65,286-290
(1982)
The Effect of Sodium Diethyldithiocarbamate Treatment Zinc Concentrations in Rat Brain E. L. LAKOMAA,* * Research
S. SATO,~
A. M. GOLDBERG,?
on Copper
AND J. M. FRAZIER?
Laboratory, Technical Research Center of Finland, Otakaari 3A, 02150 Espoo 15, Finland, TDepariment of Environmental Health Sciences, The Johns Hopkins University, 615 North Wolfe St., Baltimore, Maryland 21205
Received
July
I. 1978;
accepted
July
and
and
15. 1978
The Effect of Sodium Diethyldithiocarbamate Treatment on Copper and Zinc Concentrations in Rat Brain. LAKOMAA, E. L., SATO, S., GOLDBERG, A. M., AND FRAZIER, J. M. (1982). Toxicol. Appl. Pharmacol. 65, 286-290. Copper and zinc play important roles in the metabolic functions of the central nervous system. The effect of the chelating agent diethyldithiocarbamate (DDC) on the nervous system may be related to its effect on the kinetics and distribution of these essential metals. This study was designed to investigate the effects of DDC on copper and zinc concentrations in specific regions of the brain following both acute and repeated treatment. Acute treatment (250 mg/kg) had no effect on copper or zinc concentrations in brain regions at 24 hr while repeated treatment (250 mg/kg five times per week for 4 weeks) increased copper levels in the brain stem, cortex, hippocampus, and the rest of the brain but did not alter zinc concentrations in any brain regions. Many of the observed effects of DDC on distribution may be attributed to the formation of lipid-soluble DDC-metal complexes.
The essential metals, copper and zinc, are involved in a broad spectrum of biochemical functions necessary for normal neuronal activity. These functions range from catalytic activity in metalloenzymes involved in protein synthesis and metabolism of endogenous neuroactive chemicals to stabilization of protein structures and membranes. Regional localization of these metals in specific areas of the rat brain (Hu and Friede, 1968; Crawford and Connor, 1972; Donaldson et al., 1973; Fjerdingstad et al., 1974; Danscher et al., 1975, 1976) and spinal cord (Schroder et al., 1978) has been described. However, the functional significance of these regional localizations of essential metals has not been fully established. Regional differences may be related to the aggregation of specific cell types which employ metals and/or metalloenzymes for critical metabolic functions. 0041-008X/82/1
10286-05$02.00/O
Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
The diethyldithiocarbamates are a class of chelating agents which produce neurotoxicity. Sodium diethyldithiocarbamate (NaDDC) has been employed in the treatment of acute metal poisoning and Wilson’s disease (Sunderman, 1964). Dithiocarbamates have been identified as metabolic products following exposure to carbon disulfide (CS,) and after ingestion of disulfuram (tetraethylthiuram disulfide, Antabuse). The mechanism by which DDC produces central nervous system toxicity is thought to involve the chelation of copper at the active site of key metalloenzymes, such as dopamine+hydroxylase (McKenna and DiStefano, 1977). Koutensky et al. ( 197 1) have investigated the effects of DDC on the distribution of copper in mice. They show that a simultaneous injection of DDC and 64CuC12 in286
EFFECT
OF
DDC
ON
creases the whole body retention of copper and results in an increase in radiocopper concentrations in various tissues including the brain. In a similar study, Aaseth et al., (1979) found that simultaneous treatment with DDC caused a fivefold increase in radiocopper and a threefold increase in radiozinc concentrations in the brains of mice. In both of these studies, the investigators were observing the uptake kinetics of a tracer dose of the metal-DDC complex. Such data are not sufficient to determine the effects of DDC on the steady state concentrations of stable copper and zinc in the brain. The objective of this study was to determine the effects of DDC treatment, both acute and repeated dosing, on stable metal concentrations in the brain. Furthermore, since the effects may be confined to specific regions of the brain, five distinct regions of the brain were investigated: cortex, striatum, hippocampus, cerebellum, and brain stem. METHODS Animals. Long-Evans hooded rats (Blue Spruce Farms, Altamont, N.Y.) weighing 275 to 300 g were employed in these experiments. Animals were housed in plastic cages with filter tops and given free access to commercial rat chow (Charles River) and tap water. Dosage. A 3.5% solution of sodium diethyldithiocarbamate (J. T. Baker Chemical Co.) in distilled, deionized water was prepared fresh daily to avoid decomposition of DDC. In the acute exposure study, rats were divided into two groups of nine rats each. The control group was injected ip with a saline solution having a sodium concentration equal to the NaDDC solution. The experimental group was injected ip with a dosage of 250 mg DDC/kg. Three rats from each group were killed at 30 min, 4 hr, and 24 hr. In the repeat exposure study, rats were injected 5 days a week for 4 weeks with a DDC dosage of 250 mg DDC/kg. Control rats were injected with equimolar sodium chloride solution. On the final day of treatment, animals were killed 1, 4, and 24 hr after the last injection. Bruin dissection. Brains were removed immediately after decapitation and rinsed with distilled, deionized water to remove adhering blood. The dissection was performed by the procedure of Glowinski and Iversen (1966). After dissection, five regional samples were col-
METALS
IN
RAT
BRAIN
287
lected: cortex, striatum, hippocampus, cerebellum, brain stem (medulla oblongata and pans), plus the rest of the brain. All samples were stored in preweighed plastic vials at -20°C until they were prepared for metal analysis. Copper and zinc determination. Dried brain tissues were wet washed in concentrated nitric acid (Ultrex grade, J. T. Baker Co.) and 30% hydrogen peroxide. Zinc analyses were performed by flame atomic absorption spectrophotometry on a Varian AA5 spectrophotometer at 2 13.9 nm. Copper analyses were performed by flameless atomic absorption spectrophotometry on a Perkin-Elmer 4000 spectrophotometer equipped with a HGA 500 graphite furnace and an AS-40 automatic sampler at 324.8 nm. NBS standard reference material 1577-bovine liver was digested and analyzed concurrently with brain samples for quality control. All glass- and plasticware used during dissection, storage, and digestion was soaked overnight in 5% HNO,, rinsed with distilled, deionized water, and air dried. Stainless steel dissecting instruments were soaked in EDTA (ethylenediaminetetraacetic acid, 6 g/liter) overnight, rinsed with distilled, deionized water, and air dried. Plastic gloves used during dissection were washed similarly. Statistical analysis. The effect of time after last treatment on copper and zinc concentrations in each brain region for both the acute and repeat protocol was tested by one-way analysis of variance. Since there were no significant time effects, all animals from different termination times were grouped, i.e., following the acute treatment the animals killed at 30 min, 4 hr, and 24 hr were grouped to give nine rats per treatment group and similarly for the repeatedly dosed rats. The groups (collapsed across time) of treated rats were compared to the appropriate control rats by Student’s t test and significance was determined at the p I 0.05 level.
RESULTS The effect of a single dose of NaDDC on zinc and copper concentrations in the various brain regions is given in Table 1. Although there were significant variations in both zinc and copper concentrations between the different brain regions, there was no effect of NaDDC treatment on either metal up to 24 hr following a single dose of 250 mg/kg. Following repeated treatment with DDC for a total of 20 injections of 250 mg/kg each over 4 weeks, there were no changes in zinc concentrations in any brain regions studied (Table 2). However, there were sig-
288
LAKOMAA
EFFECT
OF SINGLE
TREATMENT
NaDDC
WITH
Zinc
ET AL.
TABLE
1
ON ZINC
AND
(pg/g
72.8 55.0 54.7 55.5 35.5 61.2
f k + * f +
CONCENTRATIONS
dry wt)
Control Cortex Striatum Hippocampus Cerebellum Brain stem Remainder of the brain
COPPER
Qvr
Treated 2.1 5.6 3.7 1.7 1.6 2.1
13.8 54.6 59.2 59.8 38.0 64.8
IN BRAIN
-c -+ + + -t +
Wg
dry wt)
Control 4.3 5.0 2.9 6.9 2.6 6.6
9.0 10.7 10.1 11.2 9.1 9.4
k + f f + rfr
REGIONS”
Treated 1.2 1.3 1.3 0.8 0.7 1.6
12.7 11.3 12.7 12.6 10.2 11.6
+ _’ f + + +
2.1 2.3 2.3 1.5 0.6 1.4
*X + SE (n = 9).
nificant effects of the repeated treatment on copper concentrations in four brain regions: cortex, hippocampus, brain stem, and the rest of the brain. In the striatum and cerebellum, repeated DDC treatment increased the mean copper concentration although the effect was not significant (p > 0.05). The largest effects, expressed as the percentage increase in copper concentration, were for the brain stem (55.2% increase), hippocampus (44.2%) cortex (33.8%) and the remainder of the brain (51.8%).
trations in any region of the brain up to 24 hr postinjection. This result appears to contradict the observations of Koutensky et al. (1971) and Aaseth et al. (1979). Their data indicate a significant elevation in both copper and zinc radioactivity in the brain 24 hr after simultaneous treatment with DDC and either radioactive copper or zinc. However, neither study evaluated the specific activity of the two metals in the brain and, therefore, it is not possible to determine whether total copper and zinc concentrations increased in the brain. It is possible in experiments using tracer doses of radioactive metals that the metal-DDC complex accumulated in the brain more rapidly than the metal alone and, thus, produced higher radioactivity levels
DISCUSSION Acute treatment with NaDDC had no effect on either the copper or zinc concenTABLE EFFECT
OF REPEATED
TREATMENT
CONCENTRATIONS
Zinc
(pg/g
WITH
a X k SE (n = 9). b Data indicated by (*) the p 5 0.05 level.
71.7 49.7 62.6 50.6 32.2 48.5
are significantly
f + k f f +
NaDDC
IN BRAIN
ON ZINC
Copper
Treated 2.7 4.6 3.4 0.7 1.3 3.7
different
68.7 48.6 58.6 48.2 32.9 49.9
from
AND
COPPER
REGION&
dry wt)
Control Cortex Striatum Hippocampus Cerebellum Brain stem Remainder of the brain
2
f 0.9 f 5.9 3~ 6.2 f 3.5 k 4.3 f 4.3
the corresponding
(a/g
Control 12.4 9.6 8.6 13.7 9.6 11.0
+ f + + + f
control
dry wt) Treated
0.8 1.7 1.6 0.9 0.3 0.8
16.6 13.1 12.4 16.6 14.9 16.7
value
f rt +_ f k f
by Student’s
2.1* 1.2 1.2* 1.8 1.9* 1.8:
t test at
EFFECT OF DDC ON METALS
without labeling the stable metalloenzyme pools which account for the majority of the total copper and zinc concentrations in the brain. This explanation is consistent with the radioisotope studies and the results reported here for the effect of DDC on stable copper and zinc concentrations. The observations of Schroder et al. (1978) also support our results. They demonstrated that short-term, high dosage of DDC (seven injections of 300 mg/kg over 2 days) had no effect on either copper or zinc concentrations in the spinal cord. Studies with other metals indicate that metal-DDC complexes have greater access to the brain. Cantilena et al. (1980) found that acute treatment with DDC increased the brain uptake of cadmium. By ruling out any effect of DDC on cerebral capillary permeability or cerebral blood flow, it was concluded that the blood-brain barrier was more permeable to the DDC-Cd complex than to ionic cadmium. Aaseth et al. ( 198 1) showed that simultaneous injection of DDC and mercuric chloride resulted in a lo-fold increase in the brain levels of mercury and also increased fetal uptake of mercury across the placenta. These data support the conclusion that the DDC-Hg complex is more lipid soluble than Hg ion. In the studies reported here, repeated treatment with NaDDC significantly affected copper concentrations in the brain. Although all brain regions tend to have elevated copper levels relative to controls, certain regions seem to be affected more than others. The brain stem seems to be particularly affected by NaDDC treatment. Differential toxicological responses of various regions of the nervous system have been reported following chronic DDC treatment. Edington and Howell ( 1966) demonstrated that ip treatment of rabbits with DDC for 7 l/2 months resulted in general incoordination and Wallerian degeneration of the spinal cord. However, no evidence of pathologic changes was observed in the corpus striatum of the brain. Their treatment re-
IN RAT BRAIN
289
sulted in a lo-fold increase in copper concentrations in the spinal cord. These data indicate that the effect of DDC on both copper kinetics and cytotoxicity demonstrates a significant degree of tissue selectivity. In this study, repeated treatment of rats with NaDDC had no effect on zinc concentrations in any brain region. A similar observation, i.e., an elevation of stable copper concentrations with no change in zinc levels, was made in peripheral nerves following carbon disulfide treatment (Lukas et al., 1974). The nature of the differential behavior of copper and zinc under these conditions of repeated DDC treatment is not known and requires additional study. The proposed mechanism of DDCs effect on the central nervous system is the inhibition of dopamine+hydroxylase due to chelation of copper at the active site of the enzyme (McKenna and DiStefano, 1977). In general, elevation of copper concentrations in the brain would be expected to activate this enzyme (Yu et al., 1976). However, if the elevated concentration of copper following repeated DDC treatment is a result of sequestration of the lipid soluble Cu-DDC complex in the lipid fraction of the brain, then higher copper concentrations would not necessarily reflect an increase in available copper. Under these conditions, higher copper concentrations in brain regions could occur concurrent with dopamine-fi-hydroxylase inhibition. An evaluation of copper distribution among intracellular pools, particularly the lipid-soluble pool, as well as the identification of the Cu-DDC complexes in brain is necessary to resolve this question. Acute treatment of rats with NaDDC, in doses that produced behavioral effects, had no effects on either copper or zinc concentrations in various regions of the brain while repeated treatment elevated copper levels with little effect on zinc concentrations. Many of the observed effects of DDC on metal kinetics may be attributable to the formation of a lipid soluble DDC-metal complex which can easily penetrate the
290
LAKOMAA
blood-brain barrier and accumulate in lipid fractions of the brain. However, the mechanistic basis of the differential behavior of copper and zinc following DDC treatment is not known. Furthermore, the relationship between selective alterations in copper distribution in the nervous system and the cytotoxic properties of DDC are not established, although the mechanism involving dopamine-/3-hydroxylase is not ruled out. ACKNOWLEDGMENT This research 00454.
was supported
by the NIH,
Grant
ES-
REFERENCES AASETH, J., ALEXANDER, J., AND WANNAG, A. (198 1). Effect of thiocarbamate derivatives on copper, zinc, and mercury distribution in rats and mice. Arch. Toxicol. 48, 2939. AASETH, J., SOLI, N. E., AND FORRE, 0. (1979). Increased brain uptake of copper and zinc in mice caused by diethyldithiocarbamate. Acta Pharmacol. Toxicol. 45, 41-44. CANTILENA, L., IRWIN, G., KLAASEN, G., AND PRESHORN, S. (1980). Effect of diethyldithiocarbamate on brain uptake of cadmium. Sot. Neurosci. Abstr. 6, 260. CRAWFORD, I. L., AND CONNOR, J. D. (1972). Zinc in maturing rat brain: Hippocampal concentration and localization. J. Neurochem. 19, 1451-1458. DANSCHER, G., FJERDINGSTAD, E. J., FJERDINGSTAD, E., AND FREDENS, K. (1976). Heavy metal content in subdivisions of the rat hippocampus (zinc, lead and copper). Bruin Res. 112, 442-446. DANSCHER, G., HALL, E., FREDENS, K., FJERDINGSTAD, E., AND FJERDINGSTAD, E. J. (1975). Heavy metals in the amygdala of the rat: Zinc, lead and copper. Brain Res. 94, 167-172.
ET AL. DONALDSON, J., ST. PIERRE, T., MINNICH, J. L., AND BAREAU, A. (1973). Determination of Na+, K+, Mg’+, CU’+, Zn’+, and Mr?* in rat brain regions. Cunad. J. Biochem. 51, 87-92. EDINGTON, N., AND HOWELL, J. McC. (1966). Changes in the nervous system of rabbits following the administration of sodium diethyldithiocarbamate. Nature (London) 210, 1060-1062. FJERDINGSTAD, E., DANSCHER, G., AND FJERDINGSTAD, E. J. (1974). Zinc content in hippocampus and whole brain of normal rats. Bruin Res. 79, 338-342. GLOWINSKI, J., AND IVERSEN, C. C. (1966). Regional studies of catecholamines in the rat brain. J. Neurochem. 13, 665-669. Hu, K. H., AND FRIEDE, R. L. (1968). Topographic determination of zinc in human brain by atomic absorption spectrophotometry. J. Neurochem. 15, 677685. KOUTENSKY, J., EYBL, V.. KOUTENSKA, M., SYKORA, J., AND MERTH, F. (1971). Influence of sodium diethyldithiocarbamate on the toxicity and distribution of copper in mice. Eur. J. Pharmacol. 14, 389392. LUKAS, E., KOTAS, P., AND OBRUSNIK, 1. (1974). Copper and zinc levels in peripheral nerve tissues of rats with experimental carbon disulphide neuropathy. Brit. J. Ind. Med. 31, 288-291. MCKENNA, M. J., AND DISTEFANO, V. (1977). Carbon disulfide. II. A proposed mechanism for the action of carbon disulfide on dopamine-fi-hydroxylase. J. Pharmucol. Exp. Ther. 202, 253-266. SCHRODER, H. D., FJERDINGSTAD, E., DANSCHER, G., AND FJERDINGSTAD, E. J. (1978). Heavy metals in the spinal cord of normal rats and of animals treated with chelating agents: A quantitative (zinc, copper, and lead) and histochemical study. Histochem. 56, l-12. SUNDERMAN, F. W. (1964). Nickel and copper mobilization by sodium diethyldithiocarbamate. J. New Drugs 4, 154-161. Yu, PH. H., BOULTON, A. A., AND Wu, P. H. (1976). Some observations on the interaction of dopamineP-hydroxylase and its natural inhibitors in the rat brain. Cunud. J. Biochem. 54, 988-991.
AND
APPLIED
PHARMACOLOGY
65,286-290
(1982)
The Effect of Sodium Diethyldithiocarbamate Treatment Zinc Concentrations in Rat Brain E. L. LAKOMAA,* * Research
S. SATO,~
A. M. GOLDBERG,?
on Copper
AND J. M. FRAZIER?
Laboratory, Technical Research Center of Finland, Otakaari 3A, 02150 Espoo 15, Finland, TDepariment of Environmental Health Sciences, The Johns Hopkins University, 615 North Wolfe St., Baltimore, Maryland 21205
Received
July
I. 1978;
accepted
July
and
and
15. 1978
The Effect of Sodium Diethyldithiocarbamate Treatment on Copper and Zinc Concentrations in Rat Brain. LAKOMAA, E. L., SATO, S., GOLDBERG, A. M., AND FRAZIER, J. M. (1982). Toxicol. Appl. Pharmacol. 65, 286-290. Copper and zinc play important roles in the metabolic functions of the central nervous system. The effect of the chelating agent diethyldithiocarbamate (DDC) on the nervous system may be related to its effect on the kinetics and distribution of these essential metals. This study was designed to investigate the effects of DDC on copper and zinc concentrations in specific regions of the brain following both acute and repeated treatment. Acute treatment (250 mg/kg) had no effect on copper or zinc concentrations in brain regions at 24 hr while repeated treatment (250 mg/kg five times per week for 4 weeks) increased copper levels in the brain stem, cortex, hippocampus, and the rest of the brain but did not alter zinc concentrations in any brain regions. Many of the observed effects of DDC on distribution may be attributed to the formation of lipid-soluble DDC-metal complexes.
The essential metals, copper and zinc, are involved in a broad spectrum of biochemical functions necessary for normal neuronal activity. These functions range from catalytic activity in metalloenzymes involved in protein synthesis and metabolism of endogenous neuroactive chemicals to stabilization of protein structures and membranes. Regional localization of these metals in specific areas of the rat brain (Hu and Friede, 1968; Crawford and Connor, 1972; Donaldson et al., 1973; Fjerdingstad et al., 1974; Danscher et al., 1975, 1976) and spinal cord (Schroder et al., 1978) has been described. However, the functional significance of these regional localizations of essential metals has not been fully established. Regional differences may be related to the aggregation of specific cell types which employ metals and/or metalloenzymes for critical metabolic functions. 0041-008X/82/1
10286-05$02.00/O
Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
The diethyldithiocarbamates are a class of chelating agents which produce neurotoxicity. Sodium diethyldithiocarbamate (NaDDC) has been employed in the treatment of acute metal poisoning and Wilson’s disease (Sunderman, 1964). Dithiocarbamates have been identified as metabolic products following exposure to carbon disulfide (CS,) and after ingestion of disulfuram (tetraethylthiuram disulfide, Antabuse). The mechanism by which DDC produces central nervous system toxicity is thought to involve the chelation of copper at the active site of key metalloenzymes, such as dopamine+hydroxylase (McKenna and DiStefano, 1977). Koutensky et al. ( 197 1) have investigated the effects of DDC on the distribution of copper in mice. They show that a simultaneous injection of DDC and 64CuC12 in286
EFFECT
OF
DDC
ON
creases the whole body retention of copper and results in an increase in radiocopper concentrations in various tissues including the brain. In a similar study, Aaseth et al., (1979) found that simultaneous treatment with DDC caused a fivefold increase in radiocopper and a threefold increase in radiozinc concentrations in the brains of mice. In both of these studies, the investigators were observing the uptake kinetics of a tracer dose of the metal-DDC complex. Such data are not sufficient to determine the effects of DDC on the steady state concentrations of stable copper and zinc in the brain. The objective of this study was to determine the effects of DDC treatment, both acute and repeated dosing, on stable metal concentrations in the brain. Furthermore, since the effects may be confined to specific regions of the brain, five distinct regions of the brain were investigated: cortex, striatum, hippocampus, cerebellum, and brain stem. METHODS Animals. Long-Evans hooded rats (Blue Spruce Farms, Altamont, N.Y.) weighing 275 to 300 g were employed in these experiments. Animals were housed in plastic cages with filter tops and given free access to commercial rat chow (Charles River) and tap water. Dosage. A 3.5% solution of sodium diethyldithiocarbamate (J. T. Baker Chemical Co.) in distilled, deionized water was prepared fresh daily to avoid decomposition of DDC. In the acute exposure study, rats were divided into two groups of nine rats each. The control group was injected ip with a saline solution having a sodium concentration equal to the NaDDC solution. The experimental group was injected ip with a dosage of 250 mg DDC/kg. Three rats from each group were killed at 30 min, 4 hr, and 24 hr. In the repeat exposure study, rats were injected 5 days a week for 4 weeks with a DDC dosage of 250 mg DDC/kg. Control rats were injected with equimolar sodium chloride solution. On the final day of treatment, animals were killed 1, 4, and 24 hr after the last injection. Bruin dissection. Brains were removed immediately after decapitation and rinsed with distilled, deionized water to remove adhering blood. The dissection was performed by the procedure of Glowinski and Iversen (1966). After dissection, five regional samples were col-
METALS
IN
RAT
BRAIN
287
lected: cortex, striatum, hippocampus, cerebellum, brain stem (medulla oblongata and pans), plus the rest of the brain. All samples were stored in preweighed plastic vials at -20°C until they were prepared for metal analysis. Copper and zinc determination. Dried brain tissues were wet washed in concentrated nitric acid (Ultrex grade, J. T. Baker Co.) and 30% hydrogen peroxide. Zinc analyses were performed by flame atomic absorption spectrophotometry on a Varian AA5 spectrophotometer at 2 13.9 nm. Copper analyses were performed by flameless atomic absorption spectrophotometry on a Perkin-Elmer 4000 spectrophotometer equipped with a HGA 500 graphite furnace and an AS-40 automatic sampler at 324.8 nm. NBS standard reference material 1577-bovine liver was digested and analyzed concurrently with brain samples for quality control. All glass- and plasticware used during dissection, storage, and digestion was soaked overnight in 5% HNO,, rinsed with distilled, deionized water, and air dried. Stainless steel dissecting instruments were soaked in EDTA (ethylenediaminetetraacetic acid, 6 g/liter) overnight, rinsed with distilled, deionized water, and air dried. Plastic gloves used during dissection were washed similarly. Statistical analysis. The effect of time after last treatment on copper and zinc concentrations in each brain region for both the acute and repeat protocol was tested by one-way analysis of variance. Since there were no significant time effects, all animals from different termination times were grouped, i.e., following the acute treatment the animals killed at 30 min, 4 hr, and 24 hr were grouped to give nine rats per treatment group and similarly for the repeatedly dosed rats. The groups (collapsed across time) of treated rats were compared to the appropriate control rats by Student’s t test and significance was determined at the p I 0.05 level.
RESULTS The effect of a single dose of NaDDC on zinc and copper concentrations in the various brain regions is given in Table 1. Although there were significant variations in both zinc and copper concentrations between the different brain regions, there was no effect of NaDDC treatment on either metal up to 24 hr following a single dose of 250 mg/kg. Following repeated treatment with DDC for a total of 20 injections of 250 mg/kg each over 4 weeks, there were no changes in zinc concentrations in any brain regions studied (Table 2). However, there were sig-
288
LAKOMAA
EFFECT
OF SINGLE
TREATMENT
NaDDC
WITH
Zinc
ET AL.
TABLE
1
ON ZINC
AND
(pg/g
72.8 55.0 54.7 55.5 35.5 61.2
f k + * f +
CONCENTRATIONS
dry wt)
Control Cortex Striatum Hippocampus Cerebellum Brain stem Remainder of the brain
COPPER
Qvr
Treated 2.1 5.6 3.7 1.7 1.6 2.1
13.8 54.6 59.2 59.8 38.0 64.8
IN BRAIN
-c -+ + + -t +
Wg
dry wt)
Control 4.3 5.0 2.9 6.9 2.6 6.6
9.0 10.7 10.1 11.2 9.1 9.4
k + f f + rfr
REGIONS”
Treated 1.2 1.3 1.3 0.8 0.7 1.6
12.7 11.3 12.7 12.6 10.2 11.6
+ _’ f + + +
2.1 2.3 2.3 1.5 0.6 1.4
*X + SE (n = 9).
nificant effects of the repeated treatment on copper concentrations in four brain regions: cortex, hippocampus, brain stem, and the rest of the brain. In the striatum and cerebellum, repeated DDC treatment increased the mean copper concentration although the effect was not significant (p > 0.05). The largest effects, expressed as the percentage increase in copper concentration, were for the brain stem (55.2% increase), hippocampus (44.2%) cortex (33.8%) and the remainder of the brain (51.8%).
trations in any region of the brain up to 24 hr postinjection. This result appears to contradict the observations of Koutensky et al. (1971) and Aaseth et al. (1979). Their data indicate a significant elevation in both copper and zinc radioactivity in the brain 24 hr after simultaneous treatment with DDC and either radioactive copper or zinc. However, neither study evaluated the specific activity of the two metals in the brain and, therefore, it is not possible to determine whether total copper and zinc concentrations increased in the brain. It is possible in experiments using tracer doses of radioactive metals that the metal-DDC complex accumulated in the brain more rapidly than the metal alone and, thus, produced higher radioactivity levels
DISCUSSION Acute treatment with NaDDC had no effect on either the copper or zinc concenTABLE EFFECT
OF REPEATED
TREATMENT
CONCENTRATIONS
Zinc
(pg/g
WITH
a X k SE (n = 9). b Data indicated by (*) the p 5 0.05 level.
71.7 49.7 62.6 50.6 32.2 48.5
are significantly
f + k f f +
NaDDC
IN BRAIN
ON ZINC
Copper
Treated 2.7 4.6 3.4 0.7 1.3 3.7
different
68.7 48.6 58.6 48.2 32.9 49.9
from
AND
COPPER
REGION&
dry wt)
Control Cortex Striatum Hippocampus Cerebellum Brain stem Remainder of the brain
2
f 0.9 f 5.9 3~ 6.2 f 3.5 k 4.3 f 4.3
the corresponding
(a/g
Control 12.4 9.6 8.6 13.7 9.6 11.0
+ f + + + f
control
dry wt) Treated
0.8 1.7 1.6 0.9 0.3 0.8
16.6 13.1 12.4 16.6 14.9 16.7
value
f rt +_ f k f
by Student’s
2.1* 1.2 1.2* 1.8 1.9* 1.8:
t test at
EFFECT OF DDC ON METALS
without labeling the stable metalloenzyme pools which account for the majority of the total copper and zinc concentrations in the brain. This explanation is consistent with the radioisotope studies and the results reported here for the effect of DDC on stable copper and zinc concentrations. The observations of Schroder et al. (1978) also support our results. They demonstrated that short-term, high dosage of DDC (seven injections of 300 mg/kg over 2 days) had no effect on either copper or zinc concentrations in the spinal cord. Studies with other metals indicate that metal-DDC complexes have greater access to the brain. Cantilena et al. (1980) found that acute treatment with DDC increased the brain uptake of cadmium. By ruling out any effect of DDC on cerebral capillary permeability or cerebral blood flow, it was concluded that the blood-brain barrier was more permeable to the DDC-Cd complex than to ionic cadmium. Aaseth et al. ( 198 1) showed that simultaneous injection of DDC and mercuric chloride resulted in a lo-fold increase in the brain levels of mercury and also increased fetal uptake of mercury across the placenta. These data support the conclusion that the DDC-Hg complex is more lipid soluble than Hg ion. In the studies reported here, repeated treatment with NaDDC significantly affected copper concentrations in the brain. Although all brain regions tend to have elevated copper levels relative to controls, certain regions seem to be affected more than others. The brain stem seems to be particularly affected by NaDDC treatment. Differential toxicological responses of various regions of the nervous system have been reported following chronic DDC treatment. Edington and Howell ( 1966) demonstrated that ip treatment of rabbits with DDC for 7 l/2 months resulted in general incoordination and Wallerian degeneration of the spinal cord. However, no evidence of pathologic changes was observed in the corpus striatum of the brain. Their treatment re-
IN RAT BRAIN
289
sulted in a lo-fold increase in copper concentrations in the spinal cord. These data indicate that the effect of DDC on both copper kinetics and cytotoxicity demonstrates a significant degree of tissue selectivity. In this study, repeated treatment of rats with NaDDC had no effect on zinc concentrations in any brain region. A similar observation, i.e., an elevation of stable copper concentrations with no change in zinc levels, was made in peripheral nerves following carbon disulfide treatment (Lukas et al., 1974). The nature of the differential behavior of copper and zinc under these conditions of repeated DDC treatment is not known and requires additional study. The proposed mechanism of DDCs effect on the central nervous system is the inhibition of dopamine+hydroxylase due to chelation of copper at the active site of the enzyme (McKenna and DiStefano, 1977). In general, elevation of copper concentrations in the brain would be expected to activate this enzyme (Yu et al., 1976). However, if the elevated concentration of copper following repeated DDC treatment is a result of sequestration of the lipid soluble Cu-DDC complex in the lipid fraction of the brain, then higher copper concentrations would not necessarily reflect an increase in available copper. Under these conditions, higher copper concentrations in brain regions could occur concurrent with dopamine-fi-hydroxylase inhibition. An evaluation of copper distribution among intracellular pools, particularly the lipid-soluble pool, as well as the identification of the Cu-DDC complexes in brain is necessary to resolve this question. Acute treatment of rats with NaDDC, in doses that produced behavioral effects, had no effects on either copper or zinc concentrations in various regions of the brain while repeated treatment elevated copper levels with little effect on zinc concentrations. Many of the observed effects of DDC on metal kinetics may be attributable to the formation of a lipid soluble DDC-metal complex which can easily penetrate the
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blood-brain barrier and accumulate in lipid fractions of the brain. However, the mechanistic basis of the differential behavior of copper and zinc following DDC treatment is not known. Furthermore, the relationship between selective alterations in copper distribution in the nervous system and the cytotoxic properties of DDC are not established, although the mechanism involving dopamine-/3-hydroxylase is not ruled out. ACKNOWLEDGMENT This research 00454.
was supported
by the NIH,
Grant
ES-
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