Toxicology, 69 (1991) 143-149 Elsevier Scientific Publishers Ireland Ltd.
143
Induction of metallothionein in mouse liver by valproic acid Masayuki Kaji* and Haruki Mikawa Department of Pediatrics, Kyoto University School of Medicine, Shogoin-Kawahara-cho, Sakyo-ku. Kyoto 606 (Japan) (Received January 30th, 1991; accepted June 4th, 1991)
Summary Intraperitoneal injection of valproic acid (VPA) induces metallothionein (MT) in mouse liver. There was a clear-cut dose-dependency and the maximal amount of MT induced by VPA was about 6 times the basal level. The hepatic MT level reached the peak value at 24 h after VPA injection. Neither Cu nor Zn concentrations showed any significant changes after VPA administration in either the whole liver, or in the mitochondrial or supernatant fractions of the liver homogenate. Gel filtration profiles for the supernatant of the liver homogenate of the VPA-treated mice, however, clearly showed that induced MT was chiefly Zn-thionein. Therefore, Zn molecules necessary for the elevated MT levels seem to originate mainly from cytosolic Zn-containing proteins except for MT.
Key words: Valproic acid; Metallothionein; Copper; Zinc; Mouse liver
Introduction Metallothionein (MT) is a low molecular weight, cysteine-rich cytosolic protein, which has several very important roles in heavy metal metabolism and detoxification [1]. MT is induced in several organs by the administration of metals [2], chelating agents [3,4], glucocorticoids [5], vitamin C [61, vitamin D 3 [7] and alcohols [8]. The protein can also be induced by X-irradiation [9], bacterial infection [10], starvation [11] and many other types of stressful conditions [12,13]. Recently, it has also been revealed that some commonly used drugs, such as acetaminophen [14], salicylate [15] and non-steroidal anti-inflammatory drugs [16], have the ability to induce MT in experimental animals. On the other hand, it has been well documented that vaiproic acid (VPA), a widely used anti-convulsant drug, induces the deterioration of trace metal homeostasis, including Zn deficiency in experimental animals [17,18]. The purpose of this investigation was to evaluate the ability of VPA to induce MT and affect the homeostasis of Cu and Zn in mouse liver. *Present address: Department of Pediatrics, Hikone City Hospital, 2-1-45, Hikone, Shiga 522, Japan. Correspondence to: Masayuki Kaji, M.D., Department of Pediatrics, Hikone City Hospital, 2-1-45, Hikone, Shiga 522, Japan. 0300-483X/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
144
Materials and methods
Animals and treatments Male C3H/HeSlc mice were obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). The mice, weighing between 24-30 g, were put in a 12-h light/dark cycle environment. They were fed a commercial diet (CE-2 from Clea Japan Inc., Osaka, Japan) with free access to tap water. Experiment 1." dose-dependency The mice (4-6 mice for each experiment) were treated with a single intraperitoneal dose of sodium valproate (Sigma Chemical Co., St.Louis, U.S.A.). The experimental animals received one dose of either 100, 250, 500,750 or 1000 mg/kg body weight. Each dose was calculated to be dissolved in 10 ml saline/kg body weight. The control animals received a saline injection of the same volume as the experimental animals. At 24 h after the injection, they were decapitated and their livers were removed for an immediate analysis of the Cu, Zn and MT levels. Experiment 11." time-course The mice (4-6 mice for each experiment) were treated with a single intraperitoneal dose of sodium valproate (500 mg/kg) and, at these time intervals (6, 12, 18, 24, 36 and 48 h), they were decapitated and their livers were removed for an immediate analysis of the Cu, Zn and MT levels. The control animals (time 0) were decapitated without any treatments. Analytical methods M T determination MT levels were determined by the varied Cd saturation method of Onosaka et al. [19]. The livers were homogenized in 4 vols. of 0.25 M sucrose in a glass homogenizer set on ice. The homogenates were centrifuged at 139 500 g for 30 min at 4°C. Resultant supernatant fractions of 0.2 ml were mixed with 10 t~g Cd (as CdCI2) and were added to a 0.02 M Tris-HNO3 buffer (pH 7.4) to total 3 ml. Subsequently, 0.2 ml of rabbit hemoglobin (Sigma Chemical Co., St. Louis, U.S.A.) solution (10 mg/ml) was added and the mixture was placed in a boiling water bath for 2 min. After being cooled in an ice bath, the mixture was centrifuged at 3000 g for 20 min at 4°C. This process (hemoglobin-heat treatment) was repeated three times. The aliquots of the supernatant fractions were used for determination of Cd by the atomic absorption spectrophotometer (AAS, Nippon-Jarrel-Ash AA-8200). MT concentrations were calculated by using purified rabbit liver MT (Sigma Chemical Co,, St.Louis, U.S.A.) as a standard in each experiment. Cu and Zn determinations (A) Whole liver. Cu and Zn concentrations of the whole liver were determined by AAS after hydrolysis with HNO3 and HCIO4. (B) Mitochondrialfraction. Isolation of mitochondria from the liver was carried out
145
by the varied m e t h o d of D a r l e y - U s m a r et al. [20]. A part o f the liver was homogenized in an ice-cold medium (0.3 M sucrose, 1 m M E G T A , 5 m M Mops, 5 m M KH2PO 4 and 0.1% bovine serum albumin ) adjusted to pH 7.4 with K O H . The h o m o g e n a t e was centrifuged at 1000 g for 10 min at 4°C, and the resultant supernatant was centrifuged again at 10 000 g for 10 rain. The resultant pellet was used as the mitochondrial fraction and assayed for Cu and Zn concentrations with A A S after hydrolysis with H N O 3 and H C I O 4.
(C) Supernatantfraction. The 139 500 g supernatants o f the liver homogenates were taken for Cu and Zn determination with A A S after hydrolysis with HNO3 and H C I O 4. (D) Gelfiltration. The 139 500 g supernatants (0.2 ml) o f liver homogenates from the VPA-treated and untreated mice were applied to a Sephadex G-75 column (1 x 45 cm) and eluted with 0 . 0 2 M T r i s - H N O 3 buffer solution (pH 7.4) at a flow rate o f 10 ml/h. Cu and Zn concentrations o f each fraction (1 ml) were determined by AAS. The protein concentrations o f the liver mitochondrial and supernatant fractions were determined by the method o f Lowry et al. [21]. Statistical analysis o f the data was performed by analysis of variance. If a significant difference was observed, the groups were c o m p a r e d by the Least Significant Difference (LSD) method. Results
Liver M T Experiment 1." dose-dependency Treatment o f mice with V P A resulted in an increase o f hepatic M T in the dosedependent manner (Fig. 1). The hepatic M T level o f the control mice was 13.6 +
I00
-~
**
m
500
750
so
b--
__1
0
100
250
VPA ( m g / k g i.p.)
Fig. 1. MT concentrations in mouse liver at 24 h after VPA injection of various dosages. Values represent the mean ± standard deviation of 4-6 animals. Asterisks indicate values significantly different from the control (**P < 0.01; *P < 0.05).
146
4.7/~g/g wet weight (mean 4- S.D.).-The hepatic MT level increased to about 6-fold (86.0 ± 20.4/zg/g ) a t 24 h after a single injection of VPA (750 mg/kg body wt) as compared to the control. Four mice that received a single injection of 1000 mg/kg dose of VPA died within 24 h, probably due to the high amount of VPA. Experiment II." time-course The time course of the hepatic MT level after the VPA administration is shown in Fig. 2. The hepatic MT level of the untreated mice was 13.0 4- 4.0/~g/g and the mice treated with a single injection of a 500 mg/kg dose of VPA showed a gradual increase of the hepatic MT, and the MT level reached a peak value of 70.5 4- 14.6 t~g/g at 24 h after the injection. Thereafter, the hepatic MT level diminished gradually and came close to the pre-treatment level at 48 h after the injection. Liver Cu and Zn
The Cu concentration of the whole liver of the untreated mice was 3.33 + 0.20 ~g/g wet weight. The Cu concentrations of mitochondrial and supernatant fractions of the liver homogenates of the untreated mice were 31.5 4- 4.8 and 46.1 4- 5.3 #g/g protein, respectively. The Zn concentration of the whole liver of the untreated mice was 26.7 4- 2.8/~g/g wet weight. The Zn concentrations of mitochondrial and supernatant fractions of the liver homogenates of the untreated mice were 93.4 4- 14.0 and 195.8 4- 23.5/~g/g protein, respectively. Neither Cu nor Zn concentrations showed any significant changes after VPA administration in either of the fractions or the whole liver (at 6, 12, 18, 24, 36 and 48 h after 500 mg/kg of VPA injection and at 24 h after one dose of either 100, 250, 500 or 750 mg/kg of VPA injection).
I00
~ 5o I--
0
I
6
I
I
12 18 24 Time (hours)
I
I
36
48
Fig. 2. MT concentrations in mouse liver at various times after VPA injection (500 mg/kg body wt). Values represent the mean 4- standard deviation of 4 - 6 animals. Asterisks indicate values significantly different from the control (**P < 0.01; *P < 0.05).
147
Gel filtration profile The gel filtration profile for the supernatant of the liver homogenate of the VPAtreated mice showed three peaks for the Zn level (Fig. 3A), while there were only two peaks for the Zn level for the untreated mice (Fig. 3B). The third peak that appeared only in the material of VPA-treated mice corresponded to the MT fraction. The Cu levels, however, showed a similar two-peak pattern in both the VPAtreated material and the control. 150-
E
100
C
N
" 0
50-
0
10
20 30 Fraction number
4o
150,
.~
lOO-
t... N
-'!
U
50
10
20 Fraction ~ r
30
4o
Fig. 3. (A) Gel filtration elution profiles for liver supernatant fractions of the VPA-treated mice. Liver supernatant fractions were obtained and eluted on Sephadex G-75 column (1 × 45 cm) with 0.02 M T r i s - H N O 3 buffer solution (pH 7.4) at a flow rate of 10 ml/h. Cu ( • - • ) and Zn ( O - O ) concentrations of each fraction (1 ml) were determined by AAS. The position of the metallothionein fraction is indicated by an arrow. (B) Gel filtration elution profiles for liver supernatant fractions of the untreated mice. Liver supernatant fractions were obtained and eluted on Sephadex G-75 column (I × 45 cm) with 0.02 M T r i s - H N O 3 buffer solution (pH 7.4) at a flow rate of 10 ml/h. Cu ( • - • ) and Zn ( O - O ) concentrations of each fraction (1 ml) were determined by AAS.
148
Discussion In this study, we demonstrated that VPA increased MT content in mouse liver. The dose-dependency of MT induction was clearly revealed (Fig. 1). The maximal amount of MT induced by VPA was about 6 times the basal level. This MT level was similar to the value of the MT induced by acetaminophen [14] and salicylate [15]. The time course of MT induction by VPA with a maximal effect at about 24 h was also similar to that of the single administration of the drugs mentioned above [14,15]. Although the mechanisms by which these drugs induce MT synthesis remain unclear, we have hypothesized that some common mechanisms might be involved, because of the similar time course and the maximal amount of MT induced by these drugs. Unlike several heavy metals which induce prolonged response of MT induction [1], these drugs produce a MT induction of a relatively short half-life. The shortlived nature of this response indicates that MT induced by these drugs would most likely be Zn-thionein [22]. Gel filtration profiles in this study clearly revealed that MT induced by VPA injection was chiefly Zn-thionein (Fig. 3A). On the other hand, the Zn concentration showed no significant changes after VPA administration in the whole liver and the supernatant fraction of the liver homogenate. Gfinther et al. reported that Zn content of livers was not significantly increased, in contrast to the increment of the MT level, after the administration of salicylate and indomethacin. Consequently, they concluded that the induction of MT synthesis by these drugs was not mediated via Zn accumulation in hepatocytes [15]. Therefore, Zn molecules necessary for the elevated MT levels seem to originate mainly from cytosolic Zncontaining proteins, excluding for MT, although there may be a little amount of Zn uptake from the bloodstream into the hepatocytes. A decline of Zn levels, however, was not observed in the mitochondrial fractions of the liver homogenate after the VPA administration. Thus, it appears that the exchange of Zn molecules between mitochondrial and other cytosolic fractions is not easily accomplished. We speculate that this may be due to the possibility of Zn deficiency in cytosolic Zn-containing proteins and, consequently, a decline of their enzymal activities. It is also speculated that some of the several side effects of VPA, salicylate and acetaminophen may be a result of these conditions. In contrast, Heilmaier et al. reported that the Zn content of rat liver was increased synchronously with the MT increment after D-penicillamine administration [4]. Since D-penicillamine has an intensive chelating action, it may have direct effects on the heavy metal status in the liver, in addition to MT induction. Therefore, some different mechanisms of the MT induction possibly apply between VPA and Dpenicillamine. The function of induced MT is a problem which needs further study.
Acknowledgements We wish to express our appreciation to Eiichiro Nakayama of the Research Center for Instrumental Analysis, Kyoto University School of Science, and to Hisaaki Kabata and Prof. Yoshinori Itokawa of the Department of Hygiene, Kyoto University School of Medicine, who gave us excellent technical suggestions for conducting analyses with the atomic absorption spectrophotometer.
149
References 1 2 3 4 5
6 7
8 9 10
11 12 13 14 15 16 17 18 19 20 21 22
I. B r e m n e r a n d J . H . Beattie, Metallothionein and the trace minerals. Annu. Rev. Nutr., 10(1990)63. M.P. Waalkes and C.D. Klaassen, Concentration of metallothionein in major organs of rats after administration of various metals. Fundam. Appl., Toxicol., 5 (1985) 473. P.L. Goering, S.K. Tandon and C.D. Klaassen, Induction of hepatic matallothionein in mouse liver following administration of chelating agents. Toxicol. Appl. Pharmacol., 80 (1985) 467. H.E. Heilmaier, J.L. Jiang, H. Greim, P. Schramel and K.H. Summer, D-Penicillamine induces rat hepatic metallothionein. Toxicology, 42 (1986) 23. M. Karin, R.D. Andersen, E. Slater, K. Smith and H.R. Hershman, Metallothionein m R N A induction in HeLa cells in response to zinc or dexamethasone is a primary induction response. Nature, 286 (1980) 295. S. Onosaka, D. Kawakami, K.-S. Min, K. Ooishi and K. Tanaka, Induced synthesis of metallothionein by ascorbic acid in mouse liver. Toxicology, 43 (1987) 251. M. Karasawa, J. Hosoi, H. Hashiba, K. Nose, C. Tohyama, E. Abe, T. Suda and T. Kuroki. Regulation of metallothionein gene expression by lc~,25-dihydroxyvitamin D 3 in cultured cells and in mice. Proc. Natl. Acad. Sci. USA, 84 (1987) 8810. M.P. Waalkes, J.J. Hjelle and C.D. Klaassen, Transient induction of hepatic metallothionein following oral ethanol administration. Toxicol. Appl. Pharmacol., 74 (1984) 230. N. Shiraishi, K. Aono and K. Utsumi, Increased metallothionein content in rat liver induced by X irradiation and exposure to high oxygen tension. Radiat. Res., 95 (1983) 298. P.Z. Sobocinski, W.J. Canterbury Jr., C.A. Mapes and R.E. Dinterman, Involvement of hepatic metallothioneins in hypozincemia associated with bacterial infection. Am. J. Physiol., 234 (1978) E399. i. Bremner and N.T. Davies, The induction of metallothionein in rat liver by zinc injection and restriction of food intake. Biochem. J. 149 (1975) 733. S.H. Oh, J.T. Deagen, P.D. Whanger and P.H. Weswig, Biological function of metallothionein. V. its induction in rats by various stresses. Am. J. Physiol., 234 (1978) E282. J. Hidalgo, M. Giralt, J.S. Garvey and A. Armario, Physiological role of glucocorticoids on rat serum and liver metallothionein in basal and stress conditions. Am. J. Physiol., 254 (1988) E71. U. Wormser and D. Calp, Increased levels of hepatic metallothionein in rat and mouse after injection of acetaminophen. Toxicology, 53 (1988) 323. T. G/inther, J. Vormann and J. Ghaida, Induction of hepatic metallothionein by salicylate in adult rats. Biol. Trace Elem. Res., 20 (1989) 243. K.H. Summer, D. Klein, N. de Ruiter and J. Abel, Metallothionein induction by nonsteroidal antiinflammatory drugs. Biol. Trace Elem. Res., 21 (1989) 165. J.C. Daffron and E.J. Kasarskis, Effect of valproic acid on zinc metabolism in the rat. Toxicol. Lett., 23 (1984) 321. C.L. Keen, J.M. Peters and L.S. Hurley, The effect of valproic acid on 65Zn distribution in the pregnant rat. J. Nutr., 119 (1989) 607. S. Onosaka and M.G. Cherian, The induced synthesis of metallothionein in various tissues of rat in response to metals, i. Effect of repeated injection of cadmium salts. Toxicology, 22 (1981) 91. V.M. Darley-Usmar, D. Rickwood and M.T. Wilson, Mitochondria - a practical approach, IRL Press, Oxford Washington DC, 1987, p. 4. O.H. Lowry, N.J. Rosebrough, A . L Farr and R.J. Randall, Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193 (1951) 265. K. Cain and D.E. Holt, Metallothionein degradation: metal composition as a controlling factor. Chem.-Biol. Interact., 28 (1979) 91.
143
Induction of metallothionein in mouse liver by valproic acid Masayuki Kaji* and Haruki Mikawa Department of Pediatrics, Kyoto University School of Medicine, Shogoin-Kawahara-cho, Sakyo-ku. Kyoto 606 (Japan) (Received January 30th, 1991; accepted June 4th, 1991)
Summary Intraperitoneal injection of valproic acid (VPA) induces metallothionein (MT) in mouse liver. There was a clear-cut dose-dependency and the maximal amount of MT induced by VPA was about 6 times the basal level. The hepatic MT level reached the peak value at 24 h after VPA injection. Neither Cu nor Zn concentrations showed any significant changes after VPA administration in either the whole liver, or in the mitochondrial or supernatant fractions of the liver homogenate. Gel filtration profiles for the supernatant of the liver homogenate of the VPA-treated mice, however, clearly showed that induced MT was chiefly Zn-thionein. Therefore, Zn molecules necessary for the elevated MT levels seem to originate mainly from cytosolic Zn-containing proteins except for MT.
Key words: Valproic acid; Metallothionein; Copper; Zinc; Mouse liver
Introduction Metallothionein (MT) is a low molecular weight, cysteine-rich cytosolic protein, which has several very important roles in heavy metal metabolism and detoxification [1]. MT is induced in several organs by the administration of metals [2], chelating agents [3,4], glucocorticoids [5], vitamin C [61, vitamin D 3 [7] and alcohols [8]. The protein can also be induced by X-irradiation [9], bacterial infection [10], starvation [11] and many other types of stressful conditions [12,13]. Recently, it has also been revealed that some commonly used drugs, such as acetaminophen [14], salicylate [15] and non-steroidal anti-inflammatory drugs [16], have the ability to induce MT in experimental animals. On the other hand, it has been well documented that vaiproic acid (VPA), a widely used anti-convulsant drug, induces the deterioration of trace metal homeostasis, including Zn deficiency in experimental animals [17,18]. The purpose of this investigation was to evaluate the ability of VPA to induce MT and affect the homeostasis of Cu and Zn in mouse liver. *Present address: Department of Pediatrics, Hikone City Hospital, 2-1-45, Hikone, Shiga 522, Japan. Correspondence to: Masayuki Kaji, M.D., Department of Pediatrics, Hikone City Hospital, 2-1-45, Hikone, Shiga 522, Japan. 0300-483X/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
144
Materials and methods
Animals and treatments Male C3H/HeSlc mice were obtained from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). The mice, weighing between 24-30 g, were put in a 12-h light/dark cycle environment. They were fed a commercial diet (CE-2 from Clea Japan Inc., Osaka, Japan) with free access to tap water. Experiment 1." dose-dependency The mice (4-6 mice for each experiment) were treated with a single intraperitoneal dose of sodium valproate (Sigma Chemical Co., St.Louis, U.S.A.). The experimental animals received one dose of either 100, 250, 500,750 or 1000 mg/kg body weight. Each dose was calculated to be dissolved in 10 ml saline/kg body weight. The control animals received a saline injection of the same volume as the experimental animals. At 24 h after the injection, they were decapitated and their livers were removed for an immediate analysis of the Cu, Zn and MT levels. Experiment 11." time-course The mice (4-6 mice for each experiment) were treated with a single intraperitoneal dose of sodium valproate (500 mg/kg) and, at these time intervals (6, 12, 18, 24, 36 and 48 h), they were decapitated and their livers were removed for an immediate analysis of the Cu, Zn and MT levels. The control animals (time 0) were decapitated without any treatments. Analytical methods M T determination MT levels were determined by the varied Cd saturation method of Onosaka et al. [19]. The livers were homogenized in 4 vols. of 0.25 M sucrose in a glass homogenizer set on ice. The homogenates were centrifuged at 139 500 g for 30 min at 4°C. Resultant supernatant fractions of 0.2 ml were mixed with 10 t~g Cd (as CdCI2) and were added to a 0.02 M Tris-HNO3 buffer (pH 7.4) to total 3 ml. Subsequently, 0.2 ml of rabbit hemoglobin (Sigma Chemical Co., St. Louis, U.S.A.) solution (10 mg/ml) was added and the mixture was placed in a boiling water bath for 2 min. After being cooled in an ice bath, the mixture was centrifuged at 3000 g for 20 min at 4°C. This process (hemoglobin-heat treatment) was repeated three times. The aliquots of the supernatant fractions were used for determination of Cd by the atomic absorption spectrophotometer (AAS, Nippon-Jarrel-Ash AA-8200). MT concentrations were calculated by using purified rabbit liver MT (Sigma Chemical Co,, St.Louis, U.S.A.) as a standard in each experiment. Cu and Zn determinations (A) Whole liver. Cu and Zn concentrations of the whole liver were determined by AAS after hydrolysis with HNO3 and HCIO4. (B) Mitochondrialfraction. Isolation of mitochondria from the liver was carried out
145
by the varied m e t h o d of D a r l e y - U s m a r et al. [20]. A part o f the liver was homogenized in an ice-cold medium (0.3 M sucrose, 1 m M E G T A , 5 m M Mops, 5 m M KH2PO 4 and 0.1% bovine serum albumin ) adjusted to pH 7.4 with K O H . The h o m o g e n a t e was centrifuged at 1000 g for 10 min at 4°C, and the resultant supernatant was centrifuged again at 10 000 g for 10 rain. The resultant pellet was used as the mitochondrial fraction and assayed for Cu and Zn concentrations with A A S after hydrolysis with H N O 3 and H C I O 4.
(C) Supernatantfraction. The 139 500 g supernatants o f the liver homogenates were taken for Cu and Zn determination with A A S after hydrolysis with HNO3 and H C I O 4. (D) Gelfiltration. The 139 500 g supernatants (0.2 ml) o f liver homogenates from the VPA-treated and untreated mice were applied to a Sephadex G-75 column (1 x 45 cm) and eluted with 0 . 0 2 M T r i s - H N O 3 buffer solution (pH 7.4) at a flow rate o f 10 ml/h. Cu and Zn concentrations o f each fraction (1 ml) were determined by AAS. The protein concentrations o f the liver mitochondrial and supernatant fractions were determined by the method o f Lowry et al. [21]. Statistical analysis o f the data was performed by analysis of variance. If a significant difference was observed, the groups were c o m p a r e d by the Least Significant Difference (LSD) method. Results
Liver M T Experiment 1." dose-dependency Treatment o f mice with V P A resulted in an increase o f hepatic M T in the dosedependent manner (Fig. 1). The hepatic M T level o f the control mice was 13.6 +
I00
-~
**
m
500
750
so
b--
__1
0
100
250
VPA ( m g / k g i.p.)
Fig. 1. MT concentrations in mouse liver at 24 h after VPA injection of various dosages. Values represent the mean ± standard deviation of 4-6 animals. Asterisks indicate values significantly different from the control (**P < 0.01; *P < 0.05).
146
4.7/~g/g wet weight (mean 4- S.D.).-The hepatic MT level increased to about 6-fold (86.0 ± 20.4/zg/g ) a t 24 h after a single injection of VPA (750 mg/kg body wt) as compared to the control. Four mice that received a single injection of 1000 mg/kg dose of VPA died within 24 h, probably due to the high amount of VPA. Experiment II." time-course The time course of the hepatic MT level after the VPA administration is shown in Fig. 2. The hepatic MT level of the untreated mice was 13.0 4- 4.0/~g/g and the mice treated with a single injection of a 500 mg/kg dose of VPA showed a gradual increase of the hepatic MT, and the MT level reached a peak value of 70.5 4- 14.6 t~g/g at 24 h after the injection. Thereafter, the hepatic MT level diminished gradually and came close to the pre-treatment level at 48 h after the injection. Liver Cu and Zn
The Cu concentration of the whole liver of the untreated mice was 3.33 + 0.20 ~g/g wet weight. The Cu concentrations of mitochondrial and supernatant fractions of the liver homogenates of the untreated mice were 31.5 4- 4.8 and 46.1 4- 5.3 #g/g protein, respectively. The Zn concentration of the whole liver of the untreated mice was 26.7 4- 2.8/~g/g wet weight. The Zn concentrations of mitochondrial and supernatant fractions of the liver homogenates of the untreated mice were 93.4 4- 14.0 and 195.8 4- 23.5/~g/g protein, respectively. Neither Cu nor Zn concentrations showed any significant changes after VPA administration in either of the fractions or the whole liver (at 6, 12, 18, 24, 36 and 48 h after 500 mg/kg of VPA injection and at 24 h after one dose of either 100, 250, 500 or 750 mg/kg of VPA injection).
I00
~ 5o I--
0
I
6
I
I
12 18 24 Time (hours)
I
I
36
48
Fig. 2. MT concentrations in mouse liver at various times after VPA injection (500 mg/kg body wt). Values represent the mean 4- standard deviation of 4 - 6 animals. Asterisks indicate values significantly different from the control (**P < 0.01; *P < 0.05).
147
Gel filtration profile The gel filtration profile for the supernatant of the liver homogenate of the VPAtreated mice showed three peaks for the Zn level (Fig. 3A), while there were only two peaks for the Zn level for the untreated mice (Fig. 3B). The third peak that appeared only in the material of VPA-treated mice corresponded to the MT fraction. The Cu levels, however, showed a similar two-peak pattern in both the VPAtreated material and the control. 150-
E
100
C
N
" 0
50-
0
10
20 30 Fraction number
4o
150,
.~
lOO-
t... N
-'!
U
50
10
20 Fraction ~ r
30
4o
Fig. 3. (A) Gel filtration elution profiles for liver supernatant fractions of the VPA-treated mice. Liver supernatant fractions were obtained and eluted on Sephadex G-75 column (1 × 45 cm) with 0.02 M T r i s - H N O 3 buffer solution (pH 7.4) at a flow rate of 10 ml/h. Cu ( • - • ) and Zn ( O - O ) concentrations of each fraction (1 ml) were determined by AAS. The position of the metallothionein fraction is indicated by an arrow. (B) Gel filtration elution profiles for liver supernatant fractions of the untreated mice. Liver supernatant fractions were obtained and eluted on Sephadex G-75 column (I × 45 cm) with 0.02 M T r i s - H N O 3 buffer solution (pH 7.4) at a flow rate of 10 ml/h. Cu ( • - • ) and Zn ( O - O ) concentrations of each fraction (1 ml) were determined by AAS.
148
Discussion In this study, we demonstrated that VPA increased MT content in mouse liver. The dose-dependency of MT induction was clearly revealed (Fig. 1). The maximal amount of MT induced by VPA was about 6 times the basal level. This MT level was similar to the value of the MT induced by acetaminophen [14] and salicylate [15]. The time course of MT induction by VPA with a maximal effect at about 24 h was also similar to that of the single administration of the drugs mentioned above [14,15]. Although the mechanisms by which these drugs induce MT synthesis remain unclear, we have hypothesized that some common mechanisms might be involved, because of the similar time course and the maximal amount of MT induced by these drugs. Unlike several heavy metals which induce prolonged response of MT induction [1], these drugs produce a MT induction of a relatively short half-life. The shortlived nature of this response indicates that MT induced by these drugs would most likely be Zn-thionein [22]. Gel filtration profiles in this study clearly revealed that MT induced by VPA injection was chiefly Zn-thionein (Fig. 3A). On the other hand, the Zn concentration showed no significant changes after VPA administration in the whole liver and the supernatant fraction of the liver homogenate. Gfinther et al. reported that Zn content of livers was not significantly increased, in contrast to the increment of the MT level, after the administration of salicylate and indomethacin. Consequently, they concluded that the induction of MT synthesis by these drugs was not mediated via Zn accumulation in hepatocytes [15]. Therefore, Zn molecules necessary for the elevated MT levels seem to originate mainly from cytosolic Zncontaining proteins, excluding for MT, although there may be a little amount of Zn uptake from the bloodstream into the hepatocytes. A decline of Zn levels, however, was not observed in the mitochondrial fractions of the liver homogenate after the VPA administration. Thus, it appears that the exchange of Zn molecules between mitochondrial and other cytosolic fractions is not easily accomplished. We speculate that this may be due to the possibility of Zn deficiency in cytosolic Zn-containing proteins and, consequently, a decline of their enzymal activities. It is also speculated that some of the several side effects of VPA, salicylate and acetaminophen may be a result of these conditions. In contrast, Heilmaier et al. reported that the Zn content of rat liver was increased synchronously with the MT increment after D-penicillamine administration [4]. Since D-penicillamine has an intensive chelating action, it may have direct effects on the heavy metal status in the liver, in addition to MT induction. Therefore, some different mechanisms of the MT induction possibly apply between VPA and Dpenicillamine. The function of induced MT is a problem which needs further study.
Acknowledgements We wish to express our appreciation to Eiichiro Nakayama of the Research Center for Instrumental Analysis, Kyoto University School of Science, and to Hisaaki Kabata and Prof. Yoshinori Itokawa of the Department of Hygiene, Kyoto University School of Medicine, who gave us excellent technical suggestions for conducting analyses with the atomic absorption spectrophotometer.
149
References 1 2 3 4 5
6 7
8 9 10
11 12 13 14 15 16 17 18 19 20 21 22
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