Chem. Res. Toxicol. 1991, 4, 27-34
27
Articles Chirality, Charge, and Chain Branching Effects on Dithiocarbamate-Induced Mobilization of Cadmium from Intracellular Deposits in Mice Mark M. Jones,* Pramod K. Singh, Shirley G. Jones, Chetan R. Mukundan, and James A. Banton Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235
Glen R. Gale, Loretta M. Atkins, and Alayne B. Smith Veterans Affairs Medical Center and Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29403 Received May 22, 1990 The effects of three major structural features on the intracellular cadmium mobilizing potency of dithiocarbamates have been examined. These features, the chirality of the groups, the total ionic charge of the chelating agent, and the extent of chain branching, would be expected t o affect the pharmacological properties of these chelating agents but to have little effect on the stability constants of the cadmium complexes involved. A total of 25 compounds (including 21 new ones) was prepared and used in animal studies designed to evaluate these effects. These included a series of amphipathic dithiocarbamates of the general type R1N(R2)CSz-Na+,where R1 is a relatively nonpolar organic group and Rz is derived from a reducing hexose. All of the factors examined influenced the potency of dithiocarbamates in the mobilization of cadmium from intracellular deposits. T h e compounds with Rz= galactose or mannose and R1 = benzyl were both more effective than the corresponding glucose derivatives in inducing the removal of' cadmium from the liver and the whole body. Increases in the net negative charge of the chelating agent uniformly decreased the observed potency in the mobilization of hepatic and renal cadmium deposits. The replacement of a normal alkyl group by a branched-chain group of the same molecular weight also led to an increase in potency for the two pairs of compounds examined. Dithiocarbamates which are not amphipathic because of the presence of similar polar substituents for both R1 and R2, such as sodium diarabitylamine carbodithioate, were relatively ineffective as agents for the mobilization of intracellular cadmium. While the three factors examined were found t o be capable of exerting an influence on the potency of the compound in vivo, these effects were not as important as the requirements of having a n amphipathic structure, a balance between the polar and nonpolar groups that slightly favors the polar part, or a molecular weight near t o or in excess of 300.
Introduction Cadmium remaining in animals 2 days after parenteral administration is present almost exclusively in intracellular sites; much of this cadmium is bound to metallothionein and is inaccessible to many typical chelating agents (1-4). For this reason, studies on the effect of chelating agent structure on the mobilization of such cadmium are of special interest in that they provide information on the effect of structural changes on the relative penetration of the cellular membranes by the chelating agents. The information obtained should thus be useful in the design of chelating agents for the mobilization of any toxic metal *Address correspondence to this author a t the Department of Chemistry, Box 1583, Station B, Vanderbilt University, Nashville, T N 37235.
which is stored primarily in intracellular sites. The recently reported increments in the potency of dithiocarbamate compounds (also designated as N-carbodithioates) specifically prepared for the mobilization of intracellular cadmium (5-8) are of considerable interest because intracellular cadmium has been considered to be inaccessible to such agents (9). Earlier studes by other investigators (10-14) have shown the critical importance of the value of the stability constant (Kstabor Keff)for the complex formed by the chelating agent with the toxic metal ion in the determination of the potency of chelating agents for the removal of extracellular toxic metals. Experiments completed previously have explored the role of the relative balance of polar and nonpolar groups, expressed as the sum of the Hansch T factors for the individual groups present ( E T )and the molecular weight values (M)of the compounds on the cadmium mobilizing potentcy (6,8,15,16). 0 1991 American Chemical Society
28 Chem. Res. Toxicol., Vol. 4, No. I , 1991 HWH2-C
"-7- p"B -C
I
I
OH OH
C
C
I
CSzNa CHZ -N-
I
OH
H
CHPh
I
I
CSzNa
1 (BGDTC)
2 (LPDTC)
+
CS2Na
CSzNa
b C O N H *
6'
COONa 4 (LPADTC)
Jones et al.
5 (PNDTC)
3 (DPDTC)
coNH2 6 (PNADTO
F i g u r e I . Structures of N-benzyl-D-glucamine-N-carbodithioate (1) and heterocyclic carbodithioates (2-6).
The variation in the mobilizing potency as the molecular structures are varied is quite striking. Thus, in one pair of these compounds, the substitution of a hydrogen on the benzene ring by a methoxy group resulted in a change in the reduction of renal cadmium levels (in a standardized experiment) from 57% of the control values to 19% (6). The purpose of the present study was to examine the role of three other structural factors on this mobilization potency: the chirality of the mobilizing agent, the degree of branching present in one of the substituents on the nitrogen atom, and the net ionic charge on the chelating agent.
Materials and Methods Melting points were determined on a Thomas-Hoover stirred liquid apparatus. 'H NMR spectra were recorded on an IBM 300-MHz NMR spectrometer in D,O with DSS as an internal standard. The chemical shifts are reported in ppm (6). Elemental analyses were performed by Galbraith Laboratories, Knoxville, Tennessee. The analyses were generally within f0.40%0;the ones not within this range have been indicated in the footnotes to Tables I and 11. All the sugars were obtained from Sigma Chemical Co., and the amines were from Aldrich Chemical Co. Sodium N-benzyl-D-glucamine-N-carbodithioate(1, BGDTC), sodium L-proline-N-carbodithioate(2, LPDTC), sodium N-(n-amyl)-Dglucamine-N-carbodithioate (20a, n-AmGDTC), and sodium N-(4-methoxybenzyl)-~-glucamine-N-carbodithioate(20h, MeOBGDTC) were prepared as reported earlier (5-7, 15). G e n e r a l P r o c e d u r e f o r t h e P r e p a r a t i o n of D i t h i o c a r b a m a t e s . A methanolic solution of amine was treated with an equivalent amount of aqueous NaOH (about 40% solution) a t 0-5 "C followed by dropwise addition of an excess of CS2 in dioxane at the same temperature under nitrogen or argon. After 12-18 h of additional stirring the solvents were mostly evaporated under vacuum. Acetone was added to the concentrated residue, whereupon the dithiocarbamate separated out as a white to hazy
white solid. Further purification was achieved by charcoal treatment of the methanolic solution of the crude product and then precipitation of the product by the addition of concentrated solution to acetone or ethyl ether. ( a ) Heterocyclic C a r b o d i t h i o a t e s (3-6). T h e heterocyclic dithiocarbamates (3-6) were prepared on the basis of the same general procedure described above (Table 11). These dithiocarbamates are illustrated in Figure 1. (b) Sodium Di-D-(-)-arabitylamine-N-carbodithioate (9). This was obtained from di-D-arabitylamine (8) by the general procedure given above. T h e reaction sequence, starting from D-(-)-arabinose (7), is shown in Figure 2. The parent secondary amine 8 was successfully synthesized following a procedure reported for its corresponding L-isomer, di-L-arabitylamine ( I 7 ) . (c) S u b s t i t u t e d D-Galactamine-N-carbodithioates(13a-4, D-Mannamine-N-carbodithioates(16a-e), a n d D - G ~ u c a m i n e - N - c a r b o d i t h i o a t e s (20b-g). All these sugar-based dithiocarbamates (Table 11)were derived, according to the general procedure given above, from their parent glycamines (12a-e, 15a-e, and 19b-g;Table I); their structures are as shown in Figures 3-5. In a typical experiment equimolar quantities of a reducing sugar and a primary amine were treated in the presence of a small quantity of water a t 60 "C in an inert atmosphere (N, or Ar) to give a gel or sometimes an oil (imine). The crude imine on subsequent hydrogenation (45-50 psi) in methanol in presence of PtO, catalyst afforded the glycamine, as based on the preparation of similar compounds (6, 7, 18). A n i m a l Studies. (a) W i t h Radioactive Cadmium. Male mice of the B6D2Fl strain were obtained from the Frederick Cancer Research Center, Frederick, MD, and were housed in a facility fully accredited by the American Association for Accreditation of Laboratory Animal Care. After a I-week acclimation, mice in the weight range of 22-26 g were closely matched (k1.0 g) and given a single ip injection of 0.03 mg of CdCl2.2.5H20 in 1.0 mL of 0.9% NaCl solution containing 1.0 pCi of lWCdC12 (DuPont NEN Products, North Billerica, MA). The "Cd used was reactor produced with a specific activity of 3.06 pCi/pg. Thus, in the cadmium loading procedure in which mice were given 30 pg of CdCl2.2.5H20, they received about 15 pg of nonradioactive cadmium ion. The radioactive cadmium which was added to this (1.0 pCi) contributed only another 2% of carrier to the system, which was disregarded in all calculations. Mice were held under routine holding conditions for several days, with Wayne Lab Blox and tap water offered ad libitum. In this model, about 10% of the administered cadmium is excreted over the first 48 h after injection, but after this interval it is excreted a t a rate of only about 0.1% per day (19). For several weeks there is only nominal redistribution of cadmium, which consists primarily of a gradual shift of hepatic cadmium to the kidneys. However, the organ cadmium response to treatment is consistent over a t least 60 days after cadmium injection (20,21). Descriptions of each treatment regimen are given in the footnotes of Tables 111-VII. Whole body y counts were performed by use of Canberra modular y detection instrumentation (19), and organ y counting was done with a Beckman Biogamma I scintillation well counter. Standard "Cd
7 @-(-)-ARABINOSE)
8
9 CSARDTO
F i g u r e 2. Preparation and structure of diarabitylamine carbodithioate (9).
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 29
Dithiocarbamate-Induced Mobilization of Cadmium H
OH
OH H
I
I
I
I I I I HOCHz-C-C-C-C-CHz-NOH H
H
R
I
I
OH
CSzNa
a, R = C H g h
13s 13b 13c 13d
(BGADTC) (McOBGADTC) (HHxGADTC) (n-HxGADTC) 13e @MBGADT€!)
b, R = CHzC6Hq.pOde c, R = (CHzkOH d, R = (CH2)sCHs e, R = CH(CH~)CHZCH(CH~~
Figure 3. Structures of substituted galactamine carbodithioates (13a-e).
i i YT" - - -
HOCHZ
C
C -C
I
I
C
I
OH OH H
16a 16b 16c 16d 16e
CHz-N-
I
R
I
CSzNa
H
a, R = CH2Ph
(BMNDTC) (MeOBMNDTC) (HHxMNDTC) (n-HxMNDTC) OMBMNDTC)
b, R = C H 2 W . , O M e R = (CH2)rjOH d, R = (CH2)5CH3 e, R = CH(CH~)CH~CH(CH~)Z C,
Figure 4. Structures of substituted mannamine carbodithioates (16a-e). HOCHz-
. C-
. C-
. C-
. C
I
I
I
I
OH OH
H
-CH2 -N-
OH
R
I
CSzNa
20a (n-AmGDTC) 2 0 b (3-AmGDTC) 2Oe (n-HpGDTC)
20d (2-HpGDTC) 20e (HRGDTC) 2 0 f (HBuGDTC) 2 0 g (HAmGDTC) 20h (MeOBGDTC)
Figure 5. Structures of substituted glucamine carbodithioates (20a- h). phantoms and vials prepared on the day cadmium was injected permitted calculation of residual cadmium in whole bodies and in individual organs. (b) With Nonradioactive Cadmium. Male ICR mice (Harlan Industries, Indianapolis, IN) weighing 30 h 2 g were allowed a 1-week acclimation period after receipt and were then given a total of 10 mg/kg CdCl2-2.5H20in a series of four injections on each of four successive days a t 1, 3, 3, and 3 mg/kg ip, dissolved in 0.5 mL of 0.9% NaC1. After a 3-day interval the mice were divided into groups and were given a daily injection of 0.40 mmol/kg ip of chelating agent each day for 5 days, in 0.5 mL total volume. After a further 2-day interval the mice were sacrificed and dissected to obtain organ samples for cadmium analyses. Tissues were digested in pure nitric acid and were analyzed for cadmium by using atomic absorption spectrometry. Throughout the experiments the mice were housed in an AAALAC-approved facility. Statistical significance of differences between experimental groups was assessed by one-way analysis of variance followed by Duncan's multiple range test. All tabulated data are given as means f standard deviations.
Results The structures of the dithiocarbamates prepared in the present study are presented in Figures 1-5. Figure 1 shows the structures of BGDTC (1) and those of the heterocyclic dithiocarbamates (3-6) prepared in one-step reactions. Figure 2 shows the preparation of the dithiocarbamate in which two arabityl groups are present (9, BARDTC), and
Table I. Characterization of Substituted Glycamines 8, 12a-3, 15a-3, and 19a-g compd no. 8 12a 12b 1 2 ~ 12d 12e 15a
formula (anal.) CioHzaNOs (C, H, N) Cl3HZ1NO5 C i & J ' J O 6(C, H, N) C12H27N06 (C, H, N) C12H27NOs (C, H, N) C12H27N05(C, H, N) C13H21N05(C, H, N) 15b C14Hz3N08 (C, H, N) 1 5 ~ C12H27NOs.O.2H20 (C, H, N) 15d CizH27N05 (C, H, N) 15e C12H27N05(C, H, N) 19a CllH25N05 19b CiiH2~N05(C, H, N) 1% Ci3Hz7N05 (C, H, N) 19d C,3HZ7NO5*0.6H20(C, H, N) 19e CgHzlNOs (C, H, N) 19f CioHz3NO6 (C, H, N) 1913 CiiHz5N06 (C, H, N)
vield." %
m n "C
60.3 60.0 83.7 69.4 79.7 61.6 55.4 80.1 79.3
177-178.5 157-158' 159-160.5 142-144 152-154 110 (foaming) 166-167 153-154.5 151-152.5
86.9 67.9 60.7 80.8 92.0 76.7
148-149 115-118 134-135' 116-117 128-129 104-106
74.7 68.6 73.5
120-121.5 120.5-122.5 133.5-136
"Refers to the yields after recrystallization. *Lit. mp 157-159 "C (Kagan et al., 1957). 'Lit. mp 131-132 "C (Gale et al., 1988).
Figures 3, 4, and 5 show the structures of the dithiocarbamate derivatives (13a-e, 16a-e, 20a-h) of glucose, mannose, and galactose, respectively. The physical and chemical data used to characterize the glycamines synthesized (8,12a-e, 15a-e, 19a-g) are presented in part in Table I. The synthetic procedures used here were effective in yielding good quantities of these intermediates in a state of excellent purity as shown by the analytical data indicated in Table I. Not shown are the 'H NMR data on the parent amines which were also in accord with the expected structures. More detailed physical and chemical data on the dithiocarbamates prepared (9, 13a-e, 16a-e, 20a-g) are shown in Table 11. Here again, the products were usually obtained in good yields and in a state of excellent purity as shown by the analytical data indicated in Table 11. The examination of compounds that differ only in the conformation of one carbon atom (1, 13a, 16a, and 2, 3) provides the results shown in Table 111. The first three compounds form one set of this sort derived from glucose (BGDTC), galactose (BGADTC), and mannose (BMNDTC). The derivatives of galactose and mannose are significantly more effective in causing a reduction in the amount of cadmium retained in the liver, though such differences are not found for the other organs examined. The dithiocarbamates derived from the two isomeric forms of proline (LPDTC and DPDTC) exhibit no significant differences under the conditions used here. It is worth noting that none of these compounds induces an increase in the cadmium content of the brain. The effect of using isomeric alkyl groups which differ only in the degree of chain branching can be seen from the data presented in Table IV, where data on two pairs of such compounds (20a,b and 20c,d) are presented and compared with both control animals and those treated with the benzyl compound, BGDTC. In each pair we note that the dithiocarbamate containing a branched-chain alkyl group, i.e., 3-AmGDTC and 2-HpGDTC, provides a compound that is significantly more effective in inducing a reduction of the cadmium content of the whole body, the kidneys, and the liver. In the case of the two amyl derivatives, n-AmGDTC and 3-AmGDTC, the significance of these differences also extends to the differences in the cadmium levels of the spleen and testes. The final two
Jones et al.
30 Chem. Res. Toxicol., Vol. 4, No. 1, 1991 Table 11. Characterization of Dithiocarbamates 3-6.9, 13a-e, 16a-e. a n d 20a-I? compd no. 3
'H NMR (D20/DSS) 4.84 (s) and 4.81 (d, J = 2.7Hz) (1 H), 3.96-3.79 (m, 2 H), 2.38-2.22 (m, 1 H), 2.08-1.95 (m, 3 H) 252-253 5.00-4.97 (dd, J = 8.6, 2.1, 2.8 Hz, 1 H), 4.04-3.83 (m, 2 H), 2.48-2.35 (m, 1 H), 2.16-2.00 (m, 3 H) 5.38 (d, J = 12.6 Hz, 2 H), 3.25 (doublets o f t , 350-353 J = 12.7, 2.6 Hz, 2 H), 2.55-2.45 (m, 1 H), 1.93-1.89 (dd, J = 13.6, 3.2 Hz, 2 H), 1.60 (doublets of q, J = 13.3, 3.7 Hz, 2 H) 5.53 (d, J = 12.8 Hz, 2 H), 3.19 (doublets o f t , 289-290 J = 12.6, 2.6 Hz, 2 H), 2.73-2.63 (m, 1 H), 1.93 (dd, J = 12.8, 2.6 Hz, 2 H), 1.65 (doublets of q, J = 12.4 Hz, 2 H) 4.66-4.61 (dd, J = 12.1, 4.9 Hz, 2 H), 4.47 (t, J >80' = 13.3 Hz, 2 H), 4.05-3.91 (dd, br, 2 H), 3.87-3.83 (2 d, J = 2.6, 2.5 Hz, 2 H), 3.77-3.75 (m, 2 H), 3.68-3.62 (m, 2 H), 3.49 (d, J = 8.5 Hz, 2 H) 7.45-7.26 (m, 5 H), 5.76 (d, J = 16.0 Hz, 1 H), 110-113' 5.23 (d, J = 16.0 Hz, 1 H), 4.48-4.05 (m, 2 H), 4.05-3.95 (m, 2 H), 3.70-3.59 (m, 4 H) 189-1 91 7.26 (d, J = 8.3 Hz, 2 H), 6.99 (d, J = 8.3 Hz, 2 H), 5.69 (d, J = 15.3 Hz, 1 H), 5.15 (d, J = 15.3 Hz, 1 H), 4.47-4.35 (m, 2 H), 4.03-3.85 (m, 2 H), 3.82-3.56 (m, 9 H) 110-1 15-f 4.49-4.38 (m, 2 H), 4.26-4.20 (m, 1 H), 4.07-3.95 (m, 2 H), 3.39-3.76 (m, 1 H), 3.71-3.67 (m, 3 H), 3.61-3.56 (m, 3 H), 1.76 (quintet, 2 H), 1.56 (quintet, 2 H), 1.42-1.30 (m, 4 HI 148-150 4.49-4.38 (m, 2 H), 4.28-4.19 (m, 1 H), 4.07-3.95 (m, 2 H), 3.88-3.78 (m, 1 H), 3.70-3.55 (m, 4 H), 1.75 (m, br, 2 H), 1.31 (9, br, 6 H), 0.86 (t, br, J = 6.4 Hz, 3 H) 5.97-5.90 (m, 1 H), 4.34-4.21 (m, 2 H), 3.98 (t, 85-f J = 6.4 Hz, 2 H), 3.69-3.65 (m, 4 H), 1.60-1.33 (m, 3 H), 1.20-1.15 (m, 3 H), 0.96-0.89 (m, 6 H) 184-185 7.45-7.26 (m, 5 H), 5.73 (d, J = 16.0 Hz, 1 H), 5.36 (d, J = 16.0 Hz, 1 H), 4.43 (d, J = 14.3 Hz, 1 H), 4.30 (t, J = 8.37 Hz, 1 H), 4.14 (q, J = 11.5 Hz, 1 H), 3.85-3.61 (m, 5 El) 7.25 (d, J = 8.6 Hz, 2 H), 7.00 (d, J = 8.6 Hz, 190-192 2 H), 5.67 (d, J = 15.5 Hz, 1 H), 5.29 (d, J = 15.5 Hz, 1 H), 4.38 (d, J = 13.9 Hz, 1 H), 4.28 (doublets o f t , J = 8.4, 2.1 Hz, 1 H), 4.13 (dd, J = 11.2, 8.7 Hz, 1 H), 3.89-3.72 (m, 7 H), 3.67-3.62 (m, 1 H) 95-100' 4.41 (d, J = 11.2 Hz, 1 H), 4.28-4.20 (m, 3 H), 3.99 (quintent, 1 H), 3.87-3.75 (m, 4 H), 3.68-3.57 (m, 3 H), 1.75 (quintet, 2 H), 1.56 (quintet, 2 H), 1.43-1.30 (m, br, 4 H) 150-1 51.5 4.41 (d, J = 11.6 Hz, 1 H), 4.29-4.16 (m, 3 H), 4.03-3.93 (m, 1 H), 3.88-3.73 (m, 4 H), 3.68-3.62 (m, 1 H), 1.73 (quintet, 2 H), 1.31 (s, br, 6 H), 0.86 (t,br, J = 7.0 Hz, 3 H) 6.00-5.86 (m, 1 H), 4.23-4.01 (m, 2 H), 90' 3.88-3.62 (m, 6 H), 1.63-1.17 (m, 3 H), 1.19 (t, J = 6.7 Hz, 3 H), 0.97-0.89 (m, 6 H) 163-165 4.46-4.42 (dd, J = 13.7, 5.6 Hz, 1 H), 4.37-4.25 (m, 2 H), 3.97-3.73 (m, 6 H), 3.67-3.62 (m, 1 H), 1.78-1.68 (quintet, 2 H), 1.37-1.26 (m, 4 H), 0.88 (t, J = 6.8 Hz, 3 H) 173-175 5.78-5.65 (m, 1 H), 4.27-4.24 (m, 1 H), 4.20-4.00 (m, 2 H), 3.87-3.56 (m, 5 H), 1.75-1.46 (m, 4 H), 0.86 (t, J = 6.0 Hz, 6 H) 4.46-4.42 (dd, J = 13.7, 4.6 Hz, 1 H), 4.36-4.25 155-157 (m, 2 H), 3.97-3.72 (m, 6 H), 3.68-3.62 (m, 1 H), 1.76-1.68 (m, 2 H), 1.29-1.27 (m, 8 H), 0.86 (t, J = 6.5 Hz, 3 H) 5.83-5.79 (m, 1 H), 4.36-4.15 (m, 2 H), 3.98-3.94 (m, 172-174 1 H), 3.83-3.75 (m, 5 H), 3.66-3.60 (m, 1 H), 1.63-1.49 (m, 1 H), 1.36-1.18 (m, 9 H), 0.85 (t, J = 6.1 Hz, 3 H)
yield, 90 mp: "C >300 83.2
abbrev. DPDTC
formula (anal.)O C6H7NNa202S2.3H20 (C, H, N, S)
4
LPADTC
C6HgN2NaO2S2.0.5H20 (C, H, N, S)
45.8
5
IPND'K
C,H9NNa2O2S2.2.9H20(C, H, N S)
89.6
6
IPNADTC
C,H,,N2Na0S2.2.2H20 (C, H, N, S)
81.0
9
BAHDTC
C,,H2zNNa08S2.2H20(C, H, N, S)
71.4
13a
BGAUI'C
C14H20NNa05S2.1.2H20 (C, H, N, Sb)
73.5
13b
MeOBCADTC
Cl5HzzNNaO6S2(C, H, N, S)
85.5
13c
HHxGADTC
13d
n-HxGADTC
Cl3Hz6NNaO5S2.lH20 (C, H, N, S)
85.6
13e
DMBCADTC
C13H2,NNa05S2.1H20(C, H, N, S)
76.2
16a
BMNUI'C
67.2
16b
MeOBMNDTC
80.4
16c
HHxMNDTC
94.9
16d
n-HxMNDTC
C13H26NNa05S2.0.5H20 (C, H, N, S)
75.9
16e
DMBMNDTC
C13H26NNa05S2.1H20 (C, H, N, S)
62.8
20a
ti- AmGDTC
C,2H24NNa05S2.1Hz0
86.3
20b
8-AmCDTC
ClZHz4NNa05S2~1.4Hz0 (C, H, N, Sc)
69.3
20C
u-HpGDTC
C14H2,NNa05S2.1H20(C, H, N, S)
93.7
20d
2-HpGDTC
C,,Hz8NNaO5S2.0.65H20 (C, H, N,d S)
72.0
83.3
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 31
D i t h i o c a r b a m a t e - I n d u c e d Mobilization of C a d m i u m
Table I1 (Continued) compd no. 20e
abbrev. HPrGD'I'C
formula (anal.)" ClnH,nNNaOfiSq*0.75H?0 (C, H, N, S) _- -_ _
20f
HBuGDTC CllH22NNaO& (C, H, N, S)
2Og
HAmGDTC C,2Hz,NNaO~Sz~0.75H20 (C, H, N, S)
yield, % mp,e "C IH NMR (D,O/DSS) 78.5 120-125 4.47-4.34 (m, 3 H), 4.03-3.88 (m, 2 H), 3.83-3.74 (m, 4 H), 3.62 (t, J = 6.0 Hz, 3 H), 2.02-1.93 (m, 2 H) 90.0 153-155 4.47-4.43 (dd, J = 13.7, 4.1 Hz, 1 H), 4.37-4.29 (m, 2 H), 3.97-3.73 (m, 6 H), 3.62 (t, J = 6.5 Hz, 3 H), 1.84-1.74 (quintet, 2 H), 1.61-1.51 (quintet, 2 H) 86.0 158-160 4.46-4.42 (dd, J = 13.5,4.3 Hz, 1 H), 4.37-4.27 (m, 2 H), 3.97-3.58 (m, 9 H). 1.81-1.71 (quintet, 2 H), 1.61-1.54 (quintet, 2 H), 1.40-1.30 iquintet, 2 H)
"Anal. within f0.4070. b S , calcd 16.40 (found 15.82). 'S,calcd 17.11 (found 16.27). d N , calcd 3.60 (found 4.03). eMelting with decomposition. 'Melts with foaming.
group control BGDTC ( I ) BGADTC (13a) BMNDTC (1Ga) LPDTC (2) DPDTC (3)
Table 111. Effect of Chirality on the Cadmium Mobilization Potency of Dithiocarbamates" whole body cadmium, % organ cadmium, pg of Cd/g of tissue wet weight cadmium administered kidneys liver spleen testes pancreas 0.201 f 0.030 2.01 f 0.17 3.55 f 0.17 6.29 f 0.17 0.639 f 0.086 86.28 f 4.51 0.140 f O.0Ogb 1.94 f 0.30 1.02 f 0.14b 2.47 f O.7gb 0.481 f 0.108b 43.55 f 6.6gb 0.159 f O.02gb 1.94 f 0.25 1.06 f 0.18b 1.68 f 0.43b 0.531 f 0.153b 38.47 f 9.13b 0.155 f 0.018b 1.89 f 0.24 1.13 f 0.12b 1.40 f 0.35b 0.467 f 0.051b 34.46 f 4.48b 0.170 f 0.012b 1.91 f 0.15 2.32 f 0.18b 6.07 f 0.38 0.456 f 0.074b 81.17 f 3.32 0.166 f O.03lb 1.91 f 0.23 2.27 f 0.1gb 6.08 f 0.67 0.429 f O.lOOb 78.00 f 3.41b interarour, Comparison kidneys liver d BGDTC (1) vs BGADTC (13a) NS' BGDTC (1) vs BMNDTC (16a) d NS NS BGADTC (13a) vs BMNDTC (16a) NS
brain 0.034 f 0.004 0.028 f 0.003b 0.031 f 0.005 0.029 f 0.003b 0.032 f 0.002 0.034 f 0.003
" 1.0 mL of a 0.9'70 NaCl solution containing 0.03 mg CdClZ.2.5Hz0and 1.0 pCi of "CdC12 was given ip to each mouse (B6D2F1) on day 0. On days 10, 12, and 14, mice received BGDTC ( l ) , BGADTC (13a), and BMNDTC (16a) a t a dose of 2.0 mmol/kg ip in 0.9% NaCl solution. r.PDTC (2) and DPDTC (3) were given at a dose of 2.0 mmol/kg on days 10, 12, 14, 16, and 18 a t the same dosage. On day 20, whole body cadmium levels were measured, and the mice were sacrificed immediately for determination of organ cadmium concentrations. Each group contained 7 mice. bSignificantly different from control; p I0.05. 'NS, not significant. d p 5 0.05. Table IV. Effect of Chain Branching on Whole Bods and SDecific Organ Cadmium Mobilization PotencY" whole body cadmium, % organ cadmium, pg of Cd/g of tissue wet weight cadmium group administered kidneys liver spleen testes pancreas brain 93.13 f 1.74 3.51 f 0.23 0.180 f 0.009 5.19 f 0.27 0.378 f 0.070 1.89 f 0.14 0.030 f 0.003 control 1.04 f 0.27b 1.84 f 0.68b 0.391 f 0.083 0.151 f 0.013b 1.99 f 0.19 BGDTC ( 1 ) 38.92 f 7.41b 0.029 f 0.002 45.83 f 4.41b 1.99 f 0.37b 2.30 f 0.62b 0.505 f 0.060b 0.170 f O.O1lb 1.90 f 0.19 0.029 f 0.003 n-AmCDTC (20a) 27.81 f 2.20b 0.66 f O . l l b 0.72 f 0.16b 0.360 f 0.077 0.147 f 0.014b 1.90 f 0.19 0.027 f 0.004 3-AmCDTC (20b) 51.66 f 10.4gb 3.00 f 0.18b 0.028 f 0.005 n-HpCDTC (20c) 2.55 f 0.80b 0.531 f 0.130b 0.155 f 0.O1gb 1.96 f 0.31 36.09 f 3.52b 2.79 f O.3Ob 1.15 f 0.31b 0.443 f O.05gb 0.161 f 0.014b 2.10 f 0.18b 0.029 f 0.003 2-HpGDTC (20d) intergroup comparison whole body kidneys liver spleen testes pancreas brain C NSd NS C C c c n-AmGDTC (20a) vs 3-AmGDTC (20b) ) L - H ~ C D T(C2 0 ~vs ) 2-HpGDTC (20d) c NS C C NS NS NS ~
~~
~~~~
1.0 mL of a 0.9% NaCl solution containing 0.03 mg CdCl2.2.5HZ0and 1.0 pCi of "T!dC12 was given ip to each mouse (B6D2F1) on day 0. On days 16, 18, and 21, each mouse was given 2.0 mmol/kg of the indicated compound ip in 0.9% NaCl solution. On day 23, whole body cadmium levels were measured, and mice were sacrificed immediately for determination of organ cadmium concentrations. There were 8 mice in each group. " p 5 0.05. dNS, not significant.
compounds in Table IV increase the cadmium levels in both the spleen and the pancreas. The effect of the ionic charge of the dithiocarbamate on its cadmium mobilizing ability has been examined in two pairs of compounds, 2,4 and 5,6 (Table V). In both cases the dithiocarbamates derived from the carboxylic acid and the corresponding amide are compared. The dithiocarbamates derived from the amides, LPADTC and IPNADTC, were significantly more effective than those derived from the corresponding carboxylic acid, LPDTC and IPNDTC, in effecting a reduction in the cadmium content of the whole body and the kidneys, and for one of these pairs there was a similar difference for the reduction in liver cadmium levels. It is of interest to note that significant differences were also noted for the spleen, testes, and pancreas for one or more pairs of such compounds, and in each case the amide was the more effective com-
pound. Also, none of these compounds induced an increase in the cadmium content of the brain. The use of enantiomeric groups with other structures was examined for the methoxybenzyl derivatives of three hexoses, MeOBGDTC, MeOBGADTC, a n d MeOBMNDTC (Table VI), to determine if the trends observed with the benzyl compounds (1,13a,16a) (Table 111)were of any marked generality. The differences found were less striking and appeared to be dose dependent, as can be seen clearly from the whole body, kidney, and liver data. The same type of comparison was undertaken for the compounds 13c-e and 16c-e (Table VII) to determine if the differences found for chain branching and the presence of hydrogen-bonding groups (6) were of any generality. Here again the results were less striking and were organ dependent. The introduction of a branched chain (DMBGADTC and DMBMNDTC) for a straight
32 Chem. Res. Toxicol., Vol. 4, No. I , 1991
Jones et a / .
Table V. Comparison of Carboxylic Acid and Carboxamide Groups on Cadmium Mobilization Potency of Dithiocarbamates" whole body cadmium, % organ cadmium, bg of Cd/g of tissue wet weight cadmium uoua administered kidnevs liver saleen testes aancreas brain 0.409 f 0.061 0.183 f 0.026 2.11 f 0.20 0.031 f 0.003 3.38 f 0.21 5.79 f 0.30 control 91.19 f 4.37 BGDTC ( I ) 18.99 f 1.94b 0.396 f 0.052b 0.300 f 0.074b 0.318 f 0.125b 0.116 f 0.012b 1.64 f 0.33b 0.024 f 0.003b 0.161 f 0.017b 1.90 f 0.17 0.029 f 0.002 5.60 f 0.57 0.363 f 0.052 LPDTC (2) 81.19 f 5.15b 2.02 f 0.02b 0.297 f 0.052b 0.112 f 0.014b 1.85 f 0.17b 0.029 f 0.003 5.88 f 0.45 LPADTC (4) 75.17 f 2.65b 1.83 f 0.20b 0.137 f O.0Ogb 1.94 f 0.16 0.030 f 0.003 5.88 f 0.41 0.366 f 0.050 82.98 f 3.62b 2.35 f 0.16b IPNDTC ( 5 ) 4.54 f 0.4gb 0.293 f 0.097b 0.118 f 0.026b 1.53 f 0.18b 0.028 f 0.003b 63.88 f 9.00b 1.08 f O.2lb IPNADTC ( 6 ) intergroup comparison whole body kidneys liver spleen testes pancreas LPDTC(Z) vs LPADTC (4) c c NSd C C NS IPNDTC ( 5 ) vs IPNADTC (6) C C c C C c a 1.0 mL of a 0.9% NaCl solution containing 0.03 mg CdCl2.2.5Hz0 and 1.0 pCi of logCdC1zwas given ip to each mouse (B6D2Fl) on day 0. On days 13, 14, 15, 16, and 17, mice in each group were given the corresponding compound a t a dose of 4.0 mmol/kg ip in 0.9% NaCl solution. On day 18, whole body cadmium levels were measured, and mice were sacrified immediately for the determination of organ cadmium concentrations. Each group contained 7 mice. bSignificantly different from control; p 5 0.05. c p 5 0.05. dNS, not significant.
Table VI. Relative Cadmium Mobilizing Potency of N-(4-Methoxybenzyl)-~-glycamine Carbodithioates" whole body cadmium, 70 organ cadmium, bg of Cd/g of tissue wet weight cadmium group administered kidneys liver spleen testes pancreas brain control 86.16 f 3.92 3.72 f 0.21 5.39 f 0.27 0.520 f 0.013 0.176 f 0.016 2.15 f 0.19 0.028 f 0.002 MeOBGDTC (20h) 33.78 f 5.02b 0.895 f 0.076b 1.28 f 0.40b 0.381 i 0.056b 0.148 f 0.024b 2.07 f 0.10 0.027 f 0.002 (1 mmol/ kg) MeOBGDTC (20h) 21.64 f 1.27b 0.487 f 0.05gb 0.512 f 0.077b 0.374 f 0.043b 0.132 f 0.009b 1.89 f 0.21 0.024 f 0.001 ( 2 mmol/kg) 0.025 f 0.002 MeOBGADTC (13b) 29.00 f 2.54b,c 1.08 f 0.075b,c 0.896 f 0.077b,c 0.360 f 0.047b 0.157 f 0.030' 1.89 f 0.13 (1 mmol/ kg) MeOBGADTC (13b) 23.48 f 1.94b 0.754 f 0.157bsd 0.450 f 0.044b 0.310 f 0.040b,d 0.141 f 0.024b 1.67 f 0.17b3d 0.026 f 0.002 ( 2 mmol/kg) 0.820 f 0.047btc 0.402 f 0.047b 0.137 f 0.007b 1.99 f 0.15 0.025 f 0.002 MeOBMNDTC (16b) 27.94 f 1.5jb>' 1.23 f 0.14b3c (1 mmol/ kg) MeOBMNDTC (16b) 22.07 f 1.41b 0.719 f 0.116b,d 0.474 f 0.020b 0.387 f 0.038b 0.124 f 0.01Ib 1.57 f 0.29b$d 0.027 f 0.001 (2 mmol/kg) 1.0 mL of a 0.9% NaCl solution containing 0.03 mg CdClz.2.5H2O and 1.0 WCi of "CdC1, was given ip to each mouse (B6D2Fl) on day 0. On days 30, 32, and 35, mice in each group were given the corresponding compound, ip, at the dosages indicated in the table. The control (I
group received saline only. On day 36, whole body cadmium levels were determined, and the mice were then sacrificed immediately for cadmium analyses on the organs. Each group contained 8 mice. bSignificantly different from control; p 5 0.05. 'Significantly different from MeOBGDTC group (1.0 mmol/kg); p 5 0.05. dSignificantly different from MeOBGDTC group (2.0 mmol/kg); p 5 0.05.
alkyl chain (n-HxGADTC and n-HxMNDTC) led to an increased effectiveness in the mobilization of cadmium from the kidneys, but reduced effectiveness for both whole body reductions and reductions in the liver cadmium content. The introduction of hydroxy groups a t the end of hexyl chains (HHxGADTC and HHxMNDTC) led to a similar but smaller effect. The significant difference between BGDTC and MeOBGDTC (6) led us to examine the effect of the chain lengths of alkyl groups (HPrGDTC, HBuGDTC, and HAmGDTC) to achieve potency comparable to that of MeOBGDTC. In none of the cases examined (Table VIII), were results obtained for the kidneys and liver that were equivalent to those obtained for MeOBGDTC. The effect of putting two sets of polar but nonionic groups on the dithiocarbamate nitrogen atoms can be seen in the data obtained for BARDTC, a compound in which two arabityl groups are present on the nitrogen. Such a structure is quite ineffective in removing cadmium from either the kidneys or the liver (Table IX). A different strain of mice was used for these experiments, which were also carried out with a greater level of cadmium loading than the other experiments using nonradioactive cadmium.
Discussion The factors examined here were selected because of their possible importance in governing the ability of a dithiocarbamate to attain intracellular sites from which it can
remove cadmium from tightly bound forms. Recent studies have revealed that the cadmium present in intracellular sites is only partially present in the form of the cadmium-metallothionein complex (22). The route by which dithiocarbamates actually reach intracellular sites in the kidneys and liver is not known, though several of the structural features which assist such a passage to intracellular sites are now reasonably well established (6,8, 15,16). One of the most important factors is the net ionic charge on the chelating agent. We furnish evidence here which indicates that structural changes which lead to a greater negative charge cause a significant reduction in the ability of the dithiocarbamate to attain such intracellular sites. The results obtained are consistent with evidence collected earlier on compounds in which there was a difference in the net charges on the dithiocarbamates. Thus the dithiocarbamates derived from iminodiacetic acid and diethanolamine differ greatly in net charge, with the compound derived from the former having a net charge of 3- while that from the latter has a net charge of 1-. The compound with a net charge of I- is considerably more effective in removing intracellular cadmium than that with a charge of 3- (20, 23). The effect of chirality on potency is clearly somewhat more complex to characterize, and the experimental data presented here do not provide a definite answer for the most suitable chirality for such chelating agents. Tables 111,VI, and VI1 contain data on sets of isomers of this sort,
Dithiocarbamate-Induced Mobilization of Cadmium Table VII. Effect of Chain Branching and the Presence of a Hydroxyl Group on the Potency of C6-Alkyl Galactamine and Mannamine Carbodithioates" whole body organ cadmium, pg of Cd/g of cadmium, % cadmium tissue wet weight group administered kidneys liver 3.71 f 0.22 6.30 f 0.55 control 91.34 f 3.13 31.09 f 2.55b 0.871 f 0.135' 1.09 f 0.20' BGDTC ( I ) n-HxGADTC 27.67 f 2.11'~~ 2.390 k 0.277'~~0.538 f 0.052b8c (13d) n-HxMNDTC 28.42 f 1.92'~~ 1.84 f 0.187bsc*d0.875 f 0.198b*d (164 DMBGADTC We) DMBMNDTC (lee) HHxGADTC (134 HHxMNDTC (164
31.12 f 4.31b
0.945 f O.20gb
42.63 f 3.42b*c*e0.900 f 0.211'
1.01 f 0.44b 1.89 f 0.34b3c*e
28.90 f 2.14'~~ 1.250 f O.lOOb*' 0.513 f 0.082'*' 34.41 f 5.60'4
1.010 f 0.100'4
0.977 f 0.288b3f
Each mouse (B6D2F1) was given 1.0 mL of a 0.9% NaCl solution containing 0.03 mg of CdCI2.25H20 and 1.0 pCi of 10gCdC12ip on day 0. On days 16, 18, and 20 each mouse received the appropriate compound a t a dose of 2.0 mmol/kg ip in 0.9% NaCl solution. On day 22, whole body cadmium levels were measured, and the mice were sacrificed immediately for determination of organ cadmium levels. There were 8 animals in each group. Significantly different from control; p < 0.05. Significantly different from BGDTC group; p 5 0.05. Significantly different from n-HxGADTC group; p 5 0.05. eSignificantly different from DMBGADTC group; p 5 0.05. 'Significantly different from HHxGADTC group; p 5 0.05. (I
'
Table VI11. Effect of Chain Length on Potency among (&, ( 7 - ,and (wHydroxyalky1)glucamine Carbodithioates" organ cadmium, pg of Cd/g of tissue wet weight group kidneys liver 20.5 f 3.3 47.6 f 4.3 control MeOBGDTC (20h) 12.5 f 1.7' 34.8 f 5.1b HPrGDTC (20e) 17.8 f 3.5 47.3 f 11.1 50.9 f 7.1 HBuGDTC (20f) 17.1 i 3.2 49.1 f 13.2 HAmGDTC (20g) 14.7 f 2.gb Each mouse (ICR) received 4 ip injections amounting to 1,3, 3, and 3 mg of CdC12.2.5H,0/kg in 0.5 mL of 0.9% saine on consecutive days. Starting 2 days later the treated groups were given MeOBGDTC, HPrGDTC, HBuGDTC, and HAmGDTC, respectively, a t a dosage of 0.40 mmol/kg ip in 0.5 mL of 0.9% saline for 5 consecutive days. The control group received saline only. Two days after the last injection all mice were sacrificed and dissected for kidney and liver cadmium levels. There were 6 animals in each group except for the control group which contained 8 animals. 'Significantly different from control; p 5 0.05.
Table IX. Relative Potency of MeOBGDTC and Diarabitylamine Carbodithioate" organ cadmium, fig of Cd/g of tissue wet weight group kidnevs liver control 20.3 i 2.5 49.5 f 8.3 MeORGDTC (20h) 11.7 f 2.0b 36.2 f 7.8b BARDTC (9) 23.0 i 4.4 47.5 i 5.6
" Each mouse
(ICR) received 4 ip injections of CdCI2.2.5H20, 1, 3, 8, and 8 mg/kg in 0.5 mL of 0.9% saline on consecutive days. After an additional 2 days the treated groups were given MeOBGDTC and BARDTC, respectively, a t a dosage of 0.40 mmol/kg ip in 0.9% saline for 5 consecutive days. The control group received saline only. Two days after the last treatment all mice were sacrificed for liver and kidney cadmium level determinations. Each group contained 8 mice. Significantly different from controls; p < 0.05.
'
and the changes in potency that are found are seen to depend very strongly on the nature of the other groups that
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 33 are present. It must be noted that these isomers will also have somewhat different polar interactions with their environment, which may also be sources of differences in behavior. The importance of an amphipathic structure in determining the potency of dithiocarbamates, which we have remarked upon earlier (24),is clearly shown in the very limited potency exhibited by a compound which contains two very polar groups, BARDTC (Table IX), which was examined in a different strain of mice. BARDTC actually has a molecular weight (383.42) close to that of MeOBGDTC (399.45), but the value of ET for this compound is much too negative (about -7.12; optimum values in previously examined compounds of analogous structures are close t,o -3.00) (8,15). This compound does show an ability to mobilize cadmium from the liver and the kidneys when administered a t a significantly higher dose, e.g., a t 4 mmol/kg (data not shown). Compounds containing two nonpolar groups such as sodium N,N-bis(.l-methoxybenzyl)dithiocarbamate, reported earlier, also show a much more limited ability to mobilize such deposits, in addition to exhibiting a greater toxicity (8, 15). It must be noted that compounds of the types examined here, when administered at a given dosage, can be expected to be more effective as cadmium mobilizing agents when used in rats than they are when used in mice (25). This is related to the differences in the ratio of body weight to body surface area and the corresponding differences in metabolic rates (26, 27). The effects of these compounds on the pancreatic cadmium levels is generally, but not always, negligible. This is probably related to the fact that the blood flow through this organ is less than that through the liver and the kidney, and these compounds may well be removed by the liver. One would also expect that the elevated zinc levels in the pancreas would compete with the cadmium for the chelating agent. The results obtained for IPNADTC in Table V and MeOBMNDTC in Table VI suggest that it might be possible to effect a substantial reduction in the cadmium levels of this organ by a more extended treatment with either of these compounds. In conclusion, it can be seen that, for the compounds examined, branched alkyl chains may provide compounds of superior potency to the corresponding isomers with straight-chain alkyl groups. It is also obvious from our results that any increase in the net negative charge on such chelating agents decreases potency significantly, presumably by rendering passage through negatively charged layers of cellular membranes appreciably more difficult (28).
Acknowledgment. We acknowledge the support of this work by the National Institute of Environmental Health Sciences via Grant ES-02638 (M.M.J.) and by the Department of Veterans Affairs (G.R.G.). We are grateful to Dr. Hurshell H. Hunt, Department of Biometry, Medical University of South Carolina, for assistance in statistical evaluations of data. Registry No. 1, 110771-92-1; 2, 7250-31-9; 3, 130792-64-2; 3 free acid, 130671-44-2; 4, 130671-22-6; 4 free acid, 130671-45-3; 5,130671-23-7; 5 free acid, 130671-46-4; 6,89970-79-6; 6 free acid, 95480-31-2; 7,10323-20-3; 8,22566-22-9; 9,130671-24-8; 9 free acid, 130792-71-1; 12a, 74410-49-4; 12b, 130851-81-9; 12c, 130671-35-1; 12d, 130851-82-0; 12e, 130671-36-2; 13a, 130851-80-8; 13a free acid, 130929-87-2; 13b, 130671-25-9; 13b free acid, 130671-47-5; 13c, 130929-86-1; 13c free acid, 130851-83-1; 13d, 130671-26-0; 13d free acid, 130671-48-6; 13e, 130929-88-3; 13e free acid, 130671-49-7; 15a, 38709-24-9; 15b, 130792-69-7; 15c, 130671-37-3; 15d, 130792-70-0; 15e, 130671-38-4; 16a, 130792-65-3; 16a free acid, 130792-72-2; 16b, 130792-67-5; 16b free acid, 130792-73-3; 16c,
34 Chem. Res. Toxicol., Vol. 4, No. 1, 1991 130792-68-6; 16c free acid, 130792-74-4; 16d, 130671-27-1; 16d free acid, 130671-50-0; 16e, 130671-28-2; 16e free acid, 130671-51-1; 19a, 74295-49-1; 19b, 130671-39-5; 19c, 130022-38-7; 19d, 130671-40-8; 19e, 130671-41-9; 19f, 130671-42-0; 19g, 130671-43-1; 20a, 180792-66-4; 20a free acid, 120908-76-1; 20b, 130671-29-3; 20b free arid, 130671-52-2; 20c, 130671-30-6; 20c free acid, 130671-53-3; 20d, 130671-31-7; 20d free acid, 130671-54-4; 20e, 130671-32-8; 20e free acid, 130671-55-5; 20f, 130671-33-9; 20f free acid, 130671-56-6; 20g, 130671-34-0; 20g free acid, 130671-57-7; 20h, 115884-16-2; 20h free acid, 115459-35-3; cadmium, 7440-43-9.
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