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Effect of thiuram sulphides on the uptake and distribution of nickel in pregnant and non-pregnant mice
Toxicology, 32 (1984) 297--313 Elsevier Scientific Publishers Ireland Ltd.
E F F E C T OF T H I U R A M SULPHIDES ON THE UPTAKE AND DISTRIBUTION OF NICKEL IN P R E G N A N T AND NONP R E G N A N T MICE
SUHAIR JASIMa and HANS TJ.~LVE b
aDepartment of Toxicology, University of Uppsala, Box 5 73, S-751 23 Uppsala (Sweden) and bDepartment of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Box 573, S-751 23 Uppsala (Sweden) (Received May 15th, 1984) {Accepted June 9th, 1984)
SUMMARY
Oral administration of 63Ni2+ together with thiuram sulphides {tetramethylthiuram disulphide, tetraethylthiuram disulphide, tetrabutylthiuram disulphide, dipentamethylenethiuram monosulphide or dipentamethylenethiuram tetrasulphide) or sodium diethyldithiocarbamate resulted in highly increased levels of 63Ni2÷ in several tissues of mice in comparison with animals given 63Ni2+ alone. Administration of these substances to pregnant animals induced increased levels of 63Ni2÷ in the fetuses. The uptake of 63Ni2÷ in the brains of both adults and fetuses was usually very markedly enhanced by these c o m p o u n d s -- dipentamethylenethiuram monosulphide and tetraethylthiuram disulphide being the most efficient c o m p o u n d s in this respect. Determination of the chloroform/water partition coefficients for nickel in the presence of thiuram sulphides or sodium diethyldithiocarbamate showed that these c o m p o u n d s are able to form lipophilic complexes with the metal. A facilitated penetration through the cellular membranes of the lipophilic complexes between nickel and these substances can explain the effects on the fate of the nickel. However, the partition coefficient for nickel in presence of sodium diethyldithiocarbamate was much higher than for the thiuram sulphides, but in spite of that, the effect of sodium diethyldithiocarbamate on the disposition of 63Ni2+ in the mice Address all correspondence to: Professor Hans Tj~ilve, Department of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Box 573, S-751 23 Uppsala, Sweden. Abbreviations: DPTM, dipentamethylenethiuram monosulphide; DPTT, dipentamethylenethiuram tetrasulphide; SDC, sodium diethyldithiocarbamate; TBTD, tetrabutylthiuram disulphide; TETD, tetraethylthiuram disulphide; TMTD, tetramethylthiuram disulphide. 0300-483X/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
297
was not more marked than for most of the thiuram sulphides. It has been shown that tetraethylthiuram disulphide undergoes a reductive fission in the gut to diethyldithiocarbamate, which is considered to be the active form of tetraethylthiuram disulphide. The marked effects on the disposition of the 63Ni2+ induced by the other thiuram sulphides examined in the present study suggest that a similar fission to chelating thiocarbamates will take place. However, the formation of lipophilic complexes with the original thiuram sulphides may contribute to the effects on the disposition of the 63Ni2+.
Key words: Nickel; Thiuram sulphides; Diethyldithiocarbamate; Chelating agents
INTRODUCTION Thiuram sulphides are widely used in industry, agriculture and medicine [1--4]. In rubber industry these substances are utilized as accelerators in the vulcanization process [2]. In agriculture they are used as fungicides and insecticides [3 ]. Tetraethylthiuram disulphide (disulfiram, Antabus® ) (TETD) is used in aversion therapy for chronic alcoholism [4]. It has been shown that TETD undergoes a reductive fission in vivo to diethyldithiocarbamate [5,6]. This probably takes place to a large extent already in the gut, and diethyldithiocarbamate is therefore considered to be the active form of TETD [6]. Diethyldithiocarbamate, as well as other dithiocarbamates, are chelating agents [1]. Based on this property sodium diethyldithiocarbamate (SDC) is used as a therapeutic agent in the treatment o f nickel carbonyl poisoning in man [7]. This substance has also been tried in human nickel hypersensitivity [ 8 ]. We have shown that SDC, given parenterally, will induce a redistribution of nickel in several tissues of mice exposed to nickel chloride or nickel carbonyl [9,10]. The complex between nickel and diethyldithiocarbamate is lipophilic [ 1] and the effects on the disposition of the nickel are probably due to a facilitated passage of the chelated nickel through the cellular membranes. Effects of SDC on the disposition of other metals, such as copper, zinc and cadmium, have also been shown [11,12]. It is possible that other thiuram sulphides, like TETD, may undergo a fission in the intestine to give chelating alkylthiocarbamates, which may influence the disposition of metals in the tissues. However, it is also possible that the thiuram sulphides by themselves may act as chelators. This has been shown for 2,2'
298
c%-c.2,
cs3-c%,
s
s... , s,
N-C
N-C-S'Na" /
c.3-cH2"
CH3-CH2 Sodium diethyldithioca rbamate
Ni
,c%-c% C-N
"s" ""s" "c%-c%
N ickel-bis { d iet hyldithioca rba mate )
(SDC)
c%.
s
s
I | / N-C-S-S-C-N
CH3
c%
CH_-CH,.
S S CH~-CH. N-C-S-S-C-N
~CH3
CH3- CH2
CH2-CH3
T e t r a e t h y l t h i u ram disulphide (TETD)
Tetramethylthiu ram disulphide (TMTD)
CH.C..C~C~
S
S
C%C%C%C%
N-C-S-S-C-N
c•-cN-cH2-c %
c~h-c.2-cN-c.3
T e t r a b u t y l t h i u r a m disulphide
(TBTD)
Q s
s
z-- x
-C-S-C- N.__]~ Dipentamethylenethiuram monosulphide (DPTM}
z--x
s
s /-- k
~__]N-C-S-S-S-S-C-N
]
Dipentamethylenethiuram tetrasulphide (DPTT)
Fig. 1. Structural formulae of the studied thiuram.sulphides and the sodium diethyldithioearbamate. The structure of the chelate between nieke| and diethyldithioearbamate is also shown.
these compounds, have been examined. Pregnant and non-pregnant mice have been used. The fate of the nickel has been studied in the non-pregnant mice and the passage of the nickel to the fetuses has been examined in the pregnant mice. To elucidate if the thiuram sulphides can form lipophilic chelates with nickel the chloroform/water partition coefficients for nickel without and together with thiuram sulphides have been determined. The substances studied are, besides TETD, tetramethylthiuram disulphide (TMTD), tetrabutylthiuram disulphide (TBTD), dipentamethylenethiuram monosulphide (DPTM) and dipentamethylenethiuram tetrasulphide (DPTT) (Fig. 1). All these compounds are used as rubber accelerators [2]. TMTD is also used as a crop fungicide [3]. As a comparison, experiments with SDC have also been included in the study. MATERIALS AND METHODS
Animals
Female C57BL mice were obtained from Anticimex AB, Stockholm, Sweden. They were fed a standard pellet diet (Ewos AB, SSdert/ilje, Sweden) 299
and were given tap water ad libitum. To obtain pregnant animals, mice were mated overnight. When a vaginal plug was found in the morning, it was considered as day 0 of pregnancy. (Delivery will usually take place on the night to day 19.)
Chemicals 63Ni2+ (spec. radioact. 10.4 mCi/mg Ni 2÷) was obtained from the Radiochemical Centre, Amersham, England. SDC, TMTD, TETD, TBTD, DPTM and DPTT were obtained from ICN Pharmaceuticals Inc., Plainview, NY, USA.
Experiments Animal experiments The mice were given 63Ni2÷ orally by gastric intubation and were then immediately given the thiuram sulphides or SDC, also orally by gastric intubation. The 63Ni2÷ was given together with non-labelled Ni 2÷ (NiC12 was used) in 0.1 ml physiological saline, so that each mouse received 10 pmol Ni2+/kg b o d y wt (0.58 mg Ni2+/kg b o d y wt). The SDC was dissolved in 0.2 ml physiological saline. The thiuram sulphides were dissolved in 0.2 ml corn oil. Each mouse was given 1 mmol/kg b o d y wt of the respective c o m p o u n d . Control mice were given the 63Ni2+ orally followed by 0.2 ml corn oil. Corn oil was also given to the mice which received the SDC, immediately after t h e administration of this substance. The following experiments were performed: 1. Non-pregnant mice were given the 63Ni2+ (2 pCi/animal together with non-labelled Ni 2*) and one o f the thiuram sulphides, SDC, or corn oil only (controls) and were then killed by CO2-asphyxiation, in groups of 4 animals, after 5 h, 24 h and 72 h. Various tissues were taken and dissolved in 1 ml o f Soluene 350® (Packard). Ten milliliters of a scintillation fluid consisting of 4.9 g PPO (2.5
300
SDC or corn oil only (controls). The mice were killed after 24 h. One animal was used per treatment. 4. Non-pregnant mice were given the 63Ni2+ (2 ~Ci/animal together with non-labelled Ni 2÷) and one o f t h e thiuram sulphides, SDC, or corn oil only (controls). They were then placed in groups of 4 animals in cages which permit collection of urine. The urine was collected after 24, 48 and 72 h and the radioactivity determined by liquid scintillation counting, using Instagel® (Packard) as scintillation fluid.
De termination o f chloroform/wa ter partition coefficien ts The chloroform/water partition coefficients of Ni 2÷ and of Ni 2+ in presence of thiuram sulphides or SDC, were determined. With the exception of SDC (see below) the following procedure was used: the 63Ni2+ was added to a solution with non-labelled NiC12 dissolved in 1/15 M phosphate buffer (pH 7.4) so t h a t the final concentration of Ni 2÷ was 0.0005 mmol/ml, and the a m o u n t of radioactivity was 0.25 ~Ci/ml. The respective thiuram sulphide was dissolved in chloroform in a concentration of 0.05 mmol/ml. Two-millilitre aliquots of the Ni2+-solutions and 2 ml of chloroform containing one of the thiuram sulphides or, for controls, chloroform without further addition, were mixed vigorously for 30 min. The samples were then kept for 4 days at room-temperature in capped test tubes. Preliminary experiments had shown that equilibrium had occurred at this interval. The a m o u n t of radioactivity in 200-~1 aliquots of both the organic and the aqueous phase was then determined by liquid scintillation counting using Instagel® (Packard) as scintillation fluid. For SDC the following procedure was used: the SDC was dissolved in the phosphate buffer in a concentration 0.055 mmol/ml. Of this solution, 1.8-ml aliquots were taken and 0.2 ml of buffer, containing 0.005 m m o l / m l of Ni 2÷ (63Ni2+ together with non-labelled NiCI2; 2.5 pCi 63Ni2+/ml) was added. The obtained 2-ml aliquots, which contained 0.05 m m o l / m l of SDC and 0.0005 m m o l / m l o f Ni 2÷ {with 0.25 pCi/ml of 63Ni2÷) were mixed with 2-ml aliquots of chloroform. The procedure described above for the thiuram sulphides was thereafter followed.
Statistical analysis For statistical analysis, Student's t-test was used. The level of significance was set at P ~ 0.0 5. RESULTS The administration of 63Ni2+ together with SDC or either of the thiuram sulphides to the non-pregnant mice resulted in very markedly increased levels of radioactivity in most tissues at all survival intervals in comparison with the animals given 63Ni2+ only (Table I). However, in some instances marked differences in the effects on the fate of the 63Ni2÷ were observed between the compounds. Generally, the levels of radioactivity in the liver, kidney, lung, fat and
301
to
¢.o
I
OF
THIURAM
SULPHIDES
AND
SDC
ON
THE
TISSUE-CONCENTRATIONS
OF
6~Ni ~÷ IN N O N - P R E G N A N T
MICE
detectable.
a M e a n ± S.E. f r o m 4 m i c e .
41±12 80 ~12 38 ~ 6 5 i 0.8 101 ± 7 19 ± 2 0 . 6 ± 0 .1
Liver Kidney Lung Brain Spinal cord Fat Serum c
72h
97 495 406 444 380 71 7
331 1874 674 436 533 245 95
1869 548 88 73 305 135
± 14 ~ 19 b ± 8b ~ 23 b ± 29 b ~ 15 b ± 0.3 b
± 52 b ± 150 b i 54 b ± 11 b ~ 58 b ± 26 b ± 28 b
± 302 b ± 174 b ± 11 b ± 9b z 48 b ± 33 b
458 ± 110 b
+3Ni2+ + SDC
86 206 146 1078 914 72 7
~ 20 ~ 34 b ~ 9b ± 139 b ± 105 b ± 11 b ± 0.5 b
59 b 84 b 28 b 89 b 73 b 30 4b
~ 142 b ± 48 b ~ 82 b ± 34 b ± 9b ± 43 b
240 ~ 752 ± 272 ~ 629 ~ 602 ± 92± 32 ~
2135 613 350 335 191 166
3776 ~ 241 l~
+3Ni2+ + TMTD
~ ± ~ ± ± ± ±
i i ± ± ± ± 164 b 304 b 81 b 208 b 147 b 120 b 45 b
294 b 107 b 130 b 59 b 128 b 59 b
334 b 253 b 11 b 9b 176 b 141 b
35 14 b 58 b 5b 6b 71 b d
~ 108 b ± 166 b • 98 b ± 7b ± 56 b ±262 b ± 22 b
~ ± ± z ~ ±
132-* 297 ~ 494 ~ 30 ± 33 ± 321 ± N .D .
828 1021 815 48 268 1033 82
2647 2726 55 43 654 1099
7886 ± 771 b
+3Ni2+ + TBTD
~ ~ ~ ~ ± ± ~
± ± ~ z z •
18 b 188 b 302 b 168 b 148 b 61 b 91 b
995 b 320 b 287 b 296 b 355 b 103 b
259 ~ 24 b 1890 ~169 b 1594 ~ 88 b 3027 • 279 b 2075 • 181 b 132 ± 29 b N.D. d
375 3701 2077 1970 1470 350 372
13641 3181 1830 1646 2705 1293
1681 ± 278 b
63Ni++ + DPTM
C The v a l u e s f o r s e r u m a r e p m o l / 1 0 0
209~ 2b 576i 25 b 614 i 25 b 1544 ~ 160 b 1533 ± 126 b 36 ~ 7b N.D. d
903 1996 688 1502 1343 555 172
2912 869 653 492 839 476
2312 i 232 b
63Ni2+ + TETD
b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s (P < 0 . 0 5 ) .
i 2. 5 ± 16 ~ 7.4 ± 0.1 ± 25 ± 9 ±0.1
38 240 102 2 100 63 3
Liver Kidney Lung Brain Spinal cord Fat Serum c
5 79 12 0.7 2. 5 3.4 7.5
24 h
± i ± ~ z ±
28 i
661 79 3 16 10 35
Liver
5 h
+3Ni2+
Tissue-concentration of ~3Ni2+ (pmol/100 m g wet tissue)a
Kidney Lung Brain Spinal cord Fat Serum c
Tissues
Survival interval
~1.
15 b
~ ~ ± ~ ~ ± ~
2b 96 b 8b 11 b 13 b 1b 6b
± 222 b z 43 b ~ 22 b z 13 b ± 17 ~ 34 b
dN.D., not
80 ± 14 292± 37 b 258 ~ 22 b 325 ~ 30 b 277 ~ 33 b 93 i 21 b N .D . d
59 1007 317 258 213 31 42
2518 445 147 76 49 445
151 t
+3Ni2+ + DPTT
Mice were given 6+Ni++ (10 u m o l [0.58 mg]/kg body wt; the a m o u n t of ++Ni ++ was 2 uCi/animal) orally by gastric intubation and were then immediately given the respective thiuram sulphide or S D C (1 m m o l / k g b o d y wt) also orally by gastric intubation. Control mice were given ++Ni 2÷ and the vehicle -- corn oil -- used to dissolve the thiuram sulphides. The mice were killed after 5, 24 and 72 h and the a m o u n t of ~+Ni ++ in the tissues was determined as described in Materials and Methods.
EFFECTS
TABLE
serum of the animals treated with these substances were the highest at the 5-h survival interval and decreased at 24 h and 72 h (Table I). In contrast, the levels of 63Ni2÷ in the brain and spinal cord generally increased with time and were the highest at 72 h. Among the studied compounds, DPTM generally induced the highest increase in the tissue-levels of Ni 2÷ at all survival intervals. Thus, at 5 h the concentrations of 63Ni2÷ in some tissues of the DPTM-treated animals were 100 times, or more, higher than in the controls. Markedly increased levels were also observed at 24 h and 72 h. At all survival intervals the highest relative increase was observed in the brain. In addition the concentrations of 63Ni2+ in the spinal cord of the DPTM-treated animals were very high. Especially TETD, and also TMTD, were in addition very efficient in increasing the tissue-levels of 63Ni2÷ at all intervals. SDC was usually somewhat less efficient in increasing the concentrations of 63Ni2÷ than the preceding compounds. DPTT was, in turn, less effective than SDC. DPTM, TETD, TMTD and SDC generally all induced the highest relative increase of 63Ni2÷ in the brain and spinal cord. TBTD, in contrast, was less efficient in increasing the concentration of 63Ni2÷ in the central nervous system. However, this c o m p o u n d was very effective in elevating the 63Ni2÷levels in fat and also in the liver and the kidney (Table I). In the pregnant mice, which were killed 24 h after the administrations, increased levels of 63Ni2÷ in the fetuses were induced by all compounds (Table II). DPTM and TETD, which were very efficient in increasing the 63Ni2÷-levels in the tissues of the non-pregnant mice, were also very efficient in increasing the levels of 63Ni2÷ in the fetal tissues. These 2 compounds induced very markedly increased levels of 63Ni2÷ in the brain of the adult animals and a corresponding marked increase in the concentration of 63Ni2÷ in the fetal brains was also observed. TMTD and SDC were generally less efficient in increasing the levels of 63Ni2÷ in the fetal tissues than DPTM and TETD. DPTT showed, in turn, a lower efficiency than these compounds. TBTD were among the least efficient compounds in increasing the fetal uptake of 63Ni2÷. The increase in concentration of 63Ni2÷ which was observed in the brains of the non-pregnant animals after the administration of TBTD was less expressed than for the other compounds and this correlated with a low increase in the levels of 63Ni2+ also in the fetal brains. The treatments with the thiuram sulphides and SDC resulted in very markedly increased amounts of urinary 63Ni2÷ (Table III). DPTM was the most efficient c o m p o u n d in this respect and DPTT was the least efficient compound. The whole-body autoradiography confirmed the results of the liquid scintillation countings (Figs. 2--4). Thus, markedly increased tissue-levels of 63Ni2÷ were observed both in the pregnant and the non-pregnant animals after the treatments with these compounds, in comparison with animals given 63Ni2÷ only. The increased concentrations of 63Ni2÷ in the central nervous system were very apparent in the animals treated with SDC, TMTD, TETD, DPTM and DPTT. An even labelling was observed in the grey and
303
SULPHIDES AND SDC ON THE TISSUE-CONCENTRATIONS
O F 63Ni~+ IN F E T U S E S A N D P L A C E N T A E
76 ± 13
Placentae
392 ± 40 b
8b 9b 35 b 12 b 9b 231 ± 22 b
115 ~ 19 b 8 1 ± 7b 159 ~ 9 b 112 i 23 b 108 ± 26 b
+ TMTD
+ SDC 165 i 90i 217 ± 140 i 174 ~
~3Ni2~-
~3Ni2+
444 ± 47 b
346, 61 b 1 7 4 ± 24 b 523 i 116 b 327 ~ 66 b 790 ± 181 b
6"Ni2+ + TETD
aMean ~ S.E. o f 4 d e t e r m i n a t i o n s o f material o b t a i n e d f r o m 4 p r e g n a n t mice. bSignificantly d i f f e r e n t f r o m c o n t r o l s (P < 0.05).
15 ± 1 20, 2 57 ~ 5 21 ~ 2 11 ~ 1
63Ni2+
T i s s u e ~ o n c e n t r a t i o n o f 63Ni2" ( p m o l / 1 0 0 m g w e t tissue) a
Whole f e t u s Liver Kidney Lung Brain
Fetal tissues
and Methods.
12 b 37 b 78 b l0 b 15 b 908 ~ 104 b
125 i 213± 402 ± 137 i 57 i
63Ni2+ + TBTD
820 • 71 b
513 z 45 b 2 3 6 ~ 22 b 685 z 88 b 403,47 b 891 + 81 b
63Ni~+ + DPTM
8b
6b 8b 7b 11 b
£
~ z i ±
240 ± 33 b
98 69 244 91 110
63Ni2+ + DPTT
Mice o n day 18 o f p r e g n a n c y w e r e given 6ZNi2* (10 ~ m o l [0.58 m g ] / k g b o d y w t ; t h e a m o u n t o f 63Ni2" was 3 ~Ci/animal) orally b y gastric i n t u b a t i o n and were t h e n i m m e d i a t e l y given t h e respective t h i u r a m s u l p h i d e or SDC (1 m m o l / k g b o d y w t ) also orally b y gastric i n t u b a t i o n . C o n t r o l mice w e r e given 63Ni2÷ and t h e vehicle - - c o r n oil - - used t o dissolve t h e t h i u r a m sulphides. T h e m i c e w e r e killed a f t e r 24 h. T h e fetuses a n d t h e p l a c e n t a e w e r e excised a n d dissected, a n d t h e a m o u n t o f 63Ni2÷ was d e t e r m i n e d as d e s c r i b e d in Materials
EFFECTS OF THIURAM OF PREGNANT MICE
T A B L E IT
O rJl
4591
Total excretion
44788
39840 2909 2039
~3Ni2+ + SDC
21099
17598 2809 692
6SNi2+ + TMTD
25459
19318 4924 1217
63Ni2+ + TETD
37665
19362 15995 2308
~Ni~, + TBTD
47276
30050 13636 3590
63Ni2+ + DPTM
11099
9988 846 265
63Ni1+ + DFIT
aMean urinary excretion per animal. Each group contained 4 animals and the combined urine excreted by these animals was collected.
4298 282 11
63Ni~÷
A m o u n t o f 63Ni2+ excreted (pmol/animal) a
24 48 72
(h)
Intervals
Mice were given 63Ni~+ (10 ~mol [0.58 mg]/kg body wt; the amount of 63Ni2+ was 2 ~Ci/animal) orally by gastric intubation and were then immediately given the respective thiuram sulphide or SDC (! mmol/kg body wt) also orally by gastric intubation. Control mice were given 63Ni2* and the vehicle - - corn off - - u s e d to dissolve the thiuram sulphides. Urine was collected after 24, 48 and 72 h and the amount of 63Ni~+ determined as described in Materials and Methods.
E F F E C T S OF THIURAM SULPHIDES AND SDC ON THE U R I N A R Y EXCRETION OF 63Ni2+
TABLE HI
Brain
Lung Contents of stomach
Tongue Brain
Lung
Tongue Brain Lung
Kidney
Intestinal contents Kidney
Liver Contents of stomach Kidney
Liver Intestinal contents Fig. 2. Whole-body autoradiograms of mice killed 24 h after oral administration of 63Ni 2+ (10 ~mol [0.58 mg]/kg body wt; the amount of ~3Ni2+ was 20 ~Ci/animal). Mouse (B) was also given DPTM and mouse (C) DPTT (1 mmol/kg body wt) orally immediately after the administration of the 63Ni2*. Mouse (A) was only given the 63Ni2+ and the vehicle -- corn oil -- used to dissolve the thiuram sulphides. In (A) most radioactivity is present in the gastro-intestinal contents. There is also a marked labelling of the kidney and a weak labelling of the lung. In (B) and (C) a marked radioactivity is present in several tissues. The labelling of the brain is very high in (B).
306
w h i t e m a t t e r o f t h e b r a i n a n d s p i n a l c o r d . T h e v e r y m a r k e d i n c r e a s e in t h e level o f 63Ni2+ in f a t w h i c h was o b s e r v e d in t h e l i q u i d s c i n t i l l a t i o n c o u n t i n g s in a n i m a l s t r e a t e d w i t h T B T D was also o b s e r v e d in t h e a u t o r a d i o g r a p h y . T h e a u t o r a d i o g r a m s o f a n i m a l s given 63Ni~+ o n l y , s h o w e d a r a d i o a c t i v i t y in t h e k i d n e y s w h i c h was l o c a l i z e d t o d i s t i n c t s p o t s in t h e c o r t e x . In t h e a n i m a l s t r e a t e d w i t h t h e t h i u r a m s u l p h i d e s o r SDC, t h e r e was a m o l e even d i s t r i b u t i o n o f r a d i o a c t i v i t y in t h e k i d n e y s . T h e a u t o r a d i o g r a p h y in t h e p r e g n a n t a n i m a l s given t h e t h i u r a m s u l p h i d e s o r SDC s h o w e d i n c r e a s e d u p t a k e o f 63Ni 2+ in t h e f e t u s e s ( F i g . 4). With DPTM and TETD a very marked labelling of the fetal central nervous system was seen, b u t t h i s was n o t o b s e r v e d w i t h T B T D . T h e c h l o r o f o r m / w a t e r p a r t i t i o n c o e f f i c i e n t s f o r Ni 2+ w i t h o u t a n d t o g e t h e r w i t h t h e t h i u r a m s u l p h i d e s a n d SDC a r e s h o w n in T a b l e IV. T h e p a r t i t i o n c o e f f i c i e n t s w e r e m u c h h i g h e r in all cases in t h e p r e s e n c e o f t h e s e c o m p o u n d s , in c o m p a r i s o n w i t h t h e d e t e r m i n a t i o n w i t h c h l o r o f o r m o n l y . In t h e p r e s e n c e o f SDC a l m o s t all o f t h e Ni 2+ was f o u n d in t h e c h l o r o f o r m . T h e t h i u r a m s u l p h i d e s s h o w e d l o w e r values: T M T D giving t h e h i g h e s t p a r t i t i o n c o e f f i c i e n t , w i t h m o r e t h a n 40% o f t h e Ni 2+ in t h e c h l o r o f o r m p h a s e ; T E T D giving t h e l o w e s t v a l u e , w i t h a b o u t 3% o f t h e Ni 2+ in t h e c h l o r o f o r m . W i t h o u t t h e s e c o m p o u n d s 9 9 . 9 5 % o f t h e Ni 2+ was p r e s e n t in t h e a q u e o u s p h a s e . TABLE IV EFFECTS OF THIURAM SULPHIDES AND PARTITION COEFFICIENT OF NICKEL
SDC ON THE CHLOROFORM:
WATER
Two-millilitre aliquots of buffer-solutions containing 0.0005 mmol/ml of Ni 2÷ were equilibrated for 4 days with 2-ml chloroform aliquots or with 2-ml chloroform aliquots containing 0.05 mmol/ml of the respective t h i u r a m s u l p h i d e s . T h e SDC was a d d e d t o the buffer-solutions in a concentration of 0.05 mmol/ml and equilibration was performed with chloroform for 4 days in the same way as for the thiuram sulphides. The amount of tracer - - 63Ni2+ -- in the buffer solutions was 0.25 uCi/ml. The ratio of the amount of nickel in the chloroform phase to the amount of nickel in the aqueous phase was considered the chloroform/water partition coefficient. The percentage of the total amount of nickel which was present in the chloroform phase was also calculated,a Substance
Partition coefficientb
% Ni 2. in the chloroform phase
c SDC TMTD TETD TBTD DPTM
0.0005 2308 0.694 0.034 0.051 0.127
0.05 99.96 40.97 3.29 4.85 11.27
± 0.0002 ± 467 ~ 0.037 ± 0.002 ~ 0.002 • 0.012
aDetermination of the chloroform/water partition coefficient of DPTT was not possible due to low solubility in chloroform. bMean ± S.E. of 4 determinations. CNo substance added to the chloroform.
307
Brain
Lung
Spinal cord
Brain
Liver Lun
Intestinal contents dney
Brain
Liver Lung
Intestinal contents Kidney
Liver
Contents of stomach and intestine ¢/!
..a .
.
Intestinal contents
308
Kidney
.
.
Abdominal fat
DISCUSSION The results of the present study have shown t h a t all t he studied thiuram sulphides have very m a r ked effects on the u p t a k e and distribution o f nickel in the mice. It was also f o u n d t h a t the thiuram sulphides are able to form lipophilic chelates with nickel. A facilitated p e n e t r a t i o n through the cellular membranes of lipophilic nickel-chelates probabl y explains the increased u p tak e in the tissues. This m a y be related directly to the f o r m a t i o n o f the lipophilic thiuram complexes. However, the possibility m e n t i o n e d in the i n t r o d u c t i o n , t h a t all the thiuram sulphides, as has already been shown for TETD, undergo a reductive fission in the intestine to chelating thiocarbamates, must also be taken into account. In s u p p o r t of the latter assumption are t he following conditions: (1) The c h l o r o f o r m / w a t e r partition coefficient was m u c h higher for the nickeld i e t h y l d i t h i o c a r b a m a t e - c o m p l e x t h a n f o r t h e c o m p l e x e s b e t w e e n nickel and the thiuram sulphides. It is well k n o w n t hat lipid solubility is a physical p r o p e r t y o f major i m p o r t a n c e f or the passage of molecules across cellular membranes [15]. However, the ability o f SDC to affect the u p t a k e and distribution o f nickel was n o t m o r e expressed than for m o s t o f the thiuram sulphides. This suggests t hat t he thiuram sulphides will undergo a reductive fission in th e intestine and t hat the f o r m a t i o n o f lipophilic complexes with the resulting thiocarbamates cont r i butes t o t he effects on the disposition o f the nickel. (2) The results showed no correlation bet w een the c h l o r o f o r m / water partition coefficients f or t he complexes between the nickel and t he thiuram sulphides and t he efficiency by which t he thiuram sulphides were able to increase the upt a ke of t he nickel in t he tissues. For example DPTM was mo r e effective in increasing t he u p t a k e o f nickel in m ost tissues than TMTD, despite a higher partition coefficient for t he latter than for t he f o r m e r c o m p o u n d . This is a f u r t h e r indication t hat the thiuram sulphides by themselves are n o t solely responsible for the effects on t he disposition o f t h e nickel -- f o r m e d t h i o c a r b a m a t e s p r o b a b l y also play an i m p o r t a n t role. Our results showed t ha t T E T D was m o r e effective than SDC in increasing the u p t a k e of nickel in m os t tissues. It is possible t h a t the effects observed with the T E T D are due to c o m p l e x - f o r m a t i o n with bot h the unchanged T E T D and the d i e t h y l d i t h i o c a r b a m a t e f o r m e d by reductive fission in the intestine and t hat the c o m b i n e d ef f ect t h e r e f o r e will be higher than when the SDC is given to t he mice.
Fig. 3. Whole-body autoradiograms of mice killed 24 h after oral administrations of 63Ni2+ (10 umol [0.58 mg]/kg body wt; the amount of 63Ni2* was 20 uCi/animal). Mouse (A) was also given SDC, mouse (B) TMTD, mouse (C) TETD and mouse (D) TBTD (1 mmol/kg body wt) orally immediately after the administration of '3Ni~÷. In (A), (B) and (C) there is a strong labelling of the central nervous system. In (D) the labelling of the brain is much lower. The labelling of fat is high in this animal. For comparison with an autoradiogram of an animal given 63Ni2" only: See Fig. 1A.
309
Llver
Fetal brain
310
Fstal liver
Fetal liver
Fetal kidney
Fetal brain
Fetal Intestines
DPTT was much less effective than the structurally related DPTM in increasing the uptake of nickel in the tissues. Conceivably the DPTM may more easily undergo a reductive fission to a chelating thiocarbamate than the DPTT. Most of the thiuram sulphides and the SDC induced a very high uptake of nickel in the central nervous system. The central nervous system is rich in lipids and has a high vascularization, and these factors m a y p r o m o t e the uptake of the lipophilic nickel chelates. TBTD showed the lowest ability of the tested compounds in increasing the uptake of nickel in the central nervous system. However, this c o m p o u n d was very effective in increasing the uptake of nickel in fat. It appears that in this case there is no correlation between the uptake in fat and in the central nervous system. This has also been shown for highly lipophilic compounds such as DDT, dieldrin and dimethyl-mercury: these substances are accumulated to a high extent in the body-fat, but they are taken up in the central nervous system only to a moderate e x t e n t [16,17]. It can be concluded that lipid solubility is n o t the only factor determinating the uptake in the central nervous system. The autoradiography showed that the pattern of labelling of the kidneys was different in the animals given the chelating agents compared with only nickel. This indicates that the nickel at least partly reaches the kidneys in a chelated form and that the renal handling of the nickel and the nickelchelates is different. The results showed that some of the thiuram sulphides in the pregnant animals induced a very high uptake of nickel in the fetuses. DPTM and TETD, which were very efficient in increasing the uptake of nickel in the central nervous system of the adult animals, were the most efficient compounds in increasing the uptake in the fetuses. TBTD, which was relatively poor in increasing the uptake of nickel in the central nervous system of the adult animals, was much less effective than the preceding compounds in increasing the uptake of nickel in the fetuses. It appears that with these compounds a high uptake in the central nervous system is correlated to a high fetal uptake, and, inversely, a low fetal uptake coincides with a low uptake in the central nervous system. As mentioned in the introduction, SDC is used as an antidote in the
Fig. 4. Whole-body autoradiograms (A--C) o f mice on day 18 o f pregnancy killed 24 h after oral administrations o f 63Ni2+ (10 u m o l [0.58 m g ] / k g body wt; the amount of 63Ni~+ was 20 uCi/animal). (D) is an enlargement of (C), showing a fetus. Mouse (B) was also given TBTD and mouse (C) TETD (1 m m o l / k g b o d y wt) orally immediately after the administration of the 63Ni2+. Mouse (A) was only given the 6~Ni2÷ and the vehicle -corn oil -- used to dissolve the thiuram sulphides. In (C) a marked radioactivity is present in the fetus. Both the fetal and the maternal brain s h o w a high radioactivity. In (B) there is a much lower fetal labelling than in (C). The labelling o f the maternal brain in (B) is also much lower than in (C). In (A) most labelling is present in the kidney and the gastrointestinal contents of the mother.
311
treatment of nickel carbonyl poisoning in man [7]. The Ni u in nickel carbonyl [Ni(CO)4] is oxidized intracellularily to Ni 2+ [18]. The lung and the brain are the main target organs for nickel carbonyl poisoning in man, and the injurious effects are probably induced when the Ni 2+ is bound intracellularily in these tissues. The beneficial effect of SDC m a y be due to a removal of the Ni 2~ from the intraceUular binding sites [10]. However, as shown in the present study, and previously [9,10], the chelated Ni 2+ will be retained in the tissues. It is known that SDC may potentiate the neurotoxicity of thallium, probably by facilitating the uptake in the central nervous system [19]. Many thiuram sulphides and dithiocarbamates can cause central nervous system malfunction [3]. It has been proposed that an increased uptake of metals in the central nervous system induced by SDC or TETD may contribute to the noxious effects of these substances towards this tissue [20]. The results of the present study suggest that an increased uptake of metals in the central nervous system may be induced by several thiuram sulphides. A combined exposure to thiuram sulphides and metals, which can occur industrially, may constitute an increased risk for metal toxicity. It is known t h a t dithiocarbamates and thiurarn sulphides m a y give rise to teratogenic effects and e m b r y o t o x i c i t y [21--23]. Aaseth et al. [20] have shown t h a t SDC can increase the uptake o f mercury in the fetuses of mice. These authors suggested t h a t the noxious effects towards the fetuses of SDC and TETD may be due to an increased transfer of metals into foetal tissues. Nickel compounds have been shown to be embryotoxic and teratogenic under certain conditions [24]. It is possible that combined exposure to thiuram sulphides or dithiocarbamates and nickel may constitute a hazard for the fetus. ACKNOWLEDGEMENTS
This s t u d y was supported by the Swedish Work Environment Fund. We t h a n k Kenneth St~hl for assistance in the determinations of the chloroform/ water partition coefficients. REFERENCES 1 G.D. Thorn and R.A. Ludwig, The dithiocarbamates and related compounds, Elsevier Publishing Company, N e w York, 1962. 2 S.H. Morrell, The chemistry and technology of vulcanisation, in C.M. Blow and C. Hepburn (Eds.), Rubber Technology and Manufacture, 2nd edn., Butterworth Scientific,London, 1982. 3 I A R C Monographs on the evaluation of carcinogenic risk of chemicals to man, Vol. 12, Some Carbamates, Thiocarbamates and Carbazides, Lyon, 1976. 4 L. Lundwall and F. Baekeland, Disulfiram treatment of alcoholism. J. Nerv. Ment. Dis., 153 (1971) 381. 5 L. Eldjarn, The metabolism of tetraethylthiuramdisulfide (Antabuse, Aversan) in rat, investigated by means of radioactive sulphur. Scand. J. Clin. Lab. Invest., 2 (1950) 198.
312
6 E.H. StrSmme, Metabolism of disulfiram and diethyldithiocarbamate in rats with demonstration of an in vivo ethanol-induced inhibition of the glucuronic acid conjugation of the thiol. Biochem. Pharmacol., 14 (1965) 391. 7 F.W. Sunderman, Efficacy of sodium diethyldithiocarbamate (dithiocarb) in acute nickel carbonyl poisoning. Ann. Clin. Lab. Sci., 9 (1979) 1. 8 D. Spruit, P.J.M. Bougaart and G.J. De Jongh, Dithiocarbamate therapy for nickel dermatitis. Contact Dermatitis, 4 (1978) 350. 9 A. Oskarsson and H. Tj~flve, Effects of diethyldithiocarbamate and penicillamine on the tissue distribution of ~3NiCI~ in mice. Arch. Toxicol., 45 (1980) 45. 10 H. Tj~flve, S. Jasim and A. Oskarsson, Nickel mobilization by sodium diethyldithiocarbamate in nickel carbonyl treated mice. I A R C Scientific Publ., in press. 11 J. Aaseth, N.E. SSli and O. FSrre, Increased brain uptake of copper and zinc in mice caused by diethyldithiocarbamate. Acta Pharmacol. Toxicol., 45 (1979)41. 12 L.R. Cantilena, Jr., G. Irwin, S. Preskorn and C.D. Klaassen, The effect of diethyldithiocarbamate on brain uptake of cadmium. Toxicol. Appl. Pharmacol., 63 (1982) 338. 13 A.Y. Caran, Vulcanization. Part VII. Kinetics of sulfur vulcanization of natural rubber in presence of delayed-action accelerators. Rubber chem. Technol., 38 (1965) 1. 14 S. Uilberg, The technique of whole body autoradiography. Cryosectioning of large specimens. Sci. Tools (Special Issue) (1977) 2. ~, 15 L.S. Schanker, Passage of drugs across body membranes. Pharmacol. Rev., 14 (1962) 501. 16 J. B~'ckstrSm, E. Hansson and S. Ullberg, Distribution of C~4-DDT and C~4-dieldrin in pregnant mice determined by whole-body autoradiography. Toxicol. Appl. Pharmacol., 7 (1965) 90. 17 K. Ostlund, Studies on the metabolism of methyl mercury and dimethyl mercury in mice. Acta Pharmacol. Toxicol., 27 (Suppl. 1) {1969) 1. 18 A. Oskarsson and H. TiC'lye, The distribution and metabolism of nickel carbonyl in mice. Br. J. Ind. Med., 36 (1979) 326. 19 H.H. Kamerbeek, A.G. Raunos, M. ten Ham and A.N.P. van Heijst, Dangerous redistribution of thallium by treatment with sodium diethyldithiocarbamate. Acta Med. Scand., 189 (1971) 149. 20 J. Aaseth, J. Alexander and A. Wannag, Effect of thiocarbamate derivatives on copper, zinc and mercury distribution in rats and mice. Arch. Toxicol., 48 (1981) 29. 21 J.F. Robeus, Teratologic studies of carbaryl, diazinon, norea, disulfiram and thiuram in small laboratory animals. Toxicol. Appl. Pharmacol., 15 ( 1969) 152. 22 K.S. Larsson, C. Arnander, E. Lekanova and M. Kjellberg, Studies of teratogenic effects of the dithiocarbamates maneb, mancozeb and propioneb. Teratology, 14 (1976) 171. 23 A. Korhonen, K. Hemminki and H. Vainio, Embryotoxicity of industrial chemicals on the chicken embryo: Dithiocarbemates. Teratogenesis, Carcinogenesis, Mutagenesis, 3 (1983) 163. 24 F.W. Sunderman, Jr, M.C. Reid, S.K. Shen and C.B. Kevorkian, Embryotoxicity and teratogenicity of nickel compounds, in T.W. Clarkson, G.F. Nordberg and P.R. Sager (Eds.), Reproductive and Development Toxicity of Metals, Plenum Publ. Corp., 1983, pp. 399--416.
313
E F F E C T OF T H I U R A M SULPHIDES ON THE UPTAKE AND DISTRIBUTION OF NICKEL IN P R E G N A N T AND NONP R E G N A N T MICE
SUHAIR JASIMa and HANS TJ.~LVE b
aDepartment of Toxicology, University of Uppsala, Box 5 73, S-751 23 Uppsala (Sweden) and bDepartment of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Box 573, S-751 23 Uppsala (Sweden) (Received May 15th, 1984) {Accepted June 9th, 1984)
SUMMARY
Oral administration of 63Ni2+ together with thiuram sulphides {tetramethylthiuram disulphide, tetraethylthiuram disulphide, tetrabutylthiuram disulphide, dipentamethylenethiuram monosulphide or dipentamethylenethiuram tetrasulphide) or sodium diethyldithiocarbamate resulted in highly increased levels of 63Ni2÷ in several tissues of mice in comparison with animals given 63Ni2+ alone. Administration of these substances to pregnant animals induced increased levels of 63Ni2÷ in the fetuses. The uptake of 63Ni2÷ in the brains of both adults and fetuses was usually very markedly enhanced by these c o m p o u n d s -- dipentamethylenethiuram monosulphide and tetraethylthiuram disulphide being the most efficient c o m p o u n d s in this respect. Determination of the chloroform/water partition coefficients for nickel in the presence of thiuram sulphides or sodium diethyldithiocarbamate showed that these c o m p o u n d s are able to form lipophilic complexes with the metal. A facilitated penetration through the cellular membranes of the lipophilic complexes between nickel and these substances can explain the effects on the fate of the nickel. However, the partition coefficient for nickel in presence of sodium diethyldithiocarbamate was much higher than for the thiuram sulphides, but in spite of that, the effect of sodium diethyldithiocarbamate on the disposition of 63Ni2+ in the mice Address all correspondence to: Professor Hans Tj~ilve, Department of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Box 573, S-751 23 Uppsala, Sweden. Abbreviations: DPTM, dipentamethylenethiuram monosulphide; DPTT, dipentamethylenethiuram tetrasulphide; SDC, sodium diethyldithiocarbamate; TBTD, tetrabutylthiuram disulphide; TETD, tetraethylthiuram disulphide; TMTD, tetramethylthiuram disulphide. 0300-483X/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
297
was not more marked than for most of the thiuram sulphides. It has been shown that tetraethylthiuram disulphide undergoes a reductive fission in the gut to diethyldithiocarbamate, which is considered to be the active form of tetraethylthiuram disulphide. The marked effects on the disposition of the 63Ni2+ induced by the other thiuram sulphides examined in the present study suggest that a similar fission to chelating thiocarbamates will take place. However, the formation of lipophilic complexes with the original thiuram sulphides may contribute to the effects on the disposition of the 63Ni2+.
Key words: Nickel; Thiuram sulphides; Diethyldithiocarbamate; Chelating agents
INTRODUCTION Thiuram sulphides are widely used in industry, agriculture and medicine [1--4]. In rubber industry these substances are utilized as accelerators in the vulcanization process [2]. In agriculture they are used as fungicides and insecticides [3 ]. Tetraethylthiuram disulphide (disulfiram, Antabus® ) (TETD) is used in aversion therapy for chronic alcoholism [4]. It has been shown that TETD undergoes a reductive fission in vivo to diethyldithiocarbamate [5,6]. This probably takes place to a large extent already in the gut, and diethyldithiocarbamate is therefore considered to be the active form of TETD [6]. Diethyldithiocarbamate, as well as other dithiocarbamates, are chelating agents [1]. Based on this property sodium diethyldithiocarbamate (SDC) is used as a therapeutic agent in the treatment o f nickel carbonyl poisoning in man [7]. This substance has also been tried in human nickel hypersensitivity [ 8 ]. We have shown that SDC, given parenterally, will induce a redistribution of nickel in several tissues of mice exposed to nickel chloride or nickel carbonyl [9,10]. The complex between nickel and diethyldithiocarbamate is lipophilic [ 1] and the effects on the disposition of the nickel are probably due to a facilitated passage of the chelated nickel through the cellular membranes. Effects of SDC on the disposition of other metals, such as copper, zinc and cadmium, have also been shown [11,12]. It is possible that other thiuram sulphides, like TETD, may undergo a fission in the intestine to give chelating alkylthiocarbamates, which may influence the disposition of metals in the tissues. However, it is also possible that the thiuram sulphides by themselves may act as chelators. This has been shown for 2,2'
298
c%-c.2,
cs3-c%,
s
s... , s,
N-C
N-C-S'Na" /
c.3-cH2"
CH3-CH2 Sodium diethyldithioca rbamate
Ni
,c%-c% C-N
"s" ""s" "c%-c%
N ickel-bis { d iet hyldithioca rba mate )
(SDC)
c%.
s
s
I | / N-C-S-S-C-N
CH3
c%
CH_-CH,.
S S CH~-CH. N-C-S-S-C-N
~CH3
CH3- CH2
CH2-CH3
T e t r a e t h y l t h i u ram disulphide (TETD)
Tetramethylthiu ram disulphide (TMTD)
CH.C..C~C~
S
S
C%C%C%C%
N-C-S-S-C-N
c•-cN-cH2-c %
c~h-c.2-cN-c.3
T e t r a b u t y l t h i u r a m disulphide
(TBTD)
Q s
s
z-- x
-C-S-C- N.__]~ Dipentamethylenethiuram monosulphide (DPTM}
z--x
s
s /-- k
~__]N-C-S-S-S-S-C-N
]
Dipentamethylenethiuram tetrasulphide (DPTT)
Fig. 1. Structural formulae of the studied thiuram.sulphides and the sodium diethyldithioearbamate. The structure of the chelate between nieke| and diethyldithioearbamate is also shown.
these compounds, have been examined. Pregnant and non-pregnant mice have been used. The fate of the nickel has been studied in the non-pregnant mice and the passage of the nickel to the fetuses has been examined in the pregnant mice. To elucidate if the thiuram sulphides can form lipophilic chelates with nickel the chloroform/water partition coefficients for nickel without and together with thiuram sulphides have been determined. The substances studied are, besides TETD, tetramethylthiuram disulphide (TMTD), tetrabutylthiuram disulphide (TBTD), dipentamethylenethiuram monosulphide (DPTM) and dipentamethylenethiuram tetrasulphide (DPTT) (Fig. 1). All these compounds are used as rubber accelerators [2]. TMTD is also used as a crop fungicide [3]. As a comparison, experiments with SDC have also been included in the study. MATERIALS AND METHODS
Animals
Female C57BL mice were obtained from Anticimex AB, Stockholm, Sweden. They were fed a standard pellet diet (Ewos AB, SSdert/ilje, Sweden) 299
and were given tap water ad libitum. To obtain pregnant animals, mice were mated overnight. When a vaginal plug was found in the morning, it was considered as day 0 of pregnancy. (Delivery will usually take place on the night to day 19.)
Chemicals 63Ni2+ (spec. radioact. 10.4 mCi/mg Ni 2÷) was obtained from the Radiochemical Centre, Amersham, England. SDC, TMTD, TETD, TBTD, DPTM and DPTT were obtained from ICN Pharmaceuticals Inc., Plainview, NY, USA.
Experiments Animal experiments The mice were given 63Ni2÷ orally by gastric intubation and were then immediately given the thiuram sulphides or SDC, also orally by gastric intubation. The 63Ni2÷ was given together with non-labelled Ni 2÷ (NiC12 was used) in 0.1 ml physiological saline, so that each mouse received 10 pmol Ni2+/kg b o d y wt (0.58 mg Ni2+/kg b o d y wt). The SDC was dissolved in 0.2 ml physiological saline. The thiuram sulphides were dissolved in 0.2 ml corn oil. Each mouse was given 1 mmol/kg b o d y wt of the respective c o m p o u n d . Control mice were given the 63Ni2+ orally followed by 0.2 ml corn oil. Corn oil was also given to the mice which received the SDC, immediately after t h e administration of this substance. The following experiments were performed: 1. Non-pregnant mice were given the 63Ni2+ (2 pCi/animal together with non-labelled Ni 2*) and one o f the thiuram sulphides, SDC, or corn oil only (controls) and were then killed by CO2-asphyxiation, in groups of 4 animals, after 5 h, 24 h and 72 h. Various tissues were taken and dissolved in 1 ml o f Soluene 350® (Packard). Ten milliliters of a scintillation fluid consisting of 4.9 g PPO (2.5
300
SDC or corn oil only (controls). The mice were killed after 24 h. One animal was used per treatment. 4. Non-pregnant mice were given the 63Ni2+ (2 ~Ci/animal together with non-labelled Ni 2÷) and one o f t h e thiuram sulphides, SDC, or corn oil only (controls). They were then placed in groups of 4 animals in cages which permit collection of urine. The urine was collected after 24, 48 and 72 h and the radioactivity determined by liquid scintillation counting, using Instagel® (Packard) as scintillation fluid.
De termination o f chloroform/wa ter partition coefficien ts The chloroform/water partition coefficients of Ni 2÷ and of Ni 2+ in presence of thiuram sulphides or SDC, were determined. With the exception of SDC (see below) the following procedure was used: the 63Ni2+ was added to a solution with non-labelled NiC12 dissolved in 1/15 M phosphate buffer (pH 7.4) so t h a t the final concentration of Ni 2÷ was 0.0005 mmol/ml, and the a m o u n t of radioactivity was 0.25 ~Ci/ml. The respective thiuram sulphide was dissolved in chloroform in a concentration of 0.05 mmol/ml. Two-millilitre aliquots of the Ni2+-solutions and 2 ml of chloroform containing one of the thiuram sulphides or, for controls, chloroform without further addition, were mixed vigorously for 30 min. The samples were then kept for 4 days at room-temperature in capped test tubes. Preliminary experiments had shown that equilibrium had occurred at this interval. The a m o u n t of radioactivity in 200-~1 aliquots of both the organic and the aqueous phase was then determined by liquid scintillation counting using Instagel® (Packard) as scintillation fluid. For SDC the following procedure was used: the SDC was dissolved in the phosphate buffer in a concentration 0.055 mmol/ml. Of this solution, 1.8-ml aliquots were taken and 0.2 ml of buffer, containing 0.005 m m o l / m l of Ni 2÷ (63Ni2+ together with non-labelled NiCI2; 2.5 pCi 63Ni2+/ml) was added. The obtained 2-ml aliquots, which contained 0.05 m m o l / m l of SDC and 0.0005 m m o l / m l o f Ni 2÷ {with 0.25 pCi/ml of 63Ni2÷) were mixed with 2-ml aliquots of chloroform. The procedure described above for the thiuram sulphides was thereafter followed.
Statistical analysis For statistical analysis, Student's t-test was used. The level of significance was set at P ~ 0.0 5. RESULTS The administration of 63Ni2+ together with SDC or either of the thiuram sulphides to the non-pregnant mice resulted in very markedly increased levels of radioactivity in most tissues at all survival intervals in comparison with the animals given 63Ni2+ only (Table I). However, in some instances marked differences in the effects on the fate of the 63Ni2÷ were observed between the compounds. Generally, the levels of radioactivity in the liver, kidney, lung, fat and
301
to
¢.o
I
OF
THIURAM
SULPHIDES
AND
SDC
ON
THE
TISSUE-CONCENTRATIONS
OF
6~Ni ~÷ IN N O N - P R E G N A N T
MICE
detectable.
a M e a n ± S.E. f r o m 4 m i c e .
41±12 80 ~12 38 ~ 6 5 i 0.8 101 ± 7 19 ± 2 0 . 6 ± 0 .1
Liver Kidney Lung Brain Spinal cord Fat Serum c
72h
97 495 406 444 380 71 7
331 1874 674 436 533 245 95
1869 548 88 73 305 135
± 14 ~ 19 b ± 8b ~ 23 b ± 29 b ~ 15 b ± 0.3 b
± 52 b ± 150 b i 54 b ± 11 b ~ 58 b ± 26 b ± 28 b
± 302 b ± 174 b ± 11 b ± 9b z 48 b ± 33 b
458 ± 110 b
+3Ni2+ + SDC
86 206 146 1078 914 72 7
~ 20 ~ 34 b ~ 9b ± 139 b ± 105 b ± 11 b ± 0.5 b
59 b 84 b 28 b 89 b 73 b 30 4b
~ 142 b ± 48 b ~ 82 b ± 34 b ± 9b ± 43 b
240 ~ 752 ± 272 ~ 629 ~ 602 ± 92± 32 ~
2135 613 350 335 191 166
3776 ~ 241 l~
+3Ni2+ + TMTD
~ ± ~ ± ± ± ±
i i ± ± ± ± 164 b 304 b 81 b 208 b 147 b 120 b 45 b
294 b 107 b 130 b 59 b 128 b 59 b
334 b 253 b 11 b 9b 176 b 141 b
35 14 b 58 b 5b 6b 71 b d
~ 108 b ± 166 b • 98 b ± 7b ± 56 b ±262 b ± 22 b
~ ± ± z ~ ±
132-* 297 ~ 494 ~ 30 ± 33 ± 321 ± N .D .
828 1021 815 48 268 1033 82
2647 2726 55 43 654 1099
7886 ± 771 b
+3Ni2+ + TBTD
~ ~ ~ ~ ± ± ~
± ± ~ z z •
18 b 188 b 302 b 168 b 148 b 61 b 91 b
995 b 320 b 287 b 296 b 355 b 103 b
259 ~ 24 b 1890 ~169 b 1594 ~ 88 b 3027 • 279 b 2075 • 181 b 132 ± 29 b N.D. d
375 3701 2077 1970 1470 350 372
13641 3181 1830 1646 2705 1293
1681 ± 278 b
63Ni++ + DPTM
C The v a l u e s f o r s e r u m a r e p m o l / 1 0 0
209~ 2b 576i 25 b 614 i 25 b 1544 ~ 160 b 1533 ± 126 b 36 ~ 7b N.D. d
903 1996 688 1502 1343 555 172
2912 869 653 492 839 476
2312 i 232 b
63Ni2+ + TETD
b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s (P < 0 . 0 5 ) .
i 2. 5 ± 16 ~ 7.4 ± 0.1 ± 25 ± 9 ±0.1
38 240 102 2 100 63 3
Liver Kidney Lung Brain Spinal cord Fat Serum c
5 79 12 0.7 2. 5 3.4 7.5
24 h
± i ± ~ z ±
28 i
661 79 3 16 10 35
Liver
5 h
+3Ni2+
Tissue-concentration of ~3Ni2+ (pmol/100 m g wet tissue)a
Kidney Lung Brain Spinal cord Fat Serum c
Tissues
Survival interval
~1.
15 b
~ ~ ± ~ ~ ± ~
2b 96 b 8b 11 b 13 b 1b 6b
± 222 b z 43 b ~ 22 b z 13 b ± 17 ~ 34 b
dN.D., not
80 ± 14 292± 37 b 258 ~ 22 b 325 ~ 30 b 277 ~ 33 b 93 i 21 b N .D . d
59 1007 317 258 213 31 42
2518 445 147 76 49 445
151 t
+3Ni2+ + DPTT
Mice were given 6+Ni++ (10 u m o l [0.58 mg]/kg body wt; the a m o u n t of ++Ni ++ was 2 uCi/animal) orally by gastric intubation and were then immediately given the respective thiuram sulphide or S D C (1 m m o l / k g b o d y wt) also orally by gastric intubation. Control mice were given ++Ni 2÷ and the vehicle -- corn oil -- used to dissolve the thiuram sulphides. The mice were killed after 5, 24 and 72 h and the a m o u n t of ~+Ni ++ in the tissues was determined as described in Materials and Methods.
EFFECTS
TABLE
serum of the animals treated with these substances were the highest at the 5-h survival interval and decreased at 24 h and 72 h (Table I). In contrast, the levels of 63Ni2÷ in the brain and spinal cord generally increased with time and were the highest at 72 h. Among the studied compounds, DPTM generally induced the highest increase in the tissue-levels of Ni 2÷ at all survival intervals. Thus, at 5 h the concentrations of 63Ni2÷ in some tissues of the DPTM-treated animals were 100 times, or more, higher than in the controls. Markedly increased levels were also observed at 24 h and 72 h. At all survival intervals the highest relative increase was observed in the brain. In addition the concentrations of 63Ni2+ in the spinal cord of the DPTM-treated animals were very high. Especially TETD, and also TMTD, were in addition very efficient in increasing the tissue-levels of 63Ni2÷ at all intervals. SDC was usually somewhat less efficient in increasing the concentrations of 63Ni2÷ than the preceding compounds. DPTT was, in turn, less effective than SDC. DPTM, TETD, TMTD and SDC generally all induced the highest relative increase of 63Ni2÷ in the brain and spinal cord. TBTD, in contrast, was less efficient in increasing the concentration of 63Ni2÷ in the central nervous system. However, this c o m p o u n d was very effective in elevating the 63Ni2÷levels in fat and also in the liver and the kidney (Table I). In the pregnant mice, which were killed 24 h after the administrations, increased levels of 63Ni2÷ in the fetuses were induced by all compounds (Table II). DPTM and TETD, which were very efficient in increasing the 63Ni2÷-levels in the tissues of the non-pregnant mice, were also very efficient in increasing the levels of 63Ni2÷ in the fetal tissues. These 2 compounds induced very markedly increased levels of 63Ni2÷ in the brain of the adult animals and a corresponding marked increase in the concentration of 63Ni2÷ in the fetal brains was also observed. TMTD and SDC were generally less efficient in increasing the levels of 63Ni2÷ in the fetal tissues than DPTM and TETD. DPTT showed, in turn, a lower efficiency than these compounds. TBTD were among the least efficient compounds in increasing the fetal uptake of 63Ni2÷. The increase in concentration of 63Ni2÷ which was observed in the brains of the non-pregnant animals after the administration of TBTD was less expressed than for the other compounds and this correlated with a low increase in the levels of 63Ni2+ also in the fetal brains. The treatments with the thiuram sulphides and SDC resulted in very markedly increased amounts of urinary 63Ni2÷ (Table III). DPTM was the most efficient c o m p o u n d in this respect and DPTT was the least efficient compound. The whole-body autoradiography confirmed the results of the liquid scintillation countings (Figs. 2--4). Thus, markedly increased tissue-levels of 63Ni2÷ were observed both in the pregnant and the non-pregnant animals after the treatments with these compounds, in comparison with animals given 63Ni2÷ only. The increased concentrations of 63Ni2÷ in the central nervous system were very apparent in the animals treated with SDC, TMTD, TETD, DPTM and DPTT. An even labelling was observed in the grey and
303
SULPHIDES AND SDC ON THE TISSUE-CONCENTRATIONS
O F 63Ni~+ IN F E T U S E S A N D P L A C E N T A E
76 ± 13
Placentae
392 ± 40 b
8b 9b 35 b 12 b 9b 231 ± 22 b
115 ~ 19 b 8 1 ± 7b 159 ~ 9 b 112 i 23 b 108 ± 26 b
+ TMTD
+ SDC 165 i 90i 217 ± 140 i 174 ~
~3Ni2~-
~3Ni2+
444 ± 47 b
346, 61 b 1 7 4 ± 24 b 523 i 116 b 327 ~ 66 b 790 ± 181 b
6"Ni2+ + TETD
aMean ~ S.E. o f 4 d e t e r m i n a t i o n s o f material o b t a i n e d f r o m 4 p r e g n a n t mice. bSignificantly d i f f e r e n t f r o m c o n t r o l s (P < 0.05).
15 ± 1 20, 2 57 ~ 5 21 ~ 2 11 ~ 1
63Ni2+
T i s s u e ~ o n c e n t r a t i o n o f 63Ni2" ( p m o l / 1 0 0 m g w e t tissue) a
Whole f e t u s Liver Kidney Lung Brain
Fetal tissues
and Methods.
12 b 37 b 78 b l0 b 15 b 908 ~ 104 b
125 i 213± 402 ± 137 i 57 i
63Ni2+ + TBTD
820 • 71 b
513 z 45 b 2 3 6 ~ 22 b 685 z 88 b 403,47 b 891 + 81 b
63Ni~+ + DPTM
8b
6b 8b 7b 11 b
£
~ z i ±
240 ± 33 b
98 69 244 91 110
63Ni2+ + DPTT
Mice o n day 18 o f p r e g n a n c y w e r e given 6ZNi2* (10 ~ m o l [0.58 m g ] / k g b o d y w t ; t h e a m o u n t o f 63Ni2" was 3 ~Ci/animal) orally b y gastric i n t u b a t i o n and were t h e n i m m e d i a t e l y given t h e respective t h i u r a m s u l p h i d e or SDC (1 m m o l / k g b o d y w t ) also orally b y gastric i n t u b a t i o n . C o n t r o l mice w e r e given 63Ni2÷ and t h e vehicle - - c o r n oil - - used t o dissolve t h e t h i u r a m sulphides. T h e m i c e w e r e killed a f t e r 24 h. T h e fetuses a n d t h e p l a c e n t a e w e r e excised a n d dissected, a n d t h e a m o u n t o f 63Ni2÷ was d e t e r m i n e d as d e s c r i b e d in Materials
EFFECTS OF THIURAM OF PREGNANT MICE
T A B L E IT
O rJl
4591
Total excretion
44788
39840 2909 2039
~3Ni2+ + SDC
21099
17598 2809 692
6SNi2+ + TMTD
25459
19318 4924 1217
63Ni2+ + TETD
37665
19362 15995 2308
~Ni~, + TBTD
47276
30050 13636 3590
63Ni2+ + DPTM
11099
9988 846 265
63Ni1+ + DFIT
aMean urinary excretion per animal. Each group contained 4 animals and the combined urine excreted by these animals was collected.
4298 282 11
63Ni~÷
A m o u n t o f 63Ni2+ excreted (pmol/animal) a
24 48 72
(h)
Intervals
Mice were given 63Ni~+ (10 ~mol [0.58 mg]/kg body wt; the amount of 63Ni2+ was 2 ~Ci/animal) orally by gastric intubation and were then immediately given the respective thiuram sulphide or SDC (! mmol/kg body wt) also orally by gastric intubation. Control mice were given 63Ni2* and the vehicle - - corn off - - u s e d to dissolve the thiuram sulphides. Urine was collected after 24, 48 and 72 h and the amount of 63Ni~+ determined as described in Materials and Methods.
E F F E C T S OF THIURAM SULPHIDES AND SDC ON THE U R I N A R Y EXCRETION OF 63Ni2+
TABLE HI
Brain
Lung Contents of stomach
Tongue Brain
Lung
Tongue Brain Lung
Kidney
Intestinal contents Kidney
Liver Contents of stomach Kidney
Liver Intestinal contents Fig. 2. Whole-body autoradiograms of mice killed 24 h after oral administration of 63Ni 2+ (10 ~mol [0.58 mg]/kg body wt; the amount of ~3Ni2+ was 20 ~Ci/animal). Mouse (B) was also given DPTM and mouse (C) DPTT (1 mmol/kg body wt) orally immediately after the administration of the 63Ni2*. Mouse (A) was only given the 63Ni2+ and the vehicle -- corn oil -- used to dissolve the thiuram sulphides. In (A) most radioactivity is present in the gastro-intestinal contents. There is also a marked labelling of the kidney and a weak labelling of the lung. In (B) and (C) a marked radioactivity is present in several tissues. The labelling of the brain is very high in (B).
306
w h i t e m a t t e r o f t h e b r a i n a n d s p i n a l c o r d . T h e v e r y m a r k e d i n c r e a s e in t h e level o f 63Ni2+ in f a t w h i c h was o b s e r v e d in t h e l i q u i d s c i n t i l l a t i o n c o u n t i n g s in a n i m a l s t r e a t e d w i t h T B T D was also o b s e r v e d in t h e a u t o r a d i o g r a p h y . T h e a u t o r a d i o g r a m s o f a n i m a l s given 63Ni~+ o n l y , s h o w e d a r a d i o a c t i v i t y in t h e k i d n e y s w h i c h was l o c a l i z e d t o d i s t i n c t s p o t s in t h e c o r t e x . In t h e a n i m a l s t r e a t e d w i t h t h e t h i u r a m s u l p h i d e s o r SDC, t h e r e was a m o l e even d i s t r i b u t i o n o f r a d i o a c t i v i t y in t h e k i d n e y s . T h e a u t o r a d i o g r a p h y in t h e p r e g n a n t a n i m a l s given t h e t h i u r a m s u l p h i d e s o r SDC s h o w e d i n c r e a s e d u p t a k e o f 63Ni 2+ in t h e f e t u s e s ( F i g . 4). With DPTM and TETD a very marked labelling of the fetal central nervous system was seen, b u t t h i s was n o t o b s e r v e d w i t h T B T D . T h e c h l o r o f o r m / w a t e r p a r t i t i o n c o e f f i c i e n t s f o r Ni 2+ w i t h o u t a n d t o g e t h e r w i t h t h e t h i u r a m s u l p h i d e s a n d SDC a r e s h o w n in T a b l e IV. T h e p a r t i t i o n c o e f f i c i e n t s w e r e m u c h h i g h e r in all cases in t h e p r e s e n c e o f t h e s e c o m p o u n d s , in c o m p a r i s o n w i t h t h e d e t e r m i n a t i o n w i t h c h l o r o f o r m o n l y . In t h e p r e s e n c e o f SDC a l m o s t all o f t h e Ni 2+ was f o u n d in t h e c h l o r o f o r m . T h e t h i u r a m s u l p h i d e s s h o w e d l o w e r values: T M T D giving t h e h i g h e s t p a r t i t i o n c o e f f i c i e n t , w i t h m o r e t h a n 40% o f t h e Ni 2+ in t h e c h l o r o f o r m p h a s e ; T E T D giving t h e l o w e s t v a l u e , w i t h a b o u t 3% o f t h e Ni 2+ in t h e c h l o r o f o r m . W i t h o u t t h e s e c o m p o u n d s 9 9 . 9 5 % o f t h e Ni 2+ was p r e s e n t in t h e a q u e o u s p h a s e . TABLE IV EFFECTS OF THIURAM SULPHIDES AND PARTITION COEFFICIENT OF NICKEL
SDC ON THE CHLOROFORM:
WATER
Two-millilitre aliquots of buffer-solutions containing 0.0005 mmol/ml of Ni 2÷ were equilibrated for 4 days with 2-ml chloroform aliquots or with 2-ml chloroform aliquots containing 0.05 mmol/ml of the respective t h i u r a m s u l p h i d e s . T h e SDC was a d d e d t o the buffer-solutions in a concentration of 0.05 mmol/ml and equilibration was performed with chloroform for 4 days in the same way as for the thiuram sulphides. The amount of tracer - - 63Ni2+ -- in the buffer solutions was 0.25 uCi/ml. The ratio of the amount of nickel in the chloroform phase to the amount of nickel in the aqueous phase was considered the chloroform/water partition coefficient. The percentage of the total amount of nickel which was present in the chloroform phase was also calculated,a Substance
Partition coefficientb
% Ni 2. in the chloroform phase
c SDC TMTD TETD TBTD DPTM
0.0005 2308 0.694 0.034 0.051 0.127
0.05 99.96 40.97 3.29 4.85 11.27
± 0.0002 ± 467 ~ 0.037 ± 0.002 ~ 0.002 • 0.012
aDetermination of the chloroform/water partition coefficient of DPTT was not possible due to low solubility in chloroform. bMean ± S.E. of 4 determinations. CNo substance added to the chloroform.
307
Brain
Lung
Spinal cord
Brain
Liver Lun
Intestinal contents dney
Brain
Liver Lung
Intestinal contents Kidney
Liver
Contents of stomach and intestine ¢/!
..a .
.
Intestinal contents
308
Kidney
.
.
Abdominal fat
DISCUSSION The results of the present study have shown t h a t all t he studied thiuram sulphides have very m a r ked effects on the u p t a k e and distribution o f nickel in the mice. It was also f o u n d t h a t the thiuram sulphides are able to form lipophilic chelates with nickel. A facilitated p e n e t r a t i o n through the cellular membranes of lipophilic nickel-chelates probabl y explains the increased u p tak e in the tissues. This m a y be related directly to the f o r m a t i o n o f the lipophilic thiuram complexes. However, the possibility m e n t i o n e d in the i n t r o d u c t i o n , t h a t all the thiuram sulphides, as has already been shown for TETD, undergo a reductive fission in the intestine to chelating thiocarbamates, must also be taken into account. In s u p p o r t of the latter assumption are t he following conditions: (1) The c h l o r o f o r m / w a t e r partition coefficient was m u c h higher for the nickeld i e t h y l d i t h i o c a r b a m a t e - c o m p l e x t h a n f o r t h e c o m p l e x e s b e t w e e n nickel and the thiuram sulphides. It is well k n o w n t hat lipid solubility is a physical p r o p e r t y o f major i m p o r t a n c e f or the passage of molecules across cellular membranes [15]. However, the ability o f SDC to affect the u p t a k e and distribution o f nickel was n o t m o r e expressed than for m o s t o f the thiuram sulphides. This suggests t hat t he thiuram sulphides will undergo a reductive fission in th e intestine and t hat the f o r m a t i o n o f lipophilic complexes with the resulting thiocarbamates cont r i butes t o t he effects on the disposition o f the nickel. (2) The results showed no correlation bet w een the c h l o r o f o r m / water partition coefficients f or t he complexes between the nickel and t he thiuram sulphides and t he efficiency by which t he thiuram sulphides were able to increase the upt a ke of t he nickel in t he tissues. For example DPTM was mo r e effective in increasing t he u p t a k e o f nickel in m ost tissues than TMTD, despite a higher partition coefficient for t he latter than for t he f o r m e r c o m p o u n d . This is a f u r t h e r indication t hat the thiuram sulphides by themselves are n o t solely responsible for the effects on t he disposition o f t h e nickel -- f o r m e d t h i o c a r b a m a t e s p r o b a b l y also play an i m p o r t a n t role. Our results showed t ha t T E T D was m o r e effective than SDC in increasing the u p t a k e of nickel in m os t tissues. It is possible t h a t the effects observed with the T E T D are due to c o m p l e x - f o r m a t i o n with bot h the unchanged T E T D and the d i e t h y l d i t h i o c a r b a m a t e f o r m e d by reductive fission in the intestine and t hat the c o m b i n e d ef f ect t h e r e f o r e will be higher than when the SDC is given to t he mice.
Fig. 3. Whole-body autoradiograms of mice killed 24 h after oral administrations of 63Ni2+ (10 umol [0.58 mg]/kg body wt; the amount of 63Ni2* was 20 uCi/animal). Mouse (A) was also given SDC, mouse (B) TMTD, mouse (C) TETD and mouse (D) TBTD (1 mmol/kg body wt) orally immediately after the administration of '3Ni~÷. In (A), (B) and (C) there is a strong labelling of the central nervous system. In (D) the labelling of the brain is much lower. The labelling of fat is high in this animal. For comparison with an autoradiogram of an animal given 63Ni2" only: See Fig. 1A.
309
Llver
Fetal brain
310
Fstal liver
Fetal liver
Fetal kidney
Fetal brain
Fetal Intestines
DPTT was much less effective than the structurally related DPTM in increasing the uptake of nickel in the tissues. Conceivably the DPTM may more easily undergo a reductive fission to a chelating thiocarbamate than the DPTT. Most of the thiuram sulphides and the SDC induced a very high uptake of nickel in the central nervous system. The central nervous system is rich in lipids and has a high vascularization, and these factors m a y p r o m o t e the uptake of the lipophilic nickel chelates. TBTD showed the lowest ability of the tested compounds in increasing the uptake of nickel in the central nervous system. However, this c o m p o u n d was very effective in increasing the uptake of nickel in fat. It appears that in this case there is no correlation between the uptake in fat and in the central nervous system. This has also been shown for highly lipophilic compounds such as DDT, dieldrin and dimethyl-mercury: these substances are accumulated to a high extent in the body-fat, but they are taken up in the central nervous system only to a moderate e x t e n t [16,17]. It can be concluded that lipid solubility is n o t the only factor determinating the uptake in the central nervous system. The autoradiography showed that the pattern of labelling of the kidneys was different in the animals given the chelating agents compared with only nickel. This indicates that the nickel at least partly reaches the kidneys in a chelated form and that the renal handling of the nickel and the nickelchelates is different. The results showed that some of the thiuram sulphides in the pregnant animals induced a very high uptake of nickel in the fetuses. DPTM and TETD, which were very efficient in increasing the uptake of nickel in the central nervous system of the adult animals, were the most efficient compounds in increasing the uptake in the fetuses. TBTD, which was relatively poor in increasing the uptake of nickel in the central nervous system of the adult animals, was much less effective than the preceding compounds in increasing the uptake of nickel in the fetuses. It appears that with these compounds a high uptake in the central nervous system is correlated to a high fetal uptake, and, inversely, a low fetal uptake coincides with a low uptake in the central nervous system. As mentioned in the introduction, SDC is used as an antidote in the
Fig. 4. Whole-body autoradiograms (A--C) o f mice on day 18 o f pregnancy killed 24 h after oral administrations o f 63Ni2+ (10 u m o l [0.58 m g ] / k g body wt; the amount of 63Ni~+ was 20 uCi/animal). (D) is an enlargement of (C), showing a fetus. Mouse (B) was also given TBTD and mouse (C) TETD (1 m m o l / k g b o d y wt) orally immediately after the administration of the 63Ni2+. Mouse (A) was only given the 6~Ni2÷ and the vehicle -corn oil -- used to dissolve the thiuram sulphides. In (C) a marked radioactivity is present in the fetus. Both the fetal and the maternal brain s h o w a high radioactivity. In (B) there is a much lower fetal labelling than in (C). The labelling o f the maternal brain in (B) is also much lower than in (C). In (A) most labelling is present in the kidney and the gastrointestinal contents of the mother.
311
treatment of nickel carbonyl poisoning in man [7]. The Ni u in nickel carbonyl [Ni(CO)4] is oxidized intracellularily to Ni 2+ [18]. The lung and the brain are the main target organs for nickel carbonyl poisoning in man, and the injurious effects are probably induced when the Ni 2+ is bound intracellularily in these tissues. The beneficial effect of SDC m a y be due to a removal of the Ni 2~ from the intraceUular binding sites [10]. However, as shown in the present study, and previously [9,10], the chelated Ni 2+ will be retained in the tissues. It is known that SDC may potentiate the neurotoxicity of thallium, probably by facilitating the uptake in the central nervous system [19]. Many thiuram sulphides and dithiocarbamates can cause central nervous system malfunction [3]. It has been proposed that an increased uptake of metals in the central nervous system induced by SDC or TETD may contribute to the noxious effects of these substances towards this tissue [20]. The results of the present study suggest that an increased uptake of metals in the central nervous system may be induced by several thiuram sulphides. A combined exposure to thiuram sulphides and metals, which can occur industrially, may constitute an increased risk for metal toxicity. It is known t h a t dithiocarbamates and thiurarn sulphides m a y give rise to teratogenic effects and e m b r y o t o x i c i t y [21--23]. Aaseth et al. [20] have shown t h a t SDC can increase the uptake o f mercury in the fetuses of mice. These authors suggested t h a t the noxious effects towards the fetuses of SDC and TETD may be due to an increased transfer of metals into foetal tissues. Nickel compounds have been shown to be embryotoxic and teratogenic under certain conditions [24]. It is possible that combined exposure to thiuram sulphides or dithiocarbamates and nickel may constitute a hazard for the fetus. ACKNOWLEDGEMENTS
This s t u d y was supported by the Swedish Work Environment Fund. We t h a n k Kenneth St~hl for assistance in the determinations of the chloroform/ water partition coefficients. REFERENCES 1 G.D. Thorn and R.A. Ludwig, The dithiocarbamates and related compounds, Elsevier Publishing Company, N e w York, 1962. 2 S.H. Morrell, The chemistry and technology of vulcanisation, in C.M. Blow and C. Hepburn (Eds.), Rubber Technology and Manufacture, 2nd edn., Butterworth Scientific,London, 1982. 3 I A R C Monographs on the evaluation of carcinogenic risk of chemicals to man, Vol. 12, Some Carbamates, Thiocarbamates and Carbazides, Lyon, 1976. 4 L. Lundwall and F. Baekeland, Disulfiram treatment of alcoholism. J. Nerv. Ment. Dis., 153 (1971) 381. 5 L. Eldjarn, The metabolism of tetraethylthiuramdisulfide (Antabuse, Aversan) in rat, investigated by means of radioactive sulphur. Scand. J. Clin. Lab. Invest., 2 (1950) 198.
312
6 E.H. StrSmme, Metabolism of disulfiram and diethyldithiocarbamate in rats with demonstration of an in vivo ethanol-induced inhibition of the glucuronic acid conjugation of the thiol. Biochem. Pharmacol., 14 (1965) 391. 7 F.W. Sunderman, Efficacy of sodium diethyldithiocarbamate (dithiocarb) in acute nickel carbonyl poisoning. Ann. Clin. Lab. Sci., 9 (1979) 1. 8 D. Spruit, P.J.M. Bougaart and G.J. De Jongh, Dithiocarbamate therapy for nickel dermatitis. Contact Dermatitis, 4 (1978) 350. 9 A. Oskarsson and H. Tj~flve, Effects of diethyldithiocarbamate and penicillamine on the tissue distribution of ~3NiCI~ in mice. Arch. Toxicol., 45 (1980) 45. 10 H. Tj~flve, S. Jasim and A. Oskarsson, Nickel mobilization by sodium diethyldithiocarbamate in nickel carbonyl treated mice. I A R C Scientific Publ., in press. 11 J. Aaseth, N.E. SSli and O. FSrre, Increased brain uptake of copper and zinc in mice caused by diethyldithiocarbamate. Acta Pharmacol. Toxicol., 45 (1979)41. 12 L.R. Cantilena, Jr., G. Irwin, S. Preskorn and C.D. Klaassen, The effect of diethyldithiocarbamate on brain uptake of cadmium. Toxicol. Appl. Pharmacol., 63 (1982) 338. 13 A.Y. Caran, Vulcanization. Part VII. Kinetics of sulfur vulcanization of natural rubber in presence of delayed-action accelerators. Rubber chem. Technol., 38 (1965) 1. 14 S. Uilberg, The technique of whole body autoradiography. Cryosectioning of large specimens. Sci. Tools (Special Issue) (1977) 2. ~, 15 L.S. Schanker, Passage of drugs across body membranes. Pharmacol. Rev., 14 (1962) 501. 16 J. B~'ckstrSm, E. Hansson and S. Ullberg, Distribution of C~4-DDT and C~4-dieldrin in pregnant mice determined by whole-body autoradiography. Toxicol. Appl. Pharmacol., 7 (1965) 90. 17 K. Ostlund, Studies on the metabolism of methyl mercury and dimethyl mercury in mice. Acta Pharmacol. Toxicol., 27 (Suppl. 1) {1969) 1. 18 A. Oskarsson and H. TiC'lye, The distribution and metabolism of nickel carbonyl in mice. Br. J. Ind. Med., 36 (1979) 326. 19 H.H. Kamerbeek, A.G. Raunos, M. ten Ham and A.N.P. van Heijst, Dangerous redistribution of thallium by treatment with sodium diethyldithiocarbamate. Acta Med. Scand., 189 (1971) 149. 20 J. Aaseth, J. Alexander and A. Wannag, Effect of thiocarbamate derivatives on copper, zinc and mercury distribution in rats and mice. Arch. Toxicol., 48 (1981) 29. 21 J.F. Robeus, Teratologic studies of carbaryl, diazinon, norea, disulfiram and thiuram in small laboratory animals. Toxicol. Appl. Pharmacol., 15 ( 1969) 152. 22 K.S. Larsson, C. Arnander, E. Lekanova and M. Kjellberg, Studies of teratogenic effects of the dithiocarbamates maneb, mancozeb and propioneb. Teratology, 14 (1976) 171. 23 A. Korhonen, K. Hemminki and H. Vainio, Embryotoxicity of industrial chemicals on the chicken embryo: Dithiocarbemates. Teratogenesis, Carcinogenesis, Mutagenesis, 3 (1983) 163. 24 F.W. Sunderman, Jr, M.C. Reid, S.K. Shen and C.B. Kevorkian, Embryotoxicity and teratogenicity of nickel compounds, in T.W. Clarkson, G.F. Nordberg and P.R. Sager (Eds.), Reproductive and Development Toxicity of Metals, Plenum Publ. Corp., 1983, pp. 399--416.
313