O HO H H H OH H OH CH2OH H HOCH2 C H OH C H O C OH H C H OH CH2 N CH2 C S–Na+ S Effect of cadmium chelating agents on organ cadmium and trace element levels in mice† Vladislav Eybl*a, Dana Kotyzov�aa, Jaroslav Koutenskya, V�era M�ý�ckov�aa, Mark M. Jonesb and Pramod K. Singhb a Department of Pharmacology and Toxicology, Charles University Faculty of Medicine, 30166 Pilsen, Czech Republic b Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37235, USA In experiments performed on male mice (CD-1, Charles River), the mobilizing effects of repeated administration of the carbodithioate analogue BLDTC [N-benzyl-4-O-(b-D-galactopyranosyl)-D-glucamine- N-carbodithioate] and CaDTPA (calcium trisodium pentetate) on cadmium deposits in the liver, kidneys, brain and testes were compared.The antidotes were injected alternately every 48 h over a period of 16 d (8 doses in total) following a previous loading with 20 doses of CdCl2·2.5 H2O (single doses of 3 mg kg21 i.p.). The experiments confirmed BLDTC to be one of the most effective cadmium mobilizing agents.The administration of CaDTPA, which is known as a useful antidote in acute cadmium intoxication, increased the mobilizing effect of BLDTC. Cadmium elevated the concentration of zinc in all organs examined and the level of copper in the liver, kidneys and testes. This accumulation of trace elements was only partially corrected by the chelators.The antidotes administered alone exert only a negligible effect on the trace element levels in the organs. Keywords: N-Benzyl-4-O-(b-d-galactopyranosyl)-dglucamine- N-carbodithioate; cadmium; chelators; DTPA; essential elements; dithiocarbamates In previous papers,1–8 the cadmium mobilizing effect of various carbodithioate analogues was studied. Among the most effective agents of this group is BLDTC [N-benzyl-4-O-(b-dgalactopyranosyl)- d-glucamine-N-carbodithioate].6 In this study, the influence of BLDTC on the cadmium deposits is compared with the effect of CaDTPA (calcium trisodium pentetate).The protective effect of this chelator in cadmium intoxication has been described previously.1 In this study the effect of combined treatment with BLDTC and DTPA was also studied. Both agents had to be given parenterally because BLDTC is unstable upon oral administration and CaDTPA is almost non-absorbable. Since the essential elements may be chelated unspecifically by metal antidotes, the effects of these compounds on the levels of zinc and copper in the tissues were examined.Experimental The experiments were performed on male mice (CD-1, Charles River; 25–30 g body mass). The following substances were administered: CdCl2·2.5 H2O (analytical-reagent grade; Lachema, Brno, Czech Republic), the dithioate analogue BLDTC [N-benzyl-4-O-(b-d-galactopyranosyl)- d-glucamine-N-carbodithioate sodium salt] (synthesized by M. M.J. and P. K. S.) (Fig. 1) and CaDTPA [calcium trisodium pentetate (Ditripentat-Heyl; DTPA)]. The animals were divided into two groups. The first group of 33 animals were injected i.p. with CdCl2·2.5 H2O at a dose of 3 mg kg21 daily for 6 d per week. A total of 20 doses were administered. The animals in the second group served as controls, receiving saline. On the 25th day of the experiment (48 h after the last injection of Cd), five animals from both groups were killed by diethyl ether anesthesia and decapitation.The liver, kidneys, brain and testes were removed, rinsed in cold saline, blotted dry and stored frozen (220 °C) until analysed. The remaining control and Cd-intoxicated animals were divided into groups of seven animals each (see tables). The chelators CaDTPA and/or BLDTC were injected s.c. alternately every 48th hour over a period of 16 days (eight doses in total) starting on the 25th day of the experiment. A single dose of the chelators corresponded to a single dose of Cd at an antidote-to- Cd molar ratio of 25 : 1.The animals were killed on the 41st day of the experiment (24 h after the eighth injection of antidotes) and tissues were removed and stored as described above. For elemental analyses the tissues were weighed, placed in platinum crucibles and dry-ashed in a muffle furnace at 460–500 °C for 18–24 h. The ash was solubilized with 3 m HCl. Appropriately diluted samples were analyzed by atomic absorption spectrometry (AAS) using a Model AAS-30 instrument (Zeiss, Jena, Germany).Tissues were analyzed for Zn and Cu using an air–acetylene flame. Electrothermal AAS was applied for Cd determination. The results were statistically evaluated using the unpaired ttest. The values in the tables are means ± s. Results and discussion The influence of chelators on cadmium concentrations in the tissues is summarized in Table 1. The levels of cadmium in the liver, testes and brain decreased from the 25th to the 41st day.However, the level of cadmium in the kidneys increased. BLDTC lowered the concentration of cadmium in the liver and kidneys. Similarly, the amount of cadmium in the liver decreased after the administration of CaDTPA. The best results were attained after treatment with both chelators together. However, neither of the agents induced cadmium mobilization from the testes. † Presented at The Sixth Nordic Symposium on Trace Elements in Human Health and Disease, Roskilde, Denmark, June 29–July 3, 1997.Fig. 1. BLDTC [N-benzyl-4-O-(b-d-galactopyranosyl)-d-glucamine-Ncarbodithioate]. Analyst, January 1998, Vol. 123 (25–26) 25The concentration of zinc in the liver, kidneys and testes was significantly elevated after the administration of cadmium. This effect was diminished in the liver on administration of CaDTPA. The concentration of copper in the liver, kidneys and testes also increased on cadmium administration. The chelators were able to correct this change only partially in the liver (Table 2).The chelators administered alone exerted no effect on the concentration of zinc in the liver, testes and brain. The level of zinc in the kidneys increased after the administration of CaDTPA and also after a combination of BLDTC and CaDTPA. The concentration of copper was enhanced in the testes after the administration of BLDTC alone and in combination with CaDTPA. This combination decreased the level of copper in the kidney and testes.BLDTC alone increased the content of Cu in the testes. No changes in trace element concentrations were found in the brain (Table 3). The results obtained in this study confirm those of our previous investigation on the effect of BLDTC in cadmium intoxication.6 Moreover, we have found that CaDTPA might increase the effect of this dithioate. This effect was especially notable in the kidneys. The carbodithioates have a higher affinity for cadmium than does metallothionein, as has been demonstrated in experiments in vitro.9 The elevation of zinc concentration in the kidneys of control animals following CaDTPA administration appears to be a consequence of the increased excretion of zinc from the body (Table 3).The increase in concentration of zinc in the liver and kidneys after the administration of cadmium was corrected to a similar extent to that given by a related drug, MeBLDTC, in previous work.6,8 However, BLDTC alone, in contrast to MeBLDTC, exerts only a negligible effect on zinc and copper level in the organs.As discussed in a previous paper,8 a systematic study of the influence of chelators on the levels of essential elements during treatment of metal poisoning is needed. Carbodithioates represent a promising group of cadmium mobilizing agents. Even though their acute toxicity is relatively low, basic pre-clinical research is needed before they could be recommended for human administration.This research was supported by grant No. 07/96 from Charles University. References 1 Eybl, V., S�ykora, J., Koutensk�y, J., Caisov�a, D., Schwartz, A., and Mertl, F., Environ. Health Perspect., 1984, 54, 267. 2 Jones, M. M., Singh, P. K., Jones, S. G., and Holscher, M. A., Pharmacol. Toxicol. (Copenhagen), 1991, 68, 115. 3 Jones, M. M., Singh, P. K., Gale, G. R., Smith, A, L. M., Pharmacol. Toxicol. (Copenhagen), 1992, 70, 336. 4 Eybl, V., Jones, M. M., Koutensk�a, M., Koutensk�y, J., S�ykora, J., Drobn�ýk, F., and � Svec, F., Arch.Toxicol., 1988, Suppl. 12, 438. 5 Eybl, V., Jones, M. M., Koutensk�a, M., Koutensk�y, J., S�ykora, J., and Smol�ýkov�a, V., Plze�n. L�ek. Sb., 1991, Suppl. 64, 25. 6 Eybl, V., Kotyzov�a, D., Koutensk�y, J., Singh, P. K., and Jones, M. M., Plze�n. L�ek. Sb., 1993, Suppl. 68, 15. 7 Eybl, V., Kotyzov�a, D., Koutensk�y, J., Jones, M. M., and Singh, P. K., Plze�n L�ek. Sb., 1996, Suppl. 70, 65. 8 Eybl, V., Kotyzov�a, D., Koutensk�y, J., Jones, M. M., and Singh, P. K., Analyst, 1995, 120, 855. 9 Gale, G. R., Smith, A. B., Atkins, L. M., and Jones, M. M., Res. Commun. Chem. Pathol. Pharmacol., 1985, 49, 423. Paper 7/04894G Received July 9, 1997 Accepted September 22, 1997 Table 1 Cadmium concentrations in the tissues after pre-treatment with Cd on the 25th day of the experiment and after treatment with chelators on the 41st day of the experiment. Results in mg g21 (means ± s) 41st day 25th day Cd + BLDTC Cd + CaDTPA Cd + BLDTC + Tissue Cd (n = 5) Cd (n = 6) (n = 6) (n = 6) CaDTPA (n = 6) Liver 81.8 ± 16.8 71.5 ± 6.7 64.1 ± 3.6* 64.9 ± 3.2* 61.8 ± 2.4** Kidneys 76.0 ± 7.9 81.0 ± 7.7 70.3 ± 6.5* 76.9 ± 6.5 64.4 ± 3.33** Testes 35.9 ± 7.3 27.1 ± 4.4 30.3 ± 5.0 27.6 ± 4.9 23.7 ± 3.6 Brain 1.0 ± 0.1 0.8 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 0.7 ± 0.1* * p < 0.05 versus control.** p < 0.01 versus control. Table 2 Zinc and copper concentrations in the tissues after treatment with Cd and chelators on the 41st day of the experiment.Results in mg g21 (means ± s, n = 7) Liver Kidneys Testes Brain Treatment Zn Cu Zn Cu Zn Cu Zn Cu Control 21.4 ± 1.1 3.7 ± 0.3 18.5 ± 1.0 3.7 ± 0.2 24.8 ± 1.7 1.3 ± 0.1 14.9 ± 0.5 3.3 ± 0.1 Cd 41.9 ± 2.8** 6.5 ± 0.4** 23.2 ± 2.1** 4.2 ± 0.5** 55.7 ± 20.4** 2.9 ± 0.9** 15.6 ± 0.8 3.3 ± 0.2 Cd + BLDTC 45.0 ± 1.9** 5.3 ± 1.0** 30.5 ± 1.8** 4.9 ± 0.6** 79.7 ± 19.3** 4.0 ± 0.9** 14.9 ± 0.9 3.2 ± 0.4 Cd + CaDTPa 37.5 ± 1.4** 6.9 ± 1.3** 27.4 ± 3.0** 4.4 ± 0.3** 47.7 ± 20.7** 2.8 ± 0.5** 16.1 ± 0.8** 3.5 ± 0.4 Cd + BLDTC + CaDTPA 41.1 ± 3.2** 6.6 ± 0.6** 33.8 ± 2.3** 4.5 ± 0.3** 42.9 ± 7.9** 3.2 ± 0.8** 15.8 ± 0.7** 3.5 ± 0.3 ** p < 0.01 versus control. Table 3 Zinc and copper concentrations in the tissues after treatment with chelators on the 41st day of the experiment. Results in mg g21 (means ± s, n = 7) Liver Kidneys Testes Brain Treatment Zn Cu Zn Cu Zn Cu Zn Cu Control 21.4 ± 1.1 3.7 ± 0.3 18.5 ± 1.0 3.7 ± 0.2 24.8 ± 1.7 1.3 ± 0.1 14.9 ± 0.5 3.3 ± 0.1 BLDTC 20.8 ± 1.1 3.3 ± 0.5 18.6 ± 0.5 3.7 ± 0.1 24.5 ± 1.2 1.5 ± 0.2* 15.0 ± 1.5 3.5 ± 0.4 CaDTPA 21.0 ± 0.9 3.8 ± 0.7 20.2 ± 0.4** 3.7 ± 0.2 26.0 ± 2.4 1.4 ± 0.2 14.6 ± 1.2 3.4 ± 0.2 BLDTC + CaDTPA 22.6 ± 1.7 4.0 ± 0.9 19.7 ± 0.8** 3.5 ± 0.1* 25.1 ± 1.4 1.5 ± 0.1* 15.1 ± 1.3 3.3 ± 0.2 * p < 0.05 versus control. ** p < 0.01 versus