TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
116,24-29
(1992)
Effects of Dithiocarbamates on Testicular Toxicity in Rats Caused by Acute Exposure to Cadmium SHOJI KOJIMA,’ Department
Of’Hygienic
Chemistry,
YOSUKE SUGIMURA, HIDEAKI HIRUKAWA, MORIO KIYOZUMI,~ HIDEAKI SHIMADA, AND TAKAYUKI FUNAKOSHI Faculty
of Pharmaceutical Received
January
Sciences,
Kumamoto
22. 1992; accepted
University, May
5-I Oe-honmachi.
N-Benzyl-D-glucamine dithiocarbamate (BGD), N-p-isopropylbenzyl-D-glucamine dithiocarbamate (PBGD), and diethyldithiocarbamate (DED) were compared for their protective effects against the testicular toxicity in rats induced by acute exposure to cadmium. Rats were injected subcutaneously with 109CdCI, (3 mg Cd and 74 kBq of lo9Cd/kg) and 30 min later, they were injected intraperitoneally with the chelating agents (0.4 or 3 mmobkg). Cadmium injection increased lipid peroxidation and concentrations of hemoglobin and Ca in the testes, decreased the testicular weight, and caused sterility. The treatment with BGD (0.4 mmobkg) did not satisfactorily protect against the testicular toxicity of cadmium. The administration of PBGD or DED at a dose of 3 mmobkg significantly prevented the increase in the lipid peroxidation and hemoglobin concentration in the testes, the decrease in the testicular weight, and the sterility caused by cadmium. PBGD and DED significantly decreased the cadmium concentration in the testes, but DED increased the cadmium concentration in the kidney and brain. Only DED significantly prevented the increase in the testicular Ca concentration after cadmium. These results indicate that PBGD and DED protect against the sterility caused by cadmium in rats and that the effect of DED to increase the brain level of cadmium is more dangerous than the lack of effect of PBGD to prevent the increase in the testicular Ca level. The protective effects of PBGD and DED against the cadmium-induced testicular toxicity. presumably result from a decrease in the cadmium . concentration in the testes. 0 1992 Academic press, hc.
20, 1992
MATERIALS Cadmium has been recognized as one of the most toxic industrial and environmental elements of which there is a continuing hazard to human exposure. In experimental animals, cadmium can cause a number of lesions in various organs such as liver, kidney, and testes (Friberg et al., 1974;
0041-008X/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
862, Japan
Stowe et al., 1972; Cook et al., 1974; Hoffmann et al., 1975). Cadmium induces a severe acute toxicity to rat testes at injection doses appreciably lower than those required for toxicity to other organs (Parizek and Zahor, 1956; Fahim and Khare, 1980). The testicular toxicity induced by cadmium injection involves hemorrhage, necrosis, and atrophy (Parizek and Zahor, 1956; Meek, 1959; Cameron and Foster, 1963; Gunn et al., 1965; Gunn and Gould, 1970). Among the various toxic effects induced by cadmium in biological systems, lipid peroxidation has been observed in numerous tissues either in vivo or in vitro (Gabor et al., 1978; Sajiki et al., 1981; Sato et al., 1983: Klimczak et al., 1984; Jamall and Smith, 1985; Muller, 1986; Hussain et al., 1987; Shukla et al., 1987). It has also been demonstrated in vitro that lipid peroxidation is an early intracellular event after cadmium exposure (Muller and Ohnesorge, 1982; Muller, 1986). Chelation therapy for cadmium has been effective in preventing cadmium-induced toxicity. Walker et al. ( 1984, 1986) studied the effects of five dithiocarbamates, dimercaptosuccinate, and diethylenetriaminepentaacetate (DTPA) on the testicular toxicity of cadmium using morphological techniques and indicated that diethyldithiocarbamate (DED) and DTPA were the most effective antagonists if given immediately after cadmium injection. The purpose of the present study was to evaluate the protective effects of N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-isopropylbenzyl-D-glucamine dithiocarbamate (PBGD), the new dithiocarbamates, and DED against the testicular damage resulting from acute exposure to cadmium in rats.
Effects of Dithiocarbamates on Testicular Toxicity in Rats Caused by Acute Exposure to Cadmium. KOJIMA, S., SUGIMURA, Y., HIRUKAWA, H., KIYOZUMI, M., SHIMADA, H., AND FUNAKOSHI, T. (1992). Toxicol. Appl. Pharmacol. 116,24-29.
’ To whom reprint requests should be addressed. ’ Present address: Department of School Health, Faculty Kumamoto University, 40-l Kurokami-2-chome. Kumamoto,
Kumamoto,
AND METHODS
Chemicals. Cadmium chloride and DED were obtained from Wako Pure Chemicals (Osaka). imCd (specific activity, 58.2 GBq/mg) was obtained from New England Nuclear Corp. (Boston, MA). BGD and PBGD were synthesized according to the procedures reported previously from our laboratory (Kojima et al., 1986, 1987). Other chemicals were of reagent grade. Animals and treatments. Male Wistar rats, weighing 180-220 g, were purchased from Kyudo Co.. Ltd. (Kumamoto) and housed in individual metabolic cages with a commercial chow (Nosan Lab Chow) and drinking water ad /i&turn in temperatureand humidity-controlled rooms and on a 12-hr light/dark cycle.
of Education, 860, Japan, 24
CADMIUM-INDUCED
TESTICULAR
The rats were injected subcutaneously with saline (0.5 ml/animal) or 3 mg Cd/kg in 0.5 ml saline. Thirty minutes later they were injected intraperitoneally with saline (control) or chelating agents (0.4 or 3 mmol/kg) in 0.5 ml saline. Three or 7 days after cadmium administration the rats were killed by cervical fracture and testes were removed. Liver and kidney were perfused by injecting ice-cold 0.01 M phosphate buffer (pH 7.4) containing 1.15% KC1 with 0.025% ethylenediaminetetraacetic acid (EDTA) into the inferior vena cava and the liver and kidney were removed. Testes, liver, and kidney were analyzed for lipid peroxidation and hemoglobin. In the experiment for determination of cadmium and essential metals (Ca, Fe, and Zn), the rats were injected intraperitoneally with chelating agents (3 mmol/kg) 30 min after subcutaneous injection of ““Cd (3 mg Cd and 74 kBq of lwCd/ kg) and testes, liver, kidney, heart, and brain were removed 3 or 7 days after cadmium injection. Lipid peroxidation. Testes, liver, and kidney were homogenized with 9 vol of ice-cold 0.0 1 M phosphate buffer (pH 7.4) containing 1.15% KC1 and 0.025% EDTA. Lipid peroxidation was assayed according to the thiobarbituric acid (TBA) method of Uchiyama and Mihara (1978) using 1,1,3,3tetraethoxypropane as the standard and expressed as nanomoles of TBA reactive substances (TBARS). Hemoglobin. Testes were homogenized with 9 vol of ice-cold 0.01 M phosphate buffer (pH 7.4) as described earlier and the homogenate was centrifuged at 2000g for 10 min at 4°C. Hemoglobin content in the supematant was determined according to the method of Sajiki et al. (1983) and expressed as mg/g wet tissue. Protein. Protein was determined according to the method of Lowry et al. (195 1). using bovine serum albumin as the standard. Metals. Cadmium content in various organs was determined by measuring the amount of lwCd radioactivity with an Aloka autowell y-scintillation counter (model ARC 300). Testes, liver, and kidney were wet-ashed by the HC104-HNOj method and essential metals (Ca, Fe, and Zn) were determined using a Hitachi Z-8000 atomic absorption instrument. Fertility test. Fertility of male rats given cadmium with and without chelating agent was determined according to the method of Jackson et al. (1961). The rat was mated with a virgin female rat for 8 days, starting 52 days after administration of cadmium with and without chelating agent. The fertility rate was expressed as the number of females impregnated versus the number of males tested. Statistical analysis. Data were analyzed by a one-way analysis of variance. When the analysis indicated that a significant difference existed, the treated groups were compared to the controls by Duncan’s new multiple range test.
RESULTS The lipid peroxidation and hemoglobin concentrations in the testes and the testicular weight of rats after cadmium administration are shown in Fig. 1. The lipid peroxidation in the testes significantly increased compared to the control at 12 hr after cadmium. Similarly, Manta et al. ( 199 1) reported that lipid peroxidation in the testes of rats significantly increased over control level within 12 hr after injection of cadmium (1 mg Cd/kg). The hemoglobin concentration in the testes also increased significantly 1 day after cadmium injection, indicating the testicular hemorrhage. The testicular weight decreased significantly after 5 days of cadmium administration. Table 1 shows the concentrations of cadmium and essential metals (Ca, Fe, and Zn) in the testes after cadmium injection. The Fe concentration significantly increased 2 days after cadmium, suggesting hemorrhage. The Ca con-
TOXICITY
o!
40
0
25
,
I
I
I
I
I
I
1
0
1
2
3
4
5
6
7
1
I
a
1
I
I
0
1
2
a
I
I
3 4 Time (day)
I
I
1
5
6
7
FIG. 1. Time course of lipid peroxidation and hemoglobin concentrations in testes and testicular weight of rats after cadmium administration. Rats were injected subcutaneously with cadmium (3 mg Cd/kg). The values represent the mean f SD for four animals. 0, control; 0, Cd. ‘Significantly different from control (a < 0.05).
centration significantly increased 1 day after cadmium and the Ca concentrations at 3 and 7 days were approximately 15 and 60 times larger, respectively, than those of the control. The Zn concentration was not affected by cadmium injection. We investigated the protective effects of BGD, PBGD, and DED against the testicular toxicity of cadmium. First when BGD (0.4 mmol/kg) was injected 30 min after cadmium, it prevented the increases in lipid peroxidation and hemoglobin concentration in the testes within 1 day after cadmium and the decrease in the testicular weight within 3
KOJIMA
26
TABLE 1 Concentration of Cadmium and EssentialMetals in Testes of Rats after Cadmium Administration Metal Days after administration Control I 2 3 5 7
concentration
0.00 0.24 0.33 0.40 0.86 0.94
k 0.00 + 0.03 i 0.05 ? 0.21 zk 0.18 * 0.14
27.8 5.09 235.6 428.9 1068.8 1696.0
f 0.3 + 7.9” + 46.3” f 30.5” k 108.6” k 245.3”
TABLE 2 Effects of Chelating Agents on Lipid Peroxidation in Testes, Liver, and Kidney of Rats after Cadmium Administration Lipid peroxidation (nmol TBARS/mg protein)
(Irp/p wet tissue)
Ca
Cd
ET AL.
Fe 16.6 16.6 32.9 30.0 28.1 32.8
?I f + t + +
Zn 0.3 2.5 13.5” 0.8” 4.1” 6.8”
19.9 18.3 17.6 17.4 20.3 21.7
* + + + + +
Treatment 0.3 2.1 1.5 0.1 2.5 2.4
Noie. Rats were injected subcutaneously with cadmium (3 mg Cd/kg). The values represent the mean f SD for four animals. a Significantly different from control (p < 0.05).
days, but did not protect against such testicular toxic changes thereafter (data not shown). Next BGD, PBGD, and DED at a dose level of 3 mmol/kg were administered 30 min after cadmium and the effects of the chelating agents on cadmiuminduced testicular toxicity were examined. As shown in Table 2, PBGD and DED completely prevented the increase in the lipid peroxidation during 7 days after cadmium injection. BGD significantly prevented the increase in the lipid peroxidation within 3 days after cadmium injection, but did not afford the protective effect at 7 days. The lipid peroxidation in the liver and kidney after cadmium did not significantly change compared to the control. As shown in Table 3, a significant change in the testicular weight as an indicator of the testicular toxicity did not occur 3 days after cadmium injection, but the testicular weight significantly decreased at 7 days. DED completely prevented the decrease in the testicular weight after cadmium injection, but BGD and PBGD failed to protect the weight loss. The increase in the hemoglobin concentration after cadmium injection was significantly prevented by PBGD and DED, but not by BGD (Table 3). The tissue concentrations of cadmium in rats treated with the chelating agents after cadmium are shown in Table 4. PBGD treatment significantly decreased the cadmium concentrations in the testes and heart 7 days after cadmium injection. DED significantly decreased the cadmium concentrations in the testes, liver, and heart, but caused a significant increase in the cadmium concentrations in the kidney and brain. BGD treatment had little effect on cadmium concentrations in the tissues. As shown in Table 5, the increases in the Ca and Fe concentrations in the testes after cadmium injection were significantly prevented by DED, but not by BGD or PBGD. The Zn concentration in the testes was affected little by the administration of cadmium with and without the chelating agents. In the liver and kidney, the Zn concentration significantly increased, but the Ca and Fe concentrations changed little.
Liver
Testes
Kidney
3 Days after cadmium Control Cd Cd + BGD Cd + PBGD Cd + DED
1.22 * 3.18 + 1.53 f 0.97 * 1.09 t
0.07 0.53” o.79b 0.04b 0.16’
0.78 0.68 0.35 0.51 0.55
f 0.19 5 0.14 z!I 0.22 + 0.10 f 0.08
0.97 1.17 0.64 0.90 0.74
+ 0.16 + 0.46 k 0.19 +_0.20 1 0.11
k + 5 f f
0.79 0.82 0.71 0.68 0.73
t + + f f
7 Days after cadmium Control Cd Cd + BGD Cd + PBGD Cd + DED
1.39 i 3.63 i 3.64 + 0.80 f 1.05 f
0.2 1 0.29” 0.54” 0.08’ 0.13b
0.52 0.41 0.43 0.43 0.61
0.09 0.01 0.01 0.10 0.08
0.14 0.16 0.03 0.17 0.03
Note. Rats were injected intraperitoneally with chelating agents (3 mmol/ kg) 30 min after subcutaneous injection of cadmium (3 mg Cd/kg). The rats were killed 3 or 7 days after cadmium and lipid peroxidation in the organs was determined. The values represent the mean * SD for four animals. a Significantly different from control (p < 0.05). b Significantly different from Cd (p < 0.05).
Furthermore, the fertility of male rats given cadmium with and without the chelating agents was examined (Table 6). The sterility produced by cadmium was completely prevented by DED and PBGD, but not by BGD. TABLE 3
Changes in Testicular Weight and Hemoglobin Concentration of Rats after Administration of Cadmium or Cadmium and Chelating Agents Hemoglobin concentration (w/g wet tissue)
Testicular weight (9%of body weight) Treatment Control Cd Cd + BGD Cd + PBGD Cd + DED
3 Days 1.04 k 0.87 k 0.94 f 1.11 + 1.22 f
0.14 0.06 0.06 0.07 0.17
7 Days 1.05 0.12 0.62 0.72 1.19
k + f f *
0.11 0.06“ 0.06” 0.09” 0.07”
3 Days 3.43 9.76 8.82 5.75 5.02
-t 0.48 + 0.4Y -+ 2.10” 2 2.10h + 0.92’
Note. Rats were injected intraperitoneally with chelating agents (3 mmol/ kg) 30 min after subcutaneous injection of cadmium (3 mg Cd/kg). The rats were killed 3 or 7 days after cadmium and testicular weight or hemoglobin concentration was determined. The values represent the mean i SD for four
animals.
LISignificantly different from control (p < 0.05). b Significantly different from Cd (p < 0.05).
CADMIUM-INDUCED
TESTICULAR
27
TOXICITY
TABLE 4 Effects of Chelating Agents on Tissue Concentration of Cadmium Concentration of cadmium (pg/g wet tissue) Treatment
Testes
Liver
Kidney
Heart
Brain
3 Days after cadmium Cd Cd + BGD Cd + PBGD Cd + DED
0.40 0.20 0.37 0.30
+ f f f
0.2 1 0.04 0.06 0.05
23.3 19.8 21.6 7.3
+ 5.6 + 1.6 f 3.9 ?I 1.2”
14.9 11.4 8.0 12.5
f f + t
1.3 0.9” 0.5” 1.9
1.61 1.09 1.85 0.73
f 0.68 f 0.27 + 0.37 zlz0.07
17.8 f 15.7 f 15.9 + 33.0 f
3.9 0.9 1.2 8.5”
2.34 + 2.06 f 1.40 f 1.08 t
0.23 0.16 0.23 0.35
f 0.08 +- 0.05 k 0.03 k 0.06
0.20 0.18 0.13 0.46
+ f + +
7 Days after cadmium Cd Cd + BGD Cd + PBGD Cd + DED
0.94 0.71 0.52 0.39
f + + +
0.14 0.28 0.10” 0.05”
24.7 22.8 18.2 9.7
+ 6.8 -c 5.7 + 1.9 zk 3.0”
0.33 0.33 0.05” 0.02“
0.09 0.04 0.06 0.03”
Note. Rats were injected intraperitoneally with chelating agents (3 mmol/kg) 30 min after subcutaneous injection of ‘@‘Cd (3 mg Cd and 74 kBo of “‘Cd/kg). The rats were killed 3 or 7 days after cadmium and cadmium concentration was determined from radioactivity. The values represent the mean + SD for four animals. ’ Significantly different from Cd (p -c 0.05).
DISCUSSION The present study was carried out to examine the protective effects of the dithiocarbamates on the testicular toxicity of cadmium in rats. It has been reported that cadmium injection can result in severe hemorrhagic necrosis (Gunn et al., 1965; Gunn and Gould, 1970) and enhancement of lipid peroxidation in the testes (Gabor et al., 1978; Sajiki et al., 1981; Sato et al., 1983; Klimczak et al., 1984). Manta et al. (199 1) have demonstrated that lipid peroxidation is an early and sensitive consequence of cadmium exposure as determined in various organs such as lung, brain, testes, liver, kidney, and heart. Our results also indicated significant increases in the lipid peroxidation in the testes at 12 hr after cadmium injection and in hemoglobin concentration in the testes at 1 day. This suggests a relationship between lipid peroxidation and hemorrhage in the testes, as reported by Sajiki et al. (198 1). The marked increase in the Ca concentration in the testes after cadmium injection was also previously reported (Sajiki et al., 1985; Yamane et al., 1990). This is considered to be due to the cadmium-induced cellular damage in the testes. However, the detailed mechanisms by which cadmium increases the testicular Ca concentration remain unknown. The decrease in the testicular weight occurred 5 days after cadmium administration, indicating that the cadmium-induced testicular damage involves the initial hemorrhagic necrosis, followed finally by the testicular atrophy. The testicular damage caused by cadmium as shown in this study is similar to what has been reported in earlier papers (Meek, 1959; Parizek, 1960; Cameron and Foster, 1963). Wong and Klaassen (1980) have reported that there
are two hypotheses for the mechanism of the toxic response of cadmium to the testes. Our results support one hypothesis that cadmium-induced testicular toxicity is due to vascular changes in the testes leading to hemorrhage, necrosis, and atrophy (Gunn et al., 1963, 1965; Niemi and Komano, 1965; Waites and Setchell, 1966; Aoki and Hoffer, 1978). The second hypothesis is that cadmium causes testicular damage by competing with Zn in Zn-requiring enzymes in the testes (Gunn et al., 196 1; Hodgen et al., 1969). However, our data showed that cadmium had little affect on the Zn concentration in the testes. The effect of cadmium on Zn-containing enzymes in the testes should be further studied. As the testicular toxicity of cadmium was not satisfactorily prevented by the treatment with BGD (0.4 mmol/kg), we further studied the protective effects of BGD, PBGD, and DED at a higher dose level (3 mmol/kg) on the testicular toxicity of injected cadmium. PBGD and DED completely prevented the increase in the lipid peroxidation in the testes during 7 days after cadmium, whereas the protective effect of BGD was less than that of PBGD or DED. PBGD and DED significantly prevented the increase in the testicular hemoglobin concentration, which is considered to be due to the hemorrhagic inflammation, and also afforded a complete protection against the sterility caused by cadmium. Among the chelating agents used only DED significantly decreased the cadmium concentrations in the testes, liver, and heart, but, on the other hand, also significantly increased the cadmium concentrations in the kidney and brain. PBGD also decreased the cadmium concentration in the testes. From these results, the protective effect of the chelating agents against the cadmium-induced testicular toxicity is thought
28
KOJIMA
ET AL.
TABLE 5 Effects of Chelating Agents on TissueConcentration of EssentialMetals
Metal
Days after cadmium
Concentration of metal (fig/g wet tissue) Control
Cd
Cd + BGD
Cd + PBGD
Cd + DED
Testes Ca
25.9 + 0.9 21.4 k 1.8 12.1 + 2.9 Il.6 k 2.6 21.3 + 0.9 18.4 f 0.9
Fe Zn
488.7 1421.7 36.0 33.1 21.7 20.0
f 74.5” + 209.9” f 10.4” + 3.8” ?I 5.5 + 6.4
421.4 1580.3 21.7 33.3 32.4 24.7
2 44.8” k 218.4” t 3.7b -i- I .4” + 6.9 k 0.6
13.4 2769.1 13.5 23.2 24.7 26.6
f 3.8ab It 404.3’,” f 2.8’ f 2.7”,h f 6.1 f 15.27
22.9 2 9.4’ 15.0 f 0.5b 12.5 f 2.7’ 13.4 f 3.9b 18.4 f 3.7 28.0 ?I 9.3
Liver Ca Fe Zn
I
7 7
5.6 f 0.7 39.4 2 9.4 18.5 f 2.0
6.2 k 1.2 37.5 f 14.9 38.6 zk 13.9”
6.9 k 37.8 f 36.7 +
0.9 2.2 5.4”
9.4 It41.8 f 36.7 +
2.lU.b 5.8 3.2”
5.5 f 0.8 39.7 zk 9.5 31.8 + 3.0”
12.8 f 24.2 f 21.2 I?
0.8 2.6 0.8”
17.0 f 13.0 f 23.2 k
2.2 5.76 2.6”
11.6 f 0.9 22.5 + I.8 24.1 IfI 1.7”
Kidney Ca Fe Zn
12.4 k 0.8 19.7 + 2.4 13.2 + 0.2
14.7 Tk 7.2 26.0 f 4.7 25.9 f 8.2”
Note. Rats were injected intraperitoneally with chelating agents (3 mmol/kg) 30 min after subcutaneous injection of ‘@‘Cd (3 mg Cd and 74 kBq of ‘09Cd/kg). The rats were killed 3 or 7 days after cadmium administration and concentration of metal was determined by atomic absorption methods. The values represent the mean f SD for four animals. ’ Significantly different from control (p < 0.05). b Significantly different from Cd (p < 0.05).
to be due to a decrease in the accumulation of cadmium in the testes. The increase in the testicular Ca concentration and the decrease in the testicular weight were completely prevented only by DED among the chelating agents tested. Thus, the marked accumulation of Ca in the testes after cadmium injection appears to be not responsible for the cadmium-induced sterility. More studies are needed to further elucidate the mechanism by which PBGD protects against the testicular toxicity caused by cadmium. TABLE 6 Effects of Chelating Agents on Fertility of Rats after Cadmium Administration Treatment
Fertility rate
Control Cd Cd + BGD Cd + PBGD Cd + DED
717 O/7 O/7 717 717
Note. Male rats were injected intraperitoneally with chelating agents (3 mmol/kg) 30 min after cadmium administration (3 mg Cd/kg). The rat was mated with a virgin female rat for 8 days, starting 52 days after cadmium. The fertility rate was expressed as the number of females impregnated versus the number of males tested.
The present study indicates that PBGD and DED protect against the sterility caused by cadmium in rats, that the effect of DED to increase the brain level of cadmium is more dangerous than the lack of effect of PBGD to prevent the increase in the testicular Ca level, and that BGD provides no satisfactory protection against the cadmium-induced testicular toxicity. REFERENCES Aoki. A., and Hoffer. A. P. (1978). Reexamination ofthe lesions in rat testis caused by cadmium. Biol. Reprod. 18, 579-59 I. Cameron, E.. and Foster, C. L. (1963). Observations on the histological effectsof sub-lethal doses of cadmium chloride in the rabbit. 1. The effect on the testis. J. .4nat. 97, 269-280. Cook. J. A., Marconic. E. A., and Diluzio, N. R. (1974). Lead, cadmium, endotoxin interaction: Effect on mortality and hepatic function. Toxicol. Appi. Pharmacol.
28,292-302.
Fahin, M. S., and Khare, N. K. (1980). Effects of subtoxic levels of lead and cadmium on urogenital organs of male rats. Arch. Androl. 4, 357-362. Friberg, L., Piscator, M., Nordberg, G. F., and Kjellstrom, T. (1974). C’admium in the environment, 2nd ed. CRC Press, Cleveland, OH. Gabor, S.. Anca, Z., and Bordas, E. (1978). Cadmium-induced lipid peroxidation in kidney and testes: Effect of zinc and copper. Rev. Roum. Biochem. 15, I 13- I 17. Gunn. S. A.. Gould, T. C.. and Anderson, W. A. D. (1961). Competition of cadmium for zinc in rat testis and dorsolateral prostate. Acta Endocrinol. 37, 24-30.
CADMIUM-INDUCED
TESTICULAR
Gunn, S. A., Gould, T. C., and Anderson, W. A. D. (1963). The selective injurious response of testicular and epididymal blood vessel to cadmium and its prevention by zinc. Am J. Pathol. 42, 685-702. Gunn, S. A., Gould, T. C., and Anderson, W. A. D. (1965). Strain differences in susceptibility of mice and rats to cadmium-induced testicular damage. J. Reprod.
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Gunn, S. A., and Gould, T. C. (1970). Cadmium and other minerals. In The Testes: Volume III: Influencing Factors (A. D. Johnson, W. R. Gomes, and N. L. Vandemark, Eds.), pp. 377-48 1. Academic Press, New York. Hodgen, G. D.. Butler, W. R., and Gomes, W. R. (1969). In vivo and in vitro effectsof cadmium chloride on carbonic anhydrase activity. J. Reprod. Fertil. 18, 156. Hoffmann. E. O., Cook, J. A., Diluzio. N. R., and Coover, J. A. (1975). The effects of acute cadmium administration in the liver and kidney of the rat. Lab. Invest. 32, 655-664. Hussain, T., Shukla, S., and Chandra, S. V. (1987). Effect of cadmium on superoxide dismutase and lipid peroxidation in liver and kidney of growing rats. Pharmacol. To.xicol. 60, 355-358. Jackson, H., Fox, B. S., and Craig. A. W. (1961). Antifertility substances and their assessment in the male rodents. J. Reprod. Fertil. 2,447-465. Jamall, I. S., and Smith, J. C. (1985). Effects of cadmium on glutathione peroxidase, superoxide dismutase, and lipid peroxidation in the rat heart: A possible mechanism of cadmium, cardiotoxicity. Toxicol. Appl. Pharmacol. 80, 33-42.
Klimczak, J., Wisniewska-Knypl, J. M., and Kolakowski, J. (1984). Stimulation of lipid peroxidation and heme oxygenase activity with inhibition of cytochrome P-450 monooxygenase in the liver of rats repeatedly exposed to cadmium. Toxicology 32, 267-276. Kojima, S., Kaminaka, K., Kiyozumi, M., and Honda, T. (1986). Comparative effectsof three chelating agents on distribution and excretion of cadmium in rats. To,xicol. Appl. Pharmacol. 83, 5 16-524. Kojima. S., Kiyozumi, M.. Honda, T., Senba, T., Kaminaka, K., and Ohnishi, M. (1987). Studies on poisonous metals. XVIII. Effects of several dithiocarbamates on tissue distribution and excretion of cadmium in rats. Cllern. Pharmacol.
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Lowry, 0. H.. Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Manta, D., Ricard, A. C., Trottier, B., and Chevalier, G. (199 I). Studies on lipid peroxidation in rat tissues following administration of low and moderate doses of cadmium chloride. Toxicology 67, 303-323. Meek, E. S. (1959). Cellular changes induced by cadmium in mouse testis and liver. Br. J. Exp. Pathol. 40, 503-507. Muller, L. (1986). Consequences of cadmium toxicity in rat hepatocytes: Mitochondrial dysfunction and lipid peroxidation. Toxicology 40, 285295.
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Muller, L., and Ohnesorge, F. K. (1982). Different responses of liver parenchymal cells from starved and fed rats to cadmium. To.xicology 25, 14 l150. Niemi, M., and Kormano, M. (1965). An angiographic study of cadmiuminduced vascular lesions in the testis and epididymis of the rat. Acta Pathol. Microbial. &and. 63, 5 13-52 I. Parizek, J. (1960). Sterilization of the male by cadmium salts. J. Reprod. Fertil.
1, 294-309.
Parizek, J., and Zahor, A. (1956). Effects of cadmium salts on testicular tissue. Nature 177, 1036-1037. Sajiki, J., Fukuda, Y., and Fukushima, E. (1981). On the lipoperoxide concentrations in the viscera of rats intoxicated by cadmium chloride. J. Appl. Biochem.
3, 467-47
I.
Sajiki, J.. Fukushima, E.. and Fujishiro, Y. (1983). Involvement of lipid peroxidation to the early stage of the injury in rat testis induced by cadmium. Eisei Kagaku 29, 389-393. Sajiki, J., Fukushima, Y., Fukuda, Y., Hirai, A., Tamura. Y.. and Yoshida, H. (1985). Effects of methyl-B,* on the inflammation induced by CdC12 in the testis of rats. Jpn. J. Injam. 5, 45-50. Sato, M., Yamanobe, K., and Nagai, Y. (1983). Sex-related differences in cadmium-induced lipid peroxidation in the rat. Life Sri. 33, 903-908. Shukla, G. S., Hussain, T., and Chandra. S. V. (1987). Possible role of regional superoxide dismutase activity and lipid peroxides levels in cadmium neurotoxicity: I~I viva and in vitro studies in growing rats. Life Sci. 41, 22 I52221. Stowe, H. D., Wilson. M., and Goyer, R. A. (1972). Clinical and morphologic effects of oral cadmium toxicity in rabbits. .4rch. Pathol. 94, 398-405. Uchiyama, M.. and Mihara, M. (1978). Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal. Biochem. 86, 27 I 278. Waites. G. M. H.. and Setchell, B. P. (1966). Changes in blood flow and vascular permeability of the testis, epididymis and accessory reproductive organs of the rat after the administration of cadmium chloride. J. Endocrinol.
34, 329-342.
Walker. E. M., Gale. G. R.. Greene, W. B., Veesely, J., Nicholson, J. H., Atkins, L. M., Smith, A. B., Jones, M. M., and Hennigar, G. R. (1984). Effects of substituted dithiocarbamates on the testicular toxicity of cadmium. Res. Commun. Chem. Pathol. Pharmacol. 46, 449-467. Walker, E. M., Gale, G. R., Fody, E. P.. Atkins, L. M., Smith, A. B.. and Jones, M. M. (I 986). Comparative antidotal effects of diethyldithiocarbamate, dimercaptosuccinate. and diethylenetriaminepentaacetate against cadmium-induced testicular toxicity in mice. Res. Common. Chem. Parhol. Pharmacol. 51,23 l-244. Wong, K.-L.. and Klaassen, C. D. (1980). Age difference in the susceptibility to cadmium-induced testicular damage in rats. Tosicol. Appl. Pharmacol. 55,456-466. Yamane. Y.. Li. Z. G., and Koizumi, T. (1990). Mechanism of cadmiuminduced rat testicular cancer. Eisei Kagaku 36, 399-405.
AND
APPLIED
PHARMACOLOGY
116,24-29
(1992)
Effects of Dithiocarbamates on Testicular Toxicity in Rats Caused by Acute Exposure to Cadmium SHOJI KOJIMA,’ Department
Of’Hygienic
Chemistry,
YOSUKE SUGIMURA, HIDEAKI HIRUKAWA, MORIO KIYOZUMI,~ HIDEAKI SHIMADA, AND TAKAYUKI FUNAKOSHI Faculty
of Pharmaceutical Received
January
Sciences,
Kumamoto
22. 1992; accepted
University, May
5-I Oe-honmachi.
N-Benzyl-D-glucamine dithiocarbamate (BGD), N-p-isopropylbenzyl-D-glucamine dithiocarbamate (PBGD), and diethyldithiocarbamate (DED) were compared for their protective effects against the testicular toxicity in rats induced by acute exposure to cadmium. Rats were injected subcutaneously with 109CdCI, (3 mg Cd and 74 kBq of lo9Cd/kg) and 30 min later, they were injected intraperitoneally with the chelating agents (0.4 or 3 mmobkg). Cadmium injection increased lipid peroxidation and concentrations of hemoglobin and Ca in the testes, decreased the testicular weight, and caused sterility. The treatment with BGD (0.4 mmobkg) did not satisfactorily protect against the testicular toxicity of cadmium. The administration of PBGD or DED at a dose of 3 mmobkg significantly prevented the increase in the lipid peroxidation and hemoglobin concentration in the testes, the decrease in the testicular weight, and the sterility caused by cadmium. PBGD and DED significantly decreased the cadmium concentration in the testes, but DED increased the cadmium concentration in the kidney and brain. Only DED significantly prevented the increase in the testicular Ca concentration after cadmium. These results indicate that PBGD and DED protect against the sterility caused by cadmium in rats and that the effect of DED to increase the brain level of cadmium is more dangerous than the lack of effect of PBGD to prevent the increase in the testicular Ca level. The protective effects of PBGD and DED against the cadmium-induced testicular toxicity. presumably result from a decrease in the cadmium . concentration in the testes. 0 1992 Academic press, hc.
20, 1992
MATERIALS Cadmium has been recognized as one of the most toxic industrial and environmental elements of which there is a continuing hazard to human exposure. In experimental animals, cadmium can cause a number of lesions in various organs such as liver, kidney, and testes (Friberg et al., 1974;
0041-008X/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
862, Japan
Stowe et al., 1972; Cook et al., 1974; Hoffmann et al., 1975). Cadmium induces a severe acute toxicity to rat testes at injection doses appreciably lower than those required for toxicity to other organs (Parizek and Zahor, 1956; Fahim and Khare, 1980). The testicular toxicity induced by cadmium injection involves hemorrhage, necrosis, and atrophy (Parizek and Zahor, 1956; Meek, 1959; Cameron and Foster, 1963; Gunn et al., 1965; Gunn and Gould, 1970). Among the various toxic effects induced by cadmium in biological systems, lipid peroxidation has been observed in numerous tissues either in vivo or in vitro (Gabor et al., 1978; Sajiki et al., 1981; Sato et al., 1983: Klimczak et al., 1984; Jamall and Smith, 1985; Muller, 1986; Hussain et al., 1987; Shukla et al., 1987). It has also been demonstrated in vitro that lipid peroxidation is an early intracellular event after cadmium exposure (Muller and Ohnesorge, 1982; Muller, 1986). Chelation therapy for cadmium has been effective in preventing cadmium-induced toxicity. Walker et al. ( 1984, 1986) studied the effects of five dithiocarbamates, dimercaptosuccinate, and diethylenetriaminepentaacetate (DTPA) on the testicular toxicity of cadmium using morphological techniques and indicated that diethyldithiocarbamate (DED) and DTPA were the most effective antagonists if given immediately after cadmium injection. The purpose of the present study was to evaluate the protective effects of N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-isopropylbenzyl-D-glucamine dithiocarbamate (PBGD), the new dithiocarbamates, and DED against the testicular damage resulting from acute exposure to cadmium in rats.
Effects of Dithiocarbamates on Testicular Toxicity in Rats Caused by Acute Exposure to Cadmium. KOJIMA, S., SUGIMURA, Y., HIRUKAWA, H., KIYOZUMI, M., SHIMADA, H., AND FUNAKOSHI, T. (1992). Toxicol. Appl. Pharmacol. 116,24-29.
’ To whom reprint requests should be addressed. ’ Present address: Department of School Health, Faculty Kumamoto University, 40-l Kurokami-2-chome. Kumamoto,
Kumamoto,
AND METHODS
Chemicals. Cadmium chloride and DED were obtained from Wako Pure Chemicals (Osaka). imCd (specific activity, 58.2 GBq/mg) was obtained from New England Nuclear Corp. (Boston, MA). BGD and PBGD were synthesized according to the procedures reported previously from our laboratory (Kojima et al., 1986, 1987). Other chemicals were of reagent grade. Animals and treatments. Male Wistar rats, weighing 180-220 g, were purchased from Kyudo Co.. Ltd. (Kumamoto) and housed in individual metabolic cages with a commercial chow (Nosan Lab Chow) and drinking water ad /i&turn in temperatureand humidity-controlled rooms and on a 12-hr light/dark cycle.
of Education, 860, Japan, 24
CADMIUM-INDUCED
TESTICULAR
The rats were injected subcutaneously with saline (0.5 ml/animal) or 3 mg Cd/kg in 0.5 ml saline. Thirty minutes later they were injected intraperitoneally with saline (control) or chelating agents (0.4 or 3 mmol/kg) in 0.5 ml saline. Three or 7 days after cadmium administration the rats were killed by cervical fracture and testes were removed. Liver and kidney were perfused by injecting ice-cold 0.01 M phosphate buffer (pH 7.4) containing 1.15% KC1 with 0.025% ethylenediaminetetraacetic acid (EDTA) into the inferior vena cava and the liver and kidney were removed. Testes, liver, and kidney were analyzed for lipid peroxidation and hemoglobin. In the experiment for determination of cadmium and essential metals (Ca, Fe, and Zn), the rats were injected intraperitoneally with chelating agents (3 mmol/kg) 30 min after subcutaneous injection of ““Cd (3 mg Cd and 74 kBq of lwCd/ kg) and testes, liver, kidney, heart, and brain were removed 3 or 7 days after cadmium injection. Lipid peroxidation. Testes, liver, and kidney were homogenized with 9 vol of ice-cold 0.0 1 M phosphate buffer (pH 7.4) containing 1.15% KC1 and 0.025% EDTA. Lipid peroxidation was assayed according to the thiobarbituric acid (TBA) method of Uchiyama and Mihara (1978) using 1,1,3,3tetraethoxypropane as the standard and expressed as nanomoles of TBA reactive substances (TBARS). Hemoglobin. Testes were homogenized with 9 vol of ice-cold 0.01 M phosphate buffer (pH 7.4) as described earlier and the homogenate was centrifuged at 2000g for 10 min at 4°C. Hemoglobin content in the supematant was determined according to the method of Sajiki et al. (1983) and expressed as mg/g wet tissue. Protein. Protein was determined according to the method of Lowry et al. (195 1). using bovine serum albumin as the standard. Metals. Cadmium content in various organs was determined by measuring the amount of lwCd radioactivity with an Aloka autowell y-scintillation counter (model ARC 300). Testes, liver, and kidney were wet-ashed by the HC104-HNOj method and essential metals (Ca, Fe, and Zn) were determined using a Hitachi Z-8000 atomic absorption instrument. Fertility test. Fertility of male rats given cadmium with and without chelating agent was determined according to the method of Jackson et al. (1961). The rat was mated with a virgin female rat for 8 days, starting 52 days after administration of cadmium with and without chelating agent. The fertility rate was expressed as the number of females impregnated versus the number of males tested. Statistical analysis. Data were analyzed by a one-way analysis of variance. When the analysis indicated that a significant difference existed, the treated groups were compared to the controls by Duncan’s new multiple range test.
RESULTS The lipid peroxidation and hemoglobin concentrations in the testes and the testicular weight of rats after cadmium administration are shown in Fig. 1. The lipid peroxidation in the testes significantly increased compared to the control at 12 hr after cadmium. Similarly, Manta et al. ( 199 1) reported that lipid peroxidation in the testes of rats significantly increased over control level within 12 hr after injection of cadmium (1 mg Cd/kg). The hemoglobin concentration in the testes also increased significantly 1 day after cadmium injection, indicating the testicular hemorrhage. The testicular weight decreased significantly after 5 days of cadmium administration. Table 1 shows the concentrations of cadmium and essential metals (Ca, Fe, and Zn) in the testes after cadmium injection. The Fe concentration significantly increased 2 days after cadmium, suggesting hemorrhage. The Ca con-
TOXICITY
o!
40
0
25
,
I
I
I
I
I
I
1
0
1
2
3
4
5
6
7
1
I
a
1
I
I
0
1
2
a
I
I
3 4 Time (day)
I
I
1
5
6
7
FIG. 1. Time course of lipid peroxidation and hemoglobin concentrations in testes and testicular weight of rats after cadmium administration. Rats were injected subcutaneously with cadmium (3 mg Cd/kg). The values represent the mean f SD for four animals. 0, control; 0, Cd. ‘Significantly different from control (a < 0.05).
centration significantly increased 1 day after cadmium and the Ca concentrations at 3 and 7 days were approximately 15 and 60 times larger, respectively, than those of the control. The Zn concentration was not affected by cadmium injection. We investigated the protective effects of BGD, PBGD, and DED against the testicular toxicity of cadmium. First when BGD (0.4 mmol/kg) was injected 30 min after cadmium, it prevented the increases in lipid peroxidation and hemoglobin concentration in the testes within 1 day after cadmium and the decrease in the testicular weight within 3
KOJIMA
26
TABLE 1 Concentration of Cadmium and EssentialMetals in Testes of Rats after Cadmium Administration Metal Days after administration Control I 2 3 5 7
concentration
0.00 0.24 0.33 0.40 0.86 0.94
k 0.00 + 0.03 i 0.05 ? 0.21 zk 0.18 * 0.14
27.8 5.09 235.6 428.9 1068.8 1696.0
f 0.3 + 7.9” + 46.3” f 30.5” k 108.6” k 245.3”
TABLE 2 Effects of Chelating Agents on Lipid Peroxidation in Testes, Liver, and Kidney of Rats after Cadmium Administration Lipid peroxidation (nmol TBARS/mg protein)
(Irp/p wet tissue)
Ca
Cd
ET AL.
Fe 16.6 16.6 32.9 30.0 28.1 32.8
?I f + t + +
Zn 0.3 2.5 13.5” 0.8” 4.1” 6.8”
19.9 18.3 17.6 17.4 20.3 21.7
* + + + + +
Treatment 0.3 2.1 1.5 0.1 2.5 2.4
Noie. Rats were injected subcutaneously with cadmium (3 mg Cd/kg). The values represent the mean f SD for four animals. a Significantly different from control (p < 0.05).
days, but did not protect against such testicular toxic changes thereafter (data not shown). Next BGD, PBGD, and DED at a dose level of 3 mmol/kg were administered 30 min after cadmium and the effects of the chelating agents on cadmiuminduced testicular toxicity were examined. As shown in Table 2, PBGD and DED completely prevented the increase in the lipid peroxidation during 7 days after cadmium injection. BGD significantly prevented the increase in the lipid peroxidation within 3 days after cadmium injection, but did not afford the protective effect at 7 days. The lipid peroxidation in the liver and kidney after cadmium did not significantly change compared to the control. As shown in Table 3, a significant change in the testicular weight as an indicator of the testicular toxicity did not occur 3 days after cadmium injection, but the testicular weight significantly decreased at 7 days. DED completely prevented the decrease in the testicular weight after cadmium injection, but BGD and PBGD failed to protect the weight loss. The increase in the hemoglobin concentration after cadmium injection was significantly prevented by PBGD and DED, but not by BGD (Table 3). The tissue concentrations of cadmium in rats treated with the chelating agents after cadmium are shown in Table 4. PBGD treatment significantly decreased the cadmium concentrations in the testes and heart 7 days after cadmium injection. DED significantly decreased the cadmium concentrations in the testes, liver, and heart, but caused a significant increase in the cadmium concentrations in the kidney and brain. BGD treatment had little effect on cadmium concentrations in the tissues. As shown in Table 5, the increases in the Ca and Fe concentrations in the testes after cadmium injection were significantly prevented by DED, but not by BGD or PBGD. The Zn concentration in the testes was affected little by the administration of cadmium with and without the chelating agents. In the liver and kidney, the Zn concentration significantly increased, but the Ca and Fe concentrations changed little.
Liver
Testes
Kidney
3 Days after cadmium Control Cd Cd + BGD Cd + PBGD Cd + DED
1.22 * 3.18 + 1.53 f 0.97 * 1.09 t
0.07 0.53” o.79b 0.04b 0.16’
0.78 0.68 0.35 0.51 0.55
f 0.19 5 0.14 z!I 0.22 + 0.10 f 0.08
0.97 1.17 0.64 0.90 0.74
+ 0.16 + 0.46 k 0.19 +_0.20 1 0.11
k + 5 f f
0.79 0.82 0.71 0.68 0.73
t + + f f
7 Days after cadmium Control Cd Cd + BGD Cd + PBGD Cd + DED
1.39 i 3.63 i 3.64 + 0.80 f 1.05 f
0.2 1 0.29” 0.54” 0.08’ 0.13b
0.52 0.41 0.43 0.43 0.61
0.09 0.01 0.01 0.10 0.08
0.14 0.16 0.03 0.17 0.03
Note. Rats were injected intraperitoneally with chelating agents (3 mmol/ kg) 30 min after subcutaneous injection of cadmium (3 mg Cd/kg). The rats were killed 3 or 7 days after cadmium and lipid peroxidation in the organs was determined. The values represent the mean * SD for four animals. a Significantly different from control (p < 0.05). b Significantly different from Cd (p < 0.05).
Furthermore, the fertility of male rats given cadmium with and without the chelating agents was examined (Table 6). The sterility produced by cadmium was completely prevented by DED and PBGD, but not by BGD. TABLE 3
Changes in Testicular Weight and Hemoglobin Concentration of Rats after Administration of Cadmium or Cadmium and Chelating Agents Hemoglobin concentration (w/g wet tissue)
Testicular weight (9%of body weight) Treatment Control Cd Cd + BGD Cd + PBGD Cd + DED
3 Days 1.04 k 0.87 k 0.94 f 1.11 + 1.22 f
0.14 0.06 0.06 0.07 0.17
7 Days 1.05 0.12 0.62 0.72 1.19
k + f f *
0.11 0.06“ 0.06” 0.09” 0.07”
3 Days 3.43 9.76 8.82 5.75 5.02
-t 0.48 + 0.4Y -+ 2.10” 2 2.10h + 0.92’
Note. Rats were injected intraperitoneally with chelating agents (3 mmol/ kg) 30 min after subcutaneous injection of cadmium (3 mg Cd/kg). The rats were killed 3 or 7 days after cadmium and testicular weight or hemoglobin concentration was determined. The values represent the mean i SD for four
animals.
LISignificantly different from control (p < 0.05). b Significantly different from Cd (p < 0.05).
CADMIUM-INDUCED
TESTICULAR
27
TOXICITY
TABLE 4 Effects of Chelating Agents on Tissue Concentration of Cadmium Concentration of cadmium (pg/g wet tissue) Treatment
Testes
Liver
Kidney
Heart
Brain
3 Days after cadmium Cd Cd + BGD Cd + PBGD Cd + DED
0.40 0.20 0.37 0.30
+ f f f
0.2 1 0.04 0.06 0.05
23.3 19.8 21.6 7.3
+ 5.6 + 1.6 f 3.9 ?I 1.2”
14.9 11.4 8.0 12.5
f f + t
1.3 0.9” 0.5” 1.9
1.61 1.09 1.85 0.73
f 0.68 f 0.27 + 0.37 zlz0.07
17.8 f 15.7 f 15.9 + 33.0 f
3.9 0.9 1.2 8.5”
2.34 + 2.06 f 1.40 f 1.08 t
0.23 0.16 0.23 0.35
f 0.08 +- 0.05 k 0.03 k 0.06
0.20 0.18 0.13 0.46
+ f + +
7 Days after cadmium Cd Cd + BGD Cd + PBGD Cd + DED
0.94 0.71 0.52 0.39
f + + +
0.14 0.28 0.10” 0.05”
24.7 22.8 18.2 9.7
+ 6.8 -c 5.7 + 1.9 zk 3.0”
0.33 0.33 0.05” 0.02“
0.09 0.04 0.06 0.03”
Note. Rats were injected intraperitoneally with chelating agents (3 mmol/kg) 30 min after subcutaneous injection of ‘@‘Cd (3 mg Cd and 74 kBo of “‘Cd/kg). The rats were killed 3 or 7 days after cadmium and cadmium concentration was determined from radioactivity. The values represent the mean + SD for four animals. ’ Significantly different from Cd (p -c 0.05).
DISCUSSION The present study was carried out to examine the protective effects of the dithiocarbamates on the testicular toxicity of cadmium in rats. It has been reported that cadmium injection can result in severe hemorrhagic necrosis (Gunn et al., 1965; Gunn and Gould, 1970) and enhancement of lipid peroxidation in the testes (Gabor et al., 1978; Sajiki et al., 1981; Sato et al., 1983; Klimczak et al., 1984). Manta et al. (199 1) have demonstrated that lipid peroxidation is an early and sensitive consequence of cadmium exposure as determined in various organs such as lung, brain, testes, liver, kidney, and heart. Our results also indicated significant increases in the lipid peroxidation in the testes at 12 hr after cadmium injection and in hemoglobin concentration in the testes at 1 day. This suggests a relationship between lipid peroxidation and hemorrhage in the testes, as reported by Sajiki et al. (198 1). The marked increase in the Ca concentration in the testes after cadmium injection was also previously reported (Sajiki et al., 1985; Yamane et al., 1990). This is considered to be due to the cadmium-induced cellular damage in the testes. However, the detailed mechanisms by which cadmium increases the testicular Ca concentration remain unknown. The decrease in the testicular weight occurred 5 days after cadmium administration, indicating that the cadmium-induced testicular damage involves the initial hemorrhagic necrosis, followed finally by the testicular atrophy. The testicular damage caused by cadmium as shown in this study is similar to what has been reported in earlier papers (Meek, 1959; Parizek, 1960; Cameron and Foster, 1963). Wong and Klaassen (1980) have reported that there
are two hypotheses for the mechanism of the toxic response of cadmium to the testes. Our results support one hypothesis that cadmium-induced testicular toxicity is due to vascular changes in the testes leading to hemorrhage, necrosis, and atrophy (Gunn et al., 1963, 1965; Niemi and Komano, 1965; Waites and Setchell, 1966; Aoki and Hoffer, 1978). The second hypothesis is that cadmium causes testicular damage by competing with Zn in Zn-requiring enzymes in the testes (Gunn et al., 196 1; Hodgen et al., 1969). However, our data showed that cadmium had little affect on the Zn concentration in the testes. The effect of cadmium on Zn-containing enzymes in the testes should be further studied. As the testicular toxicity of cadmium was not satisfactorily prevented by the treatment with BGD (0.4 mmol/kg), we further studied the protective effects of BGD, PBGD, and DED at a higher dose level (3 mmol/kg) on the testicular toxicity of injected cadmium. PBGD and DED completely prevented the increase in the lipid peroxidation in the testes during 7 days after cadmium, whereas the protective effect of BGD was less than that of PBGD or DED. PBGD and DED significantly prevented the increase in the testicular hemoglobin concentration, which is considered to be due to the hemorrhagic inflammation, and also afforded a complete protection against the sterility caused by cadmium. Among the chelating agents used only DED significantly decreased the cadmium concentrations in the testes, liver, and heart, but, on the other hand, also significantly increased the cadmium concentrations in the kidney and brain. PBGD also decreased the cadmium concentration in the testes. From these results, the protective effect of the chelating agents against the cadmium-induced testicular toxicity is thought
28
KOJIMA
ET AL.
TABLE 5 Effects of Chelating Agents on TissueConcentration of EssentialMetals
Metal
Days after cadmium
Concentration of metal (fig/g wet tissue) Control
Cd
Cd + BGD
Cd + PBGD
Cd + DED
Testes Ca
25.9 + 0.9 21.4 k 1.8 12.1 + 2.9 Il.6 k 2.6 21.3 + 0.9 18.4 f 0.9
Fe Zn
488.7 1421.7 36.0 33.1 21.7 20.0
f 74.5” + 209.9” f 10.4” + 3.8” ?I 5.5 + 6.4
421.4 1580.3 21.7 33.3 32.4 24.7
2 44.8” k 218.4” t 3.7b -i- I .4” + 6.9 k 0.6
13.4 2769.1 13.5 23.2 24.7 26.6
f 3.8ab It 404.3’,” f 2.8’ f 2.7”,h f 6.1 f 15.27
22.9 2 9.4’ 15.0 f 0.5b 12.5 f 2.7’ 13.4 f 3.9b 18.4 f 3.7 28.0 ?I 9.3
Liver Ca Fe Zn
I
7 7
5.6 f 0.7 39.4 2 9.4 18.5 f 2.0
6.2 k 1.2 37.5 f 14.9 38.6 zk 13.9”
6.9 k 37.8 f 36.7 +
0.9 2.2 5.4”
9.4 It41.8 f 36.7 +
2.lU.b 5.8 3.2”
5.5 f 0.8 39.7 zk 9.5 31.8 + 3.0”
12.8 f 24.2 f 21.2 I?
0.8 2.6 0.8”
17.0 f 13.0 f 23.2 k
2.2 5.76 2.6”
11.6 f 0.9 22.5 + I.8 24.1 IfI 1.7”
Kidney Ca Fe Zn
12.4 k 0.8 19.7 + 2.4 13.2 + 0.2
14.7 Tk 7.2 26.0 f 4.7 25.9 f 8.2”
Note. Rats were injected intraperitoneally with chelating agents (3 mmol/kg) 30 min after subcutaneous injection of ‘@‘Cd (3 mg Cd and 74 kBq of ‘09Cd/kg). The rats were killed 3 or 7 days after cadmium administration and concentration of metal was determined by atomic absorption methods. The values represent the mean f SD for four animals. ’ Significantly different from control (p < 0.05). b Significantly different from Cd (p < 0.05).
to be due to a decrease in the accumulation of cadmium in the testes. The increase in the testicular Ca concentration and the decrease in the testicular weight were completely prevented only by DED among the chelating agents tested. Thus, the marked accumulation of Ca in the testes after cadmium injection appears to be not responsible for the cadmium-induced sterility. More studies are needed to further elucidate the mechanism by which PBGD protects against the testicular toxicity caused by cadmium. TABLE 6 Effects of Chelating Agents on Fertility of Rats after Cadmium Administration Treatment
Fertility rate
Control Cd Cd + BGD Cd + PBGD Cd + DED
717 O/7 O/7 717 717
Note. Male rats were injected intraperitoneally with chelating agents (3 mmol/kg) 30 min after cadmium administration (3 mg Cd/kg). The rat was mated with a virgin female rat for 8 days, starting 52 days after cadmium. The fertility rate was expressed as the number of females impregnated versus the number of males tested.
The present study indicates that PBGD and DED protect against the sterility caused by cadmium in rats, that the effect of DED to increase the brain level of cadmium is more dangerous than the lack of effect of PBGD to prevent the increase in the testicular Ca level, and that BGD provides no satisfactory protection against the cadmium-induced testicular toxicity. REFERENCES Aoki. A., and Hoffer. A. P. (1978). Reexamination ofthe lesions in rat testis caused by cadmium. Biol. Reprod. 18, 579-59 I. Cameron, E.. and Foster, C. L. (1963). Observations on the histological effectsof sub-lethal doses of cadmium chloride in the rabbit. 1. The effect on the testis. J. .4nat. 97, 269-280. Cook. J. A., Marconic. E. A., and Diluzio, N. R. (1974). Lead, cadmium, endotoxin interaction: Effect on mortality and hepatic function. Toxicol. Appi. Pharmacol.
28,292-302.
Fahin, M. S., and Khare, N. K. (1980). Effects of subtoxic levels of lead and cadmium on urogenital organs of male rats. Arch. Androl. 4, 357-362. Friberg, L., Piscator, M., Nordberg, G. F., and Kjellstrom, T. (1974). C’admium in the environment, 2nd ed. CRC Press, Cleveland, OH. Gabor, S.. Anca, Z., and Bordas, E. (1978). Cadmium-induced lipid peroxidation in kidney and testes: Effect of zinc and copper. Rev. Roum. Biochem. 15, I 13- I 17. Gunn. S. A.. Gould, T. C.. and Anderson, W. A. D. (1961). Competition of cadmium for zinc in rat testis and dorsolateral prostate. Acta Endocrinol. 37, 24-30.
CADMIUM-INDUCED
TESTICULAR
Gunn, S. A., Gould, T. C., and Anderson, W. A. D. (1963). The selective injurious response of testicular and epididymal blood vessel to cadmium and its prevention by zinc. Am J. Pathol. 42, 685-702. Gunn, S. A., Gould, T. C., and Anderson, W. A. D. (1965). Strain differences in susceptibility of mice and rats to cadmium-induced testicular damage. J. Reprod.
Fertil.
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