TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
SHORT Protective
Effect
(1975)
%,517-520
COMMUNICATION
of Phenobarbital
on Cadmium
Toxicity
in Mice
Protective Effect of Phenobarbital on Cadmium Toxicity in Mice. H. AND OHSAWA, M. (1975). Toxicol. Appl. Pharmacol. 34, 517-520. Prior administration of phenobarbital (30 mg/kg, sc, for 5 days) produced a significant reduction in mortality of male mice after cadmium, lead, and inorganic mercury and a trend toward reduced mortality after copper and methylmercury. Phenobarbital also increased cadmium accumulation in the liver, but decreased it in the kidney. No difference in cadmium excretion wasfound between the control and the treated groups. These findings suggest that phenobarbital may exert its protective effect by increasing cadmium deposition in the liver.
YOSHIKAWA,
The prior or simultaneous administration of cobalt (Gabbiani et al., 1967), selenium (Gunn et al., 1968), or zinc (Webb, 1972a) has prevented gonadal atrophy induced by cadmium. It also has been found that a small dose of cadmium before the subsequent lethal dose can remarkably reduce mortality in mice (Yoshikawa, 1970). Rats pretreated with a small dose of cadmium accumulate significantly higher amounts of cadmium in the liver 24 hr after a subsequent large dose (Yoshikawa, 1973), but not in the kidney, lung, and testis. Moreover, pretreatment with a small dose of cadmium increases the amount of cadmium bound to a low-molecular-weight protein in the liver (Suzuki and Yoshikawa, 1974), which corresponds to metallothionein (KBgi and Vallee, 1960). It, therefore, is probable that such a protective effect is due partially to the induction of cadmium-binding protein. The protective effect of pretreatment with metals also occurs with other metals like indium and lead (Yoshikawa, 1970) which are unlikely to induce metallothionein (Webb, 1972b). Recently, Fowler et al. (1973) reported that phenobarbital was able to prevent the nephrotoxicity of methylmercury which does not form a metallothionein complex (Chen et al., 1973). The present study investigates the possibility that phenobarbital could affect cadmium mortality and distribution of cadmium among various organs in mice. METHODS Mortality test. Male ICR mice weighing 30-35 g were given 30 mg/kg phenobarbital, SC,once a day for 5 days, followed by a single ip injection of a sublethal dose of a heavy metal, as shown in Table I, 24 hr after the last phenobarbital injection. The metals used were the chloride salts of cadmium, manganese, mercury, and methylmercury, the sulfate salts of copper and zinc, and lead nitrate. Control mice not treated with phenobarbital were given the corresponding single dose of heavy metals on the same day. The number of the dead animals was counted for 7 days after administration of the heavy metals. Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
18
517
518
SHORT
Determination
COMMUNICATION
of cadmium in organs, feces, and urine. Twenty male ICR mice weighing
36-46 g were kept in metabolic cages with a device for separate collection of feces and urine. The treated group of animals was given phenobarbital for 5 days. On Day 6 they were injected ip with 3 mg Cd/kg. Feces and urine samples were collected over 24 hr from each of three animals and pooled. On Days 1 and 4 after cadmium administration the animals were killed and liver and kidneys were obtained. Cadmium in these samples was determined by atomic absorption spectrometry after wet-ashing of samples with nitric acid and hydrogen peroxide. RESULTS Table 1 shows the mortality
metals. Phenobarbital
AND
DISCUSSION
in mice 7 days after the administration
of the heavy
significantly protected mice against the lethal effects of cadmium, TABLE
1
EFFECT OF PHENOBARBITAL ON MORTALITY BY HEAVY METALS IN MICE’
Treatment Cd Phenobarbital-Cd Pb Phenobarbital-Pb CU Phenobarbital-Cu Zn Phenobarbital-Zn Mn Phenobarbital-Mn Hg Phenobarbital-Hg MeHgC Phenobarbital-MeHg
Dose (mg/kg)
Mortality (dead/total)
4 4 60 60 4 4 15 15 50 50 4.5 4.5 7.5 7.5
8110 l/10 9/10 4/10 9110 5/10 8/10 7110 8110 8110 9/10 4110 8/10 4/10
Significanceb
p < 0.005 p < 0.05 NS NS NS p < 0.05 NS
0After daily administrationof phenobarbital(30 mg/kg, SC)for 5 days malemiceweregivenan ip injectionof eachmetalcompound.Numberof deadanimals7 daysafter administration of themetalsisshown. bp Valueswerecalculated.by Fischer’sexactmethod.NS meansp> 0.05. c Methylmercury.
lead, and inorganic mercury, and tended to reduce the mortality due to copper and methylmercury, but not manganese. Fecal and urinary cadmium content 24 hr after the administration of cadmium was 19.8 f 0.1 and 0.28 + 0.01 pg/3 mice in control (mean &SE, N= 3), and 20.0 f 0.1 and 0.28 + 0.03 pg/3 mice in the treated (N = 3), respectively. Although phenobarbital has been shown to enhance the biliary excretion of methylmercury (Magos et al., 1974), the drug seems to have no effect on fecal and urinary excretion of cadmium. Table 2 shows that phenobarbital significantly increased the cadmium content in the liver 4 days after Cd administration but decreasedit in the kidney. In a preliminary
Phenobarbital-Cd Cd
Kidney
32.9 + 1.3 25.5 + 1.0 p < 0.01 8.07 + 0.27 10.33 + 0.66 p < 0.025 59.5 + 2.4 42.8 -t 1.4 p < 0.005 4.68 + 0.46 6.30 + 0.44 p < 0.05
burden per organ (%)
of body
OF CADMIUM
Percentage
Day lb
DISTRIBUTION
Cd content bdg wet wt)
ON THE
2 IN
+_2.1 & 1.4 0.005 + 0.87 -+ 1.32
NS’
35.2 21.1 p< 9.92 13.18
Cd content hdi3 wet w0
THE
KIDNEY’
60.6k 3.1 44.8 f 1.7 p < 0.01 5.21 + 0.56 7.48 f 0.49 p < 0.05
Percentageof body burden per organ (%)
Day 4
THE LIVER AND
(1Mice were given 3 mg/kg of cadmium, ip, after daily administration of phenobarbital (30 mg/kg, SC)for 5 days; 1 and 4 days later cadmium content in organs was determined. All values in the table represent the mean +_SE (N = 5); p values were calculated by Student’s t test. h Day after the cadmium injection. CNS means p z 0.05.
Phenobarbital-Cd Cd
Group
OF PHENOBARBITAL
Liver
Organ
EFFECT
TABLE
520
SHORTCOMMUNICATION
experiment in mice, no marked increasein cadmium bound to hepatic microsomeswas found in animals pretreated with phenobarbital when compared with controls. The amount of cadmium bound to a low-molecular-weight cadmium-binding protein in the liver slightly increased in the group treated with phenobarbital but not as much as observed in rats pretreated with a small dose of cadmium (Suzuki and Yoshikawa, 1974). Thesefindings suggestthat augmentation of cadmium accumulation in the liver may contribute to a decreased cadmium concentration in critical organs, thereby reducing mortality in mice. The shift in cadmium to the liver was similar to that observed in rats pretreated with a small dose of cadmium (Yoshikawa, 1973). The results of the present experiment suggest a mechanism other than induction of metallothionein for the protective effect of phenobarbital on metal toxicity. REFERENCES CHEN, R. W., GANTHER, H. E. AND HOEKSTRA, W. G. (1973).Studieson the binding of methylmercury by thionein. Biochem.Biophys.Res.Commun.51, 383-390. FOWLER, B. A., LUCIER, G. W., FOLSOM, M. D. AND BROWN, H. W. (1973).Phenobarbital
protection againstmethylmercury nephrotoxicity. Environ. Hlth. Perspect.4, 100-101. GABBIANI, G., BASIC, D. AND DEZIEL, G. (1967).Studieson toleranceand ionic antagonism
for cadmiumor mercury. Can.J. Physiol. Pharmacol.45,443-450. GUNN, S. A., GOULD, T. C. AND ANDERSON, W. A. D. (1968).Selectivity of organ responseto
cadmiun injury and various protective measures.J. Pathol. Bacterial. %, 89-96. J. H. R. AND VALLEE, B. L. (1960).Metallothionein: A cadmium-and zinc-containing protein from equinerenal cortex. J. Biol. Chem.235, 3460-3465. MACKIS, L., MACGREGOR, J. T. AND CLARKSON, T. W. (1974).The effect of phenobarbitaland sodium dehydrocholate on the biliary excretion of methylmercury in the rat. Toxicol. Appl. Pharmacol.30, l-6. SUZLJKI,~. ANDYOSHIKAWA, H. (1974).Roleofmetallothionein in theliver in protection against cadmiumtoxicity. Znd.Health 12, 141-151. WEBB, M. (1972a).Protection by zinc againstcadmium.Biochem.Pharmacol.21,2767-2771. WEBB, M. (1972b). Binding of cadmium ions by rat liver and kidney. Biochem.Pharmacol. K&q
21,2751-2765. YOSHIKAWA, H. (1970).Preventive effect of pretreatmentwith low doseof metalson the acute
toxicity of metalsin mice. Znd.Health 8, 184-191. YOSHIKAWA, H. (1973). Preventive effectsof pretreatment with cadmiumon acute cadmium poisoningin rats. Znd.Health 11, 113-l 19. HIROSHI YOSHIKAWA MOTOYASU OHSAWA
Departmentof OccupationalDiseases National Institute of Industrial Health Kizukisumiyoshi,Nakahara-ku, Kawasaki,Japan ReceivedApril 19, 1975,acceptedJuly IS,1975
AND
APPLIED
PHARMACOLOGY
SHORT Protective
Effect
(1975)
%,517-520
COMMUNICATION
of Phenobarbital
on Cadmium
Toxicity
in Mice
Protective Effect of Phenobarbital on Cadmium Toxicity in Mice. H. AND OHSAWA, M. (1975). Toxicol. Appl. Pharmacol. 34, 517-520. Prior administration of phenobarbital (30 mg/kg, sc, for 5 days) produced a significant reduction in mortality of male mice after cadmium, lead, and inorganic mercury and a trend toward reduced mortality after copper and methylmercury. Phenobarbital also increased cadmium accumulation in the liver, but decreased it in the kidney. No difference in cadmium excretion wasfound between the control and the treated groups. These findings suggest that phenobarbital may exert its protective effect by increasing cadmium deposition in the liver.
YOSHIKAWA,
The prior or simultaneous administration of cobalt (Gabbiani et al., 1967), selenium (Gunn et al., 1968), or zinc (Webb, 1972a) has prevented gonadal atrophy induced by cadmium. It also has been found that a small dose of cadmium before the subsequent lethal dose can remarkably reduce mortality in mice (Yoshikawa, 1970). Rats pretreated with a small dose of cadmium accumulate significantly higher amounts of cadmium in the liver 24 hr after a subsequent large dose (Yoshikawa, 1973), but not in the kidney, lung, and testis. Moreover, pretreatment with a small dose of cadmium increases the amount of cadmium bound to a low-molecular-weight protein in the liver (Suzuki and Yoshikawa, 1974), which corresponds to metallothionein (KBgi and Vallee, 1960). It, therefore, is probable that such a protective effect is due partially to the induction of cadmium-binding protein. The protective effect of pretreatment with metals also occurs with other metals like indium and lead (Yoshikawa, 1970) which are unlikely to induce metallothionein (Webb, 1972b). Recently, Fowler et al. (1973) reported that phenobarbital was able to prevent the nephrotoxicity of methylmercury which does not form a metallothionein complex (Chen et al., 1973). The present study investigates the possibility that phenobarbital could affect cadmium mortality and distribution of cadmium among various organs in mice. METHODS Mortality test. Male ICR mice weighing 30-35 g were given 30 mg/kg phenobarbital, SC,once a day for 5 days, followed by a single ip injection of a sublethal dose of a heavy metal, as shown in Table I, 24 hr after the last phenobarbital injection. The metals used were the chloride salts of cadmium, manganese, mercury, and methylmercury, the sulfate salts of copper and zinc, and lead nitrate. Control mice not treated with phenobarbital were given the corresponding single dose of heavy metals on the same day. The number of the dead animals was counted for 7 days after administration of the heavy metals. Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
18
517
518
SHORT
Determination
COMMUNICATION
of cadmium in organs, feces, and urine. Twenty male ICR mice weighing
36-46 g were kept in metabolic cages with a device for separate collection of feces and urine. The treated group of animals was given phenobarbital for 5 days. On Day 6 they were injected ip with 3 mg Cd/kg. Feces and urine samples were collected over 24 hr from each of three animals and pooled. On Days 1 and 4 after cadmium administration the animals were killed and liver and kidneys were obtained. Cadmium in these samples was determined by atomic absorption spectrometry after wet-ashing of samples with nitric acid and hydrogen peroxide. RESULTS Table 1 shows the mortality
metals. Phenobarbital
AND
DISCUSSION
in mice 7 days after the administration
of the heavy
significantly protected mice against the lethal effects of cadmium, TABLE
1
EFFECT OF PHENOBARBITAL ON MORTALITY BY HEAVY METALS IN MICE’
Treatment Cd Phenobarbital-Cd Pb Phenobarbital-Pb CU Phenobarbital-Cu Zn Phenobarbital-Zn Mn Phenobarbital-Mn Hg Phenobarbital-Hg MeHgC Phenobarbital-MeHg
Dose (mg/kg)
Mortality (dead/total)
4 4 60 60 4 4 15 15 50 50 4.5 4.5 7.5 7.5
8110 l/10 9/10 4/10 9110 5/10 8/10 7110 8110 8110 9/10 4110 8/10 4/10
Significanceb
p < 0.005 p < 0.05 NS NS NS p < 0.05 NS
0After daily administrationof phenobarbital(30 mg/kg, SC)for 5 days malemiceweregivenan ip injectionof eachmetalcompound.Numberof deadanimals7 daysafter administration of themetalsisshown. bp Valueswerecalculated.by Fischer’sexactmethod.NS meansp> 0.05. c Methylmercury.
lead, and inorganic mercury, and tended to reduce the mortality due to copper and methylmercury, but not manganese. Fecal and urinary cadmium content 24 hr after the administration of cadmium was 19.8 f 0.1 and 0.28 + 0.01 pg/3 mice in control (mean &SE, N= 3), and 20.0 f 0.1 and 0.28 + 0.03 pg/3 mice in the treated (N = 3), respectively. Although phenobarbital has been shown to enhance the biliary excretion of methylmercury (Magos et al., 1974), the drug seems to have no effect on fecal and urinary excretion of cadmium. Table 2 shows that phenobarbital significantly increased the cadmium content in the liver 4 days after Cd administration but decreasedit in the kidney. In a preliminary
Phenobarbital-Cd Cd
Kidney
32.9 + 1.3 25.5 + 1.0 p < 0.01 8.07 + 0.27 10.33 + 0.66 p < 0.025 59.5 + 2.4 42.8 -t 1.4 p < 0.005 4.68 + 0.46 6.30 + 0.44 p < 0.05
burden per organ (%)
of body
OF CADMIUM
Percentage
Day lb
DISTRIBUTION
Cd content bdg wet wt)
ON THE
2 IN
+_2.1 & 1.4 0.005 + 0.87 -+ 1.32
NS’
35.2 21.1 p< 9.92 13.18
Cd content hdi3 wet w0
THE
KIDNEY’
60.6k 3.1 44.8 f 1.7 p < 0.01 5.21 + 0.56 7.48 f 0.49 p < 0.05
Percentageof body burden per organ (%)
Day 4
THE LIVER AND
(1Mice were given 3 mg/kg of cadmium, ip, after daily administration of phenobarbital (30 mg/kg, SC)for 5 days; 1 and 4 days later cadmium content in organs was determined. All values in the table represent the mean +_SE (N = 5); p values were calculated by Student’s t test. h Day after the cadmium injection. CNS means p z 0.05.
Phenobarbital-Cd Cd
Group
OF PHENOBARBITAL
Liver
Organ
EFFECT
TABLE
520
SHORTCOMMUNICATION
experiment in mice, no marked increasein cadmium bound to hepatic microsomeswas found in animals pretreated with phenobarbital when compared with controls. The amount of cadmium bound to a low-molecular-weight cadmium-binding protein in the liver slightly increased in the group treated with phenobarbital but not as much as observed in rats pretreated with a small dose of cadmium (Suzuki and Yoshikawa, 1974). Thesefindings suggestthat augmentation of cadmium accumulation in the liver may contribute to a decreased cadmium concentration in critical organs, thereby reducing mortality in mice. The shift in cadmium to the liver was similar to that observed in rats pretreated with a small dose of cadmium (Yoshikawa, 1973). The results of the present experiment suggest a mechanism other than induction of metallothionein for the protective effect of phenobarbital on metal toxicity. REFERENCES CHEN, R. W., GANTHER, H. E. AND HOEKSTRA, W. G. (1973).Studieson the binding of methylmercury by thionein. Biochem.Biophys.Res.Commun.51, 383-390. FOWLER, B. A., LUCIER, G. W., FOLSOM, M. D. AND BROWN, H. W. (1973).Phenobarbital
protection againstmethylmercury nephrotoxicity. Environ. Hlth. Perspect.4, 100-101. GABBIANI, G., BASIC, D. AND DEZIEL, G. (1967).Studieson toleranceand ionic antagonism
for cadmiumor mercury. Can.J. Physiol. Pharmacol.45,443-450. GUNN, S. A., GOULD, T. C. AND ANDERSON, W. A. D. (1968).Selectivity of organ responseto
cadmiun injury and various protective measures.J. Pathol. Bacterial. %, 89-96. J. H. R. AND VALLEE, B. L. (1960).Metallothionein: A cadmium-and zinc-containing protein from equinerenal cortex. J. Biol. Chem.235, 3460-3465. MACKIS, L., MACGREGOR, J. T. AND CLARKSON, T. W. (1974).The effect of phenobarbitaland sodium dehydrocholate on the biliary excretion of methylmercury in the rat. Toxicol. Appl. Pharmacol.30, l-6. SUZLJKI,~. ANDYOSHIKAWA, H. (1974).Roleofmetallothionein in theliver in protection against cadmiumtoxicity. Znd.Health 12, 141-151. WEBB, M. (1972a).Protection by zinc againstcadmium.Biochem.Pharmacol.21,2767-2771. WEBB, M. (1972b). Binding of cadmium ions by rat liver and kidney. Biochem.Pharmacol. K&q
21,2751-2765. YOSHIKAWA, H. (1970).Preventive effect of pretreatmentwith low doseof metalson the acute
toxicity of metalsin mice. Znd.Health 8, 184-191. YOSHIKAWA, H. (1973). Preventive effectsof pretreatment with cadmiumon acute cadmium poisoningin rats. Znd.Health 11, 113-l 19. HIROSHI YOSHIKAWA MOTOYASU OHSAWA
Departmentof OccupationalDiseases National Institute of Industrial Health Kizukisumiyoshi,Nakahara-ku, Kawasaki,Japan ReceivedApril 19, 1975,acceptedJuly IS,1975
