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Change in ratio of the two hepatic isometallothioneins with development from prenatal to neonatal rats
Camp. Biochem. Physiol. Vol. 76C, pp. 33-38, Printed in Great Britain
0306.4492/83$3.00 + 0.00 %> 1983 Pergamon Press Ltd
1983
CHANGE IN RATIO ISOMETALLOTHIONEINS FROM PRENATAL KAZUO T.
SUZUKI,
MASATAKA
OF THE TWO HEPATIC WITH DEVELOPMENT TO NEONATAL RATS
YOSHIYUKI
National Institute for Environmental
EBIHARA,
HIROYUKI
AKITOMI
and RYOKO
KAWAM~JRA Studies, Yatabe, Tsukuba, Ibaraki 305, Japan
NISHIKAWA
(Rewired 4 January 1983) Abstract-l. Concentrations and contents of essential metals in the livers of prenatal and neonatal rats were determined simultaneously by inductively coupled plasma-atomic emission spectrometry. 2. The concentrations and contents changed with development in a mode characteristic to the respective metals. 3. Changes in metallothionein. 4. The relative predominant in
the concentrations and contents of zinc and copper were correlated to those bound to the two metals being high in concentration during prenatal and neonatal periods. ratio of the two isoforms of metallothionein changed with development. the form I being prenatal livers.
INTRODUCTION
Recently we observed that the two isoforms of metallothionein (MT) were induced in the liver of the frog Rana catesheiana (bullfrog) by loading of cadmium (Cd) and that the relative ratio of the two isoforms was different between the larva and adult (Suzuki and Akitomi, 1983). Among several essential metals in the liver of the frog, the concentration of copper (Cu) was also shown to be significantly different between the two developmental stages. All mammalian species thus far investigated have been shown to contain large amounts of MT in their livers during prenatal and neonatal periods (Bakka and Webb, 1981; Bakka et al., 1981; Bell, 1979; Bell and Waalkes, 1982; Brady et al., 1982; Mason et al., 1980; Ohtake et al., 1978; Riordan and Richards, 1980; Waalkes et al., 1982; Webb et ul., 1979; Wong and Klaassen, 1979). In some cases, neonatal MT has been shown to consist of the two isoforms as in the case of MT induced in the adult. However, the relative ratio of the two isoforms has not been studied due to lack in the convenient analytical method and it has not been recognized whether there are any differences in the ratio between the prenatal and neonatal MTs. The present experiment was intended to examine whether or not the relative &-MT ratio changes with development of mammalian species by using prenatal and neonatal rat livers. Although the developmental stage of the frog can be easily recognized by the metamorphosis from larva to adult, it is not distinct in the case of mammals. Therefore, the livers were analyzed in detail from those of the 16th day of gestation to those of the 33rd day after birth (in total 18 different developmental stages). In this experiment, several essential metals other than Zn and Cu were also determined to trace the developmental change and to compare with the changes of Zn and Cu.
MATERIALS
AND METHODS
The same experiment was repeated twice by using different lots of rats (JCL, Wistar strain). In the first experiment, the pregnant rats (12 weeks old, 20 rats) were purchased from a breeder (Clea Japan, Tokyo) and fed with a laboratory chow (MF diet, Oriental Yeast Co., Tokyo) and distilled water ad libitum. Two rats were killed every day at the 1420th day of gestation. Newborn rats were nursed by their mothers until weaned at 27 days, and were killed every day at O-4 days and at 8, 20 and 30 days after birth (5 rats each). A 100 mg portion of the pooled livers of the embryos and a part of the neonatal livers were wet-washed with 1 ml of mixed acid (HNO,:HCIO, = 5: 1 v/v). Concentrations of the metals were determined after dilution to 3 ml with twicedistilled water by inductively coupled plasma-atomic emission spectrometry (ICP-AES) (JarelI-Ash Model 975 Plasma Atomcomp). The concentrations and contents of metals were expressed as means i SD of 5 samples. The rest of the livers at each developmental stage was pooled and homogenized in 3 vol. of 0. I M Tris-HCI buffer (pH 7.4, 0.25 M glucose, bubbled through with nitrogen gas before use) using a Teflon homogenizer in an atmosphere of nitrogen. The homogenates were centrifuged at 170,OOOg for 1 hr at 2°C. In the second experiment, male and female rats of the Wistar strain (I 2 weeks old) were purchased from the same breeder and fed as mentioned above. The first day of gestation was confirmed by the plug formation. Two rats were killed every day at 1621st day of gestation and the livers of embryos were excised. Newborn pups were killed every day at O-6 days and at 8, 1 I. 19, 27 and 33 days after birth. Concentrations of metals were determined as described for the first experiment and the data were expressed as means + SD of 5 samples. The data presented in this report are those of the second experiment. Distribution profiles of Zn and Cu in the supernatants were determined on an SW column by high performance liquid chromatograph-atomic absorption spectrophotometry (HPLC-AAS) (Suzuki, 1980) by elution with 50 mM Tris-HCI buffer (pH 8.6 at 25’C, 0.1% NaN,, dissolved gases were removed at 80 C under reduced pressure) at a flow rate of I ml/min. The concentrations of the two
KAZIJO T.
34
,
1
16
17
k+days Fig. 1. Changes
I
18
I
I
19
20
/
21,
I
0
I
1
1
2
I
56
Of 9estation~
days
in hepatic Zn concentration indicates that the range
The concentration of Zn in the livers of embryos increased with days of gestation. The abrupt increase of the Zn concentration at the time of birth is possibly due to decrease in liver weight; 302 + 54 mg at 21st day of gestation and 241 k 10 mg at the day
Fig. 2. Changes
in hepatic Cu concentration indicates that the range
after
(,
al.
1
::
1
8
11
blrth
-4
I;
I
19
::
1
,
27
and content with development of rats. Absence is less than the size of the symbols.
RESULTS
T T
et
I
34
metals in the two iso-MTs were estimated from the peak heights relative to standard MT peaks. The distribution profiles and concentrations of the two metals were also determined and estimated after reducing the oxidized MTs by adding neat mercaptoethanol at 74;, (v/v) concentration and replacing Zn in MT with Cd by adding Cd’+ at a concentration of IO ng Cd/lOOnl of supernatant. The solution was allowed to stand at room temperature for I5 min after mixing mercaptoethanol and Cd’+ with the supernatant. A 100 ~1 portion of the reaction mixture was applied to an SW column (HPLC-AAS method).
I
SUZUKI
of a bar
of birth. The hepatic Zn concentration in newborn rats retained a high plateau level for about 10 days after birth and then started to decrease. The concentration remained at a low constant level after about 3 weeks as shown in Fig. I. Although the concentration of Zn decreased after weaning, the content of Zn (pgg/whole liver) increased due to rapid growth and increase in liver weight. As a whole, hepatic Zn was high in late prenatal and early neonatal rats when it was expressed in concentration, while the metal increased steadily with development of the liver when it was expressed in content. The concentration and content of Cu in the livers of embryos and newborn rats changed as shown in Fig. 2. The concentration of Cu also showed an abrupt increase at the time of birth as observed in the case of Zn. The Cu concentration in the newborn rats was high for about 2 weeks after birth and then
1
and content with development of rats. Absence is less than the size of the symbols.
of a bar
Pre- and neonatal
metallothionein
35
Fig. 3. Changes in hepatic concentration and content of thionein-bound Zn with development of rats. The pooled livers at each developmental stage were homogenized in 3 vol. of 0. I M Tris-HCI buffer. The concentrations of thionein-bound Zn in the supernatants were estimated by HPLC-AAS after addition of mercaptoethanol and Cd’+ to the supernatants.
started to decrease, the concentration being a low constant level after weaning. Cu in the whole liver increased steadily with growth throughout prenatal and early neonatal periods. In contrast to the Zn content, the Cu content attained the highest level at about 2 weeks after birth and then decreased to the lowest constant level. This decrease in Cu content at weaning was different from changes observed for other elements such as Zn, iron (Fe), magnesium (Mg) and calcium (Ca) as shown in Fig. 7. The amounts of thionein-bound Zn and Cu in the soluble fraction of livers were estimated on an SW column by HPLC-AAS. The pooled livers at each developmental stage were carefully homogenized in 3 vol. of nitrogen gas-filled buffer in an atmosphere of nitrogen in order to prevent oxidation of MT. However, Zn and Cu-containing MT in the supernatants was easily oxidized (mainly intramolecular oxidation) despite the supernatants being stored at -80 C in a nitrogen gas-filled tube. As MT-I is less unstable compared to MT-II (Suzuki and Maitani, 1983; Winge and Miklossy, 1982), the relative ratio of the two iso-MTs (MT-I/MT-II) decreased during storage and analytical procedures. Therefore, MT was analyzed after reduction and replacement of Zn with Cd by adding mercaptoethanol and Cd’+ to the supernatants. The concentration of thionein-bound Zn in the soluble fraction changed as shown in Fig. 3 and the overall pattern was similar to that of the Zn concentration in Fig. 1. The content of thionein-bound Zn is also presented in Fig. 3. The concentration and content of thionein-bound Cu in the soluble fraction of the livers were determined and plotted vs days of gestation and days after birth. The concentration changed in a similar pattern to that of the Zn concentration and a maximum was observed just before the concentration started to
decrease. The increase of thionein-bound Cu at about 2 weeks after birth to attain a maximum is more evidently shown in the graph plotted vs the content rather than that plotted vs the concentration as shown in Fig. 4. The relative ratio of the two iso-forms of MT was determined from the peak heights on an SW column after the reduction and replacement. The relative ratio of the two ivo-MTs (MT-I/MT-II) in the livers of embryos decreased continuously with days of gestation, and then the ratio remained at a constant level for about 2 weeks after birth as shown in Fig. 5. Thus, the relative ratio of the two iso-MTs was shown to change with development, especially in the livers of embryos at the late gestational period. The ratio of the thionein-bound Cu (MT-I/MT-II) also changed with development of rats as shown in Fig. 5. Figure 6 demonstrates typical elution profiles determined in this experiment. As the relative ratio of Zn bound to the iso-MTs (MT-I/MT-II) decreased during repeated determination and storage for the same supernatant and MT-I has been shown less stable compared to MT-II (Suzuki and Maitani, 1983; Winge and Miklossy, 1982), MT in the supernatant was analyzed after reduction of oxidized MT with mercaptoethanol and replacement of Zn in MT with Cd. The reduction and replacement reactions gave highly reproducible results. Excess Cd not bound to MT was eluted at the void volume of the column as non-selectively bound Cd to the high molecular weight proteins and, at a retention time of 26.4 min, as Cd-mercaptoethanol complex. In contrast to the sharp Cd peaks of MT, the thioneinbound Cu was eluted as broad peaks on an SW column (Fig. 6). The changes in the concentrations and contents of elements other than Zn and Cu were also determined
KAZU~ T. SUZUKI et ul.
36
16 17
t+dws Fig. 4. Changes
18 19 20 21, 0 of gestotlonv
in hepatic
The concentrations
concentration
of thionein-bound addition
1
2
3
4
5 6 days after blrth -4
and content
of thionein-bound Cu with development of rats, were determined by HPLC-AAS after and Cd’+ to the supernatants.
Cu in the supernatants
of mercaptoethanol
simultaneously by ICP-AES and compared with those of Zn and Cu. Among those elements, changes in the concentrations of Fe, Mg and Ca which showed characteristic patterns are represented in Fig. 7. Potassium (K) and phosphorus (P) changed in concentration in a similar pattern to that of Mg. DISCUSSION
The principal aim of the present experiment was to examine whether or not the relative ratio of the two iso-MTs changes with development of mammalian species. The isoform I was shown to be a dominant peak in the livers of prenatal rats, while the isoform II was present as an abundant peak in the livers of newborn rats. Thus, the ratio of the two iso-MTs was shown to change with development of rats as ob-
served in the case of the frog Ram catesbeianu (Suzuki and Akitomi, 1983). However, the two isoMTs were present as native MT in the livers of prenatal and neonatal rats. On the other hand, only one iso-MT was present as native MT in the livers of larva and adult bullfrogs, and the other iso-MT (MT-II) was induced by loading of Cd. The relative ratio of the two induced iso-MTs was different between the larva and adult bullfrogs, the &-form I1 being a dominant &o-MT in the larva. Therefore, it may not be justified to compare the change of the relative iso-MT ratio directly between the bullfrog and the rat. However, the change of the &o-MT ratio observed in the present study suggests that the two iso-MTs are synthesized at a differing ratio with development in the livers of fetuses and degraded at a different ratio selectivity in the livers of newborn
Fig. 5. Changes in the relative ratios of the two iso-MTs with development of rdtS. of Zn and Cu bound to the two iso-forms were determined by HPLCAAS mercaptoethanol and Cd’+ to the supernatants.
The relative ratios after addition of
Pre- and neonatal
31
metallothionein 1 6.U
6.4
119.4
”
l118.2
Cd
-I
CU
Retentlon
time
15.0 --L
(
mln
_
1 -L!
k
a= cu
Retentlon tlm
(
mln
)
Fig. 6. Typical elution profiles of the two iso-MTs in the supernatants after addition of mercaptoethanol and Cd’+. The oxidized forms of MT were reduced with mercaptoethanol and the thionein-bound Zn was replaced with Cd as described in Materials and Methods. The elution profiles were determined by HPLC-AAS. The left and right panels correspond to the profiles of the embryos of 17th day of gestation and of newborns 3 days after birth, respectively. The void volume of the column and mercaptoethanol-Cd complex correspond to peaks at retention times of Il.2 and 26.4mit-1, respectively. I (19.4min) and II (18.2 min) indicate MT-I and MT-II, respectively. The vertical bars show the detector levels of Cd and cu.
rats about IO days after birth. The present observation also suggests the possibility that the gene expression of the two iso-MTs depends on the development and MT-I may be a primary &-form in a gene duplication throughout the course of molecular evolution as also suggested in the case of bullfrog MT (Suzuki and Akitomi, 1983). Although MT-I may be the primary &-form, the protein has been shown to be less stable compared with the other &-form (MT-II) (Suzuki and Maitani, 1983; Winge and Miklossy, 1982). The &-form I was also easily oxidized during the storage and
Fig. 7. Changes
in hepatic
concentrations
analytical procedures in this study and the relative ratio of the two iso-MTs (MT-I/MT-II) was found to decrease for every repetition of the procedures. Therefore, MT was analyzed after the reduction and replacement reactions. Zn in MT was quantitatively replaced with Cd and the Cd was eluted as sharp peaks in the present analytical procedure. However, Cu in the reduced and replaced solutions was eluted as broad peaks. This may indicate that Cu was not bound to the same molecule as that of Zn or at least Cu and Zn were not bound to MT at the same metal composition.
of Fe, Mg and Ca with development
of rats.
KAZUO
38
T.
The changes in the concentrations of total and thionein-bound Zn and Cu were similar to those already reported for rats (Bakka et a/., 1981; Brady et d., 1982; Mason et (II., 1980) and the changes have been discussed in relation to MT. Although it was not the aim of the present study to explain the changes of other elements, the changes in the concentrations of several elements were shown to compare with those of Zn and Cu. Although K and P changed in concentration in a similar pattern as that of Ca, other elements showed characteristic patterns to the respective elements as shown in Fig. 7. However, further investigations were not conducted in the present study. Acknowledgements~The authors express their thanks to Dr K. Kubota of this Institute, Professors K. Taki (Kitasato Univ.) and M. Takimoto (Toho Univ.) for their encouragement. REFERENCES
Bakka A. and Webb M. (1981) Metabolism of zinc and copper in the neonate: changes in the concentrations and contents of thionein-bound Zn and Cu with age in the livers of the newborn of various mammalian species. Biochem. Phurmu. 30, 721-725. Bakka A., Samarawickrama G. P. and Webb M. (1981) Metabolism of zinc and copper in the neonate: effect of cadmium administration during late gestation in the rat on the zinc and copper metabolism of the newborn. Chem. Biol. Interrrct. 34, 16lll71. Bell J. U. (1979) A metallothionein-like protein in the hepatic cytosol of the term rat fetus. Toxic. rrppl. Phurmat. 48, 1399144. Bell J. U. and Waalkcs M. P. (1982) Role of hepatic metallothionein during perinatal development in the rat. In Biolugicul Roles o/ Mriullolhionein (Edited by Foulkes E. C.) pp. 9991 I I. Elsevier-North Holland, Amsterdam.
SUZUKl
et d.
Brady F. 0.. Webb M. and Mason R. (1982) Zinc and copper metabolism in neonates: role of metallothionein in growth and development in the rat. In Biologiccrl Roles of Merullothionein (Edited by Foulkes E. C.) pp. 77-98. Elsevier-North Holland. Amsterdam. Mason R., Bakka A., Samarawickrama G. P. and Webb M. (1980) Metabolism of zinc and copper in the neonate: accumulation and function of (Zn, Cu)-metallothionein in the liver of the newborn rat. Br. J. Nurr. 45, 375-389. Ohtake H., Hasegawa K. and Koga M. (1978) Zinc-binding protein in the livers of neonatal, normal and partially hepatectonized rats. Biochem. J. 174, 999%1005. Riordan J. R. and Richards V. (1980) Human fetal liver contains both zinc- and copper-rich forms of metallothionein. J. hiol. Chem. 255, 5380-5383. Suzuki K. T. (I 980) Direct connection of high speed liquid chromatograph (equipped with gel permeation column) to atomic absorption spectrophotometer for metalloprotein analysis: Metallothionein. Anul~t. Biochem. 102, 3 l-34. Suzuki K. T. and Akitomi H. (1983) Difference in relative isometallothionein ratio between adult and larva of cadmium loaded bullfrog Rana catesheiana. Comp. Biochem. Physiol. 75C, 21 I-215. Suzuki K. T. and Maitani T. (1983) Comparative properties of the two isometallothioneins in oxidation and metal substitution reactions, submitted. Waalkes M. P., Thomas J. A. and Bell J. U. (1982) Induction of hepatic metallothionein in the rabbit fetus following maternal cadmium exposure. Toxic. appl. Pharmuc. 62, 21 l-218. Webb M.. Plastow S. R. and Magos L. (1979) (Copper. zinc)-thionein in pig liver. Lift Sci. 24, 1901-1906. Winge D. R. and Miklossy K. A. (1982) Differences in the polymorphic forms of metallothionein. Archs Biochem. Biophys. 214, 8G-88. Wong K.-L. and Klaassen C. D. (1979) Isolation and characterization of metallothionein which is highly concentrated in newborn rat liver. J. hiol. C/tern. 254, 12399912403.
0306.4492/83$3.00 + 0.00 %> 1983 Pergamon Press Ltd
1983
CHANGE IN RATIO ISOMETALLOTHIONEINS FROM PRENATAL KAZUO T.
SUZUKI,
MASATAKA
OF THE TWO HEPATIC WITH DEVELOPMENT TO NEONATAL RATS
YOSHIYUKI
National Institute for Environmental
EBIHARA,
HIROYUKI
AKITOMI
and RYOKO
KAWAM~JRA Studies, Yatabe, Tsukuba, Ibaraki 305, Japan
NISHIKAWA
(Rewired 4 January 1983) Abstract-l. Concentrations and contents of essential metals in the livers of prenatal and neonatal rats were determined simultaneously by inductively coupled plasma-atomic emission spectrometry. 2. The concentrations and contents changed with development in a mode characteristic to the respective metals. 3. Changes in metallothionein. 4. The relative predominant in
the concentrations and contents of zinc and copper were correlated to those bound to the two metals being high in concentration during prenatal and neonatal periods. ratio of the two isoforms of metallothionein changed with development. the form I being prenatal livers.
INTRODUCTION
Recently we observed that the two isoforms of metallothionein (MT) were induced in the liver of the frog Rana catesheiana (bullfrog) by loading of cadmium (Cd) and that the relative ratio of the two isoforms was different between the larva and adult (Suzuki and Akitomi, 1983). Among several essential metals in the liver of the frog, the concentration of copper (Cu) was also shown to be significantly different between the two developmental stages. All mammalian species thus far investigated have been shown to contain large amounts of MT in their livers during prenatal and neonatal periods (Bakka and Webb, 1981; Bakka et al., 1981; Bell, 1979; Bell and Waalkes, 1982; Brady et al., 1982; Mason et al., 1980; Ohtake et al., 1978; Riordan and Richards, 1980; Waalkes et al., 1982; Webb et ul., 1979; Wong and Klaassen, 1979). In some cases, neonatal MT has been shown to consist of the two isoforms as in the case of MT induced in the adult. However, the relative ratio of the two isoforms has not been studied due to lack in the convenient analytical method and it has not been recognized whether there are any differences in the ratio between the prenatal and neonatal MTs. The present experiment was intended to examine whether or not the relative &-MT ratio changes with development of mammalian species by using prenatal and neonatal rat livers. Although the developmental stage of the frog can be easily recognized by the metamorphosis from larva to adult, it is not distinct in the case of mammals. Therefore, the livers were analyzed in detail from those of the 16th day of gestation to those of the 33rd day after birth (in total 18 different developmental stages). In this experiment, several essential metals other than Zn and Cu were also determined to trace the developmental change and to compare with the changes of Zn and Cu.
MATERIALS
AND METHODS
The same experiment was repeated twice by using different lots of rats (JCL, Wistar strain). In the first experiment, the pregnant rats (12 weeks old, 20 rats) were purchased from a breeder (Clea Japan, Tokyo) and fed with a laboratory chow (MF diet, Oriental Yeast Co., Tokyo) and distilled water ad libitum. Two rats were killed every day at the 1420th day of gestation. Newborn rats were nursed by their mothers until weaned at 27 days, and were killed every day at O-4 days and at 8, 20 and 30 days after birth (5 rats each). A 100 mg portion of the pooled livers of the embryos and a part of the neonatal livers were wet-washed with 1 ml of mixed acid (HNO,:HCIO, = 5: 1 v/v). Concentrations of the metals were determined after dilution to 3 ml with twicedistilled water by inductively coupled plasma-atomic emission spectrometry (ICP-AES) (JarelI-Ash Model 975 Plasma Atomcomp). The concentrations and contents of metals were expressed as means i SD of 5 samples. The rest of the livers at each developmental stage was pooled and homogenized in 3 vol. of 0. I M Tris-HCI buffer (pH 7.4, 0.25 M glucose, bubbled through with nitrogen gas before use) using a Teflon homogenizer in an atmosphere of nitrogen. The homogenates were centrifuged at 170,OOOg for 1 hr at 2°C. In the second experiment, male and female rats of the Wistar strain (I 2 weeks old) were purchased from the same breeder and fed as mentioned above. The first day of gestation was confirmed by the plug formation. Two rats were killed every day at 1621st day of gestation and the livers of embryos were excised. Newborn pups were killed every day at O-6 days and at 8, 1 I. 19, 27 and 33 days after birth. Concentrations of metals were determined as described for the first experiment and the data were expressed as means + SD of 5 samples. The data presented in this report are those of the second experiment. Distribution profiles of Zn and Cu in the supernatants were determined on an SW column by high performance liquid chromatograph-atomic absorption spectrophotometry (HPLC-AAS) (Suzuki, 1980) by elution with 50 mM Tris-HCI buffer (pH 8.6 at 25’C, 0.1% NaN,, dissolved gases were removed at 80 C under reduced pressure) at a flow rate of I ml/min. The concentrations of the two
KAZIJO T.
34
,
1
16
17
k+days Fig. 1. Changes
I
18
I
I
19
20
/
21,
I
0
I
1
1
2
I
56
Of 9estation~
days
in hepatic Zn concentration indicates that the range
The concentration of Zn in the livers of embryos increased with days of gestation. The abrupt increase of the Zn concentration at the time of birth is possibly due to decrease in liver weight; 302 + 54 mg at 21st day of gestation and 241 k 10 mg at the day
Fig. 2. Changes
in hepatic Cu concentration indicates that the range
after
(,
al.
1
::
1
8
11
blrth
-4
I;
I
19
::
1
,
27
and content with development of rats. Absence is less than the size of the symbols.
RESULTS
T T
et
I
34
metals in the two iso-MTs were estimated from the peak heights relative to standard MT peaks. The distribution profiles and concentrations of the two metals were also determined and estimated after reducing the oxidized MTs by adding neat mercaptoethanol at 74;, (v/v) concentration and replacing Zn in MT with Cd by adding Cd’+ at a concentration of IO ng Cd/lOOnl of supernatant. The solution was allowed to stand at room temperature for I5 min after mixing mercaptoethanol and Cd’+ with the supernatant. A 100 ~1 portion of the reaction mixture was applied to an SW column (HPLC-AAS method).
I
SUZUKI
of a bar
of birth. The hepatic Zn concentration in newborn rats retained a high plateau level for about 10 days after birth and then started to decrease. The concentration remained at a low constant level after about 3 weeks as shown in Fig. I. Although the concentration of Zn decreased after weaning, the content of Zn (pgg/whole liver) increased due to rapid growth and increase in liver weight. As a whole, hepatic Zn was high in late prenatal and early neonatal rats when it was expressed in concentration, while the metal increased steadily with development of the liver when it was expressed in content. The concentration and content of Cu in the livers of embryos and newborn rats changed as shown in Fig. 2. The concentration of Cu also showed an abrupt increase at the time of birth as observed in the case of Zn. The Cu concentration in the newborn rats was high for about 2 weeks after birth and then
1
and content with development of rats. Absence is less than the size of the symbols.
of a bar
Pre- and neonatal
metallothionein
35
Fig. 3. Changes in hepatic concentration and content of thionein-bound Zn with development of rats. The pooled livers at each developmental stage were homogenized in 3 vol. of 0. I M Tris-HCI buffer. The concentrations of thionein-bound Zn in the supernatants were estimated by HPLC-AAS after addition of mercaptoethanol and Cd’+ to the supernatants.
started to decrease, the concentration being a low constant level after weaning. Cu in the whole liver increased steadily with growth throughout prenatal and early neonatal periods. In contrast to the Zn content, the Cu content attained the highest level at about 2 weeks after birth and then decreased to the lowest constant level. This decrease in Cu content at weaning was different from changes observed for other elements such as Zn, iron (Fe), magnesium (Mg) and calcium (Ca) as shown in Fig. 7. The amounts of thionein-bound Zn and Cu in the soluble fraction of livers were estimated on an SW column by HPLC-AAS. The pooled livers at each developmental stage were carefully homogenized in 3 vol. of nitrogen gas-filled buffer in an atmosphere of nitrogen in order to prevent oxidation of MT. However, Zn and Cu-containing MT in the supernatants was easily oxidized (mainly intramolecular oxidation) despite the supernatants being stored at -80 C in a nitrogen gas-filled tube. As MT-I is less unstable compared to MT-II (Suzuki and Maitani, 1983; Winge and Miklossy, 1982), the relative ratio of the two iso-MTs (MT-I/MT-II) decreased during storage and analytical procedures. Therefore, MT was analyzed after reduction and replacement of Zn with Cd by adding mercaptoethanol and Cd’+ to the supernatants. The concentration of thionein-bound Zn in the soluble fraction changed as shown in Fig. 3 and the overall pattern was similar to that of the Zn concentration in Fig. 1. The content of thionein-bound Zn is also presented in Fig. 3. The concentration and content of thionein-bound Cu in the soluble fraction of the livers were determined and plotted vs days of gestation and days after birth. The concentration changed in a similar pattern to that of the Zn concentration and a maximum was observed just before the concentration started to
decrease. The increase of thionein-bound Cu at about 2 weeks after birth to attain a maximum is more evidently shown in the graph plotted vs the content rather than that plotted vs the concentration as shown in Fig. 4. The relative ratio of the two iso-forms of MT was determined from the peak heights on an SW column after the reduction and replacement. The relative ratio of the two ivo-MTs (MT-I/MT-II) in the livers of embryos decreased continuously with days of gestation, and then the ratio remained at a constant level for about 2 weeks after birth as shown in Fig. 5. Thus, the relative ratio of the two iso-MTs was shown to change with development, especially in the livers of embryos at the late gestational period. The ratio of the thionein-bound Cu (MT-I/MT-II) also changed with development of rats as shown in Fig. 5. Figure 6 demonstrates typical elution profiles determined in this experiment. As the relative ratio of Zn bound to the iso-MTs (MT-I/MT-II) decreased during repeated determination and storage for the same supernatant and MT-I has been shown less stable compared to MT-II (Suzuki and Maitani, 1983; Winge and Miklossy, 1982), MT in the supernatant was analyzed after reduction of oxidized MT with mercaptoethanol and replacement of Zn in MT with Cd. The reduction and replacement reactions gave highly reproducible results. Excess Cd not bound to MT was eluted at the void volume of the column as non-selectively bound Cd to the high molecular weight proteins and, at a retention time of 26.4 min, as Cd-mercaptoethanol complex. In contrast to the sharp Cd peaks of MT, the thioneinbound Cu was eluted as broad peaks on an SW column (Fig. 6). The changes in the concentrations and contents of elements other than Zn and Cu were also determined
KAZU~ T. SUZUKI et ul.
36
16 17
t+dws Fig. 4. Changes
18 19 20 21, 0 of gestotlonv
in hepatic
The concentrations
concentration
of thionein-bound addition
1
2
3
4
5 6 days after blrth -4
and content
of thionein-bound Cu with development of rats, were determined by HPLC-AAS after and Cd’+ to the supernatants.
Cu in the supernatants
of mercaptoethanol
simultaneously by ICP-AES and compared with those of Zn and Cu. Among those elements, changes in the concentrations of Fe, Mg and Ca which showed characteristic patterns are represented in Fig. 7. Potassium (K) and phosphorus (P) changed in concentration in a similar pattern to that of Mg. DISCUSSION
The principal aim of the present experiment was to examine whether or not the relative ratio of the two iso-MTs changes with development of mammalian species. The isoform I was shown to be a dominant peak in the livers of prenatal rats, while the isoform II was present as an abundant peak in the livers of newborn rats. Thus, the ratio of the two iso-MTs was shown to change with development of rats as ob-
served in the case of the frog Ram catesbeianu (Suzuki and Akitomi, 1983). However, the two isoMTs were present as native MT in the livers of prenatal and neonatal rats. On the other hand, only one iso-MT was present as native MT in the livers of larva and adult bullfrogs, and the other iso-MT (MT-II) was induced by loading of Cd. The relative ratio of the two induced iso-MTs was different between the larva and adult bullfrogs, the &-form I1 being a dominant &o-MT in the larva. Therefore, it may not be justified to compare the change of the relative iso-MT ratio directly between the bullfrog and the rat. However, the change of the &o-MT ratio observed in the present study suggests that the two iso-MTs are synthesized at a differing ratio with development in the livers of fetuses and degraded at a different ratio selectivity in the livers of newborn
Fig. 5. Changes in the relative ratios of the two iso-MTs with development of rdtS. of Zn and Cu bound to the two iso-forms were determined by HPLCAAS mercaptoethanol and Cd’+ to the supernatants.
The relative ratios after addition of
Pre- and neonatal
31
metallothionein 1 6.U
6.4
119.4
”
l118.2
Cd
-I
CU
Retentlon
time
15.0 --L
(
mln
_
1 -L!
k
a= cu
Retentlon tlm
(
mln
)
Fig. 6. Typical elution profiles of the two iso-MTs in the supernatants after addition of mercaptoethanol and Cd’+. The oxidized forms of MT were reduced with mercaptoethanol and the thionein-bound Zn was replaced with Cd as described in Materials and Methods. The elution profiles were determined by HPLC-AAS. The left and right panels correspond to the profiles of the embryos of 17th day of gestation and of newborns 3 days after birth, respectively. The void volume of the column and mercaptoethanol-Cd complex correspond to peaks at retention times of Il.2 and 26.4mit-1, respectively. I (19.4min) and II (18.2 min) indicate MT-I and MT-II, respectively. The vertical bars show the detector levels of Cd and cu.
rats about IO days after birth. The present observation also suggests the possibility that the gene expression of the two iso-MTs depends on the development and MT-I may be a primary &-form in a gene duplication throughout the course of molecular evolution as also suggested in the case of bullfrog MT (Suzuki and Akitomi, 1983). Although MT-I may be the primary &-form, the protein has been shown to be less stable compared with the other &-form (MT-II) (Suzuki and Maitani, 1983; Winge and Miklossy, 1982). The &-form I was also easily oxidized during the storage and
Fig. 7. Changes
in hepatic
concentrations
analytical procedures in this study and the relative ratio of the two iso-MTs (MT-I/MT-II) was found to decrease for every repetition of the procedures. Therefore, MT was analyzed after the reduction and replacement reactions. Zn in MT was quantitatively replaced with Cd and the Cd was eluted as sharp peaks in the present analytical procedure. However, Cu in the reduced and replaced solutions was eluted as broad peaks. This may indicate that Cu was not bound to the same molecule as that of Zn or at least Cu and Zn were not bound to MT at the same metal composition.
of Fe, Mg and Ca with development
of rats.
KAZUO
38
T.
The changes in the concentrations of total and thionein-bound Zn and Cu were similar to those already reported for rats (Bakka et a/., 1981; Brady et d., 1982; Mason et (II., 1980) and the changes have been discussed in relation to MT. Although it was not the aim of the present study to explain the changes of other elements, the changes in the concentrations of several elements were shown to compare with those of Zn and Cu. Although K and P changed in concentration in a similar pattern as that of Ca, other elements showed characteristic patterns to the respective elements as shown in Fig. 7. However, further investigations were not conducted in the present study. Acknowledgements~The authors express their thanks to Dr K. Kubota of this Institute, Professors K. Taki (Kitasato Univ.) and M. Takimoto (Toho Univ.) for their encouragement. REFERENCES
Bakka A. and Webb M. (1981) Metabolism of zinc and copper in the neonate: changes in the concentrations and contents of thionein-bound Zn and Cu with age in the livers of the newborn of various mammalian species. Biochem. Phurmu. 30, 721-725. Bakka A., Samarawickrama G. P. and Webb M. (1981) Metabolism of zinc and copper in the neonate: effect of cadmium administration during late gestation in the rat on the zinc and copper metabolism of the newborn. Chem. Biol. Interrrct. 34, 16lll71. Bell J. U. (1979) A metallothionein-like protein in the hepatic cytosol of the term rat fetus. Toxic. rrppl. Phurmat. 48, 1399144. Bell J. U. and Waalkcs M. P. (1982) Role of hepatic metallothionein during perinatal development in the rat. In Biolugicul Roles o/ Mriullolhionein (Edited by Foulkes E. C.) pp. 9991 I I. Elsevier-North Holland, Amsterdam.
SUZUKl
et d.
Brady F. 0.. Webb M. and Mason R. (1982) Zinc and copper metabolism in neonates: role of metallothionein in growth and development in the rat. In Biologiccrl Roles of Merullothionein (Edited by Foulkes E. C.) pp. 77-98. Elsevier-North Holland. Amsterdam. Mason R., Bakka A., Samarawickrama G. P. and Webb M. (1980) Metabolism of zinc and copper in the neonate: accumulation and function of (Zn, Cu)-metallothionein in the liver of the newborn rat. Br. J. Nurr. 45, 375-389. Ohtake H., Hasegawa K. and Koga M. (1978) Zinc-binding protein in the livers of neonatal, normal and partially hepatectonized rats. Biochem. J. 174, 999%1005. Riordan J. R. and Richards V. (1980) Human fetal liver contains both zinc- and copper-rich forms of metallothionein. J. hiol. Chem. 255, 5380-5383. Suzuki K. T. (I 980) Direct connection of high speed liquid chromatograph (equipped with gel permeation column) to atomic absorption spectrophotometer for metalloprotein analysis: Metallothionein. Anul~t. Biochem. 102, 3 l-34. Suzuki K. T. and Akitomi H. (1983) Difference in relative isometallothionein ratio between adult and larva of cadmium loaded bullfrog Rana catesheiana. Comp. Biochem. Physiol. 75C, 21 I-215. Suzuki K. T. and Maitani T. (1983) Comparative properties of the two isometallothioneins in oxidation and metal substitution reactions, submitted. Waalkes M. P., Thomas J. A. and Bell J. U. (1982) Induction of hepatic metallothionein in the rabbit fetus following maternal cadmium exposure. Toxic. appl. Pharmuc. 62, 21 l-218. Webb M.. Plastow S. R. and Magos L. (1979) (Copper. zinc)-thionein in pig liver. Lift Sci. 24, 1901-1906. Winge D. R. and Miklossy K. A. (1982) Differences in the polymorphic forms of metallothionein. Archs Biochem. Biophys. 214, 8G-88. Wong K.-L. and Klaassen C. D. (1979) Isolation and characterization of metallothionein which is highly concentrated in newborn rat liver. J. hiol. C/tern. 254, 12399912403.