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Postnatal changes in subcellular distribution of copper, zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferation
Camp. Biochem.Physiol. Vol. 103C,No. 2, PP. 285-290, 1992 Printed in Great Britain
0306~4492/92 $5.00+ 0.00 0 1992Pergamon Press Ltd
POSTNATAL CHANGES IN SUBCELLULAR DISTRIBUTION OF COPPER, ZINC AND METALLOTHIONEIN IN THE LIVER OF BANK VOLE (CLETHRIONOMYS GLAREOLUS): A POSSIBLE INVOLVEMENT OF METALLOTHIONEIN AND COPPER IN CELL PROLIFERATION T. WLOSTOWSKI Institute of Biology, Bialystok Branch of Warsaw University, Swierkowa 20B, 15-950 Bialystok, Poland (Received 28 April 1992; accepted for publication 5 June 1992)
Abstract-l. Dramatic interdependent changes in the intracellular concentrations of copper (Cu), zinc (Zn) and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were observed. 2. The post-mitochondrial Cu, Zn and MT (Zn-MT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (C&MT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex Cu-MT was translocated to the nuclei. 3. The nuclear Cu concentration reached the highest level (62-7 1% of the total tissue Cu) in the period
from day 3 to day 20 post-partum, just prior to and during a rapidly growing liver. 4. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.
INTRODUCTION
Essential elements such as zinc (Zn) and copper (Cu) are indispensable for normal growth, development and differentiation (Walravens, 1980; Prasad, 1984; Cousins, 1985; Prohaska, 1987). The perinatal liver of most mammalian species thus far studied contains higher concentrations of these metals than do extrahepatic tissues (Shiraishi et al., 1991). The concentration of the two metals in the liver undergoes marked age-related changes during development and the peak levels of hepatic Zn and Cu in this period are several-fold higher than those in the adult liver (Terao and Owen, 1977; Riordan and Richards, 1980; Bakka and Webb, 1981; Klein et al., 1991). However, the precise role for these high levels of Zn and Cu during fetal and postnatal development remains to be determined. It has been shown that appreciable amounts of Zn and/or Cu concentrated in the cytosol of the perinatal liver are bound to metallothionein (MT), a lowmolecular-weight sulfhydryl-rich protein with a high affinity for divalent metals (Wang and Klaassen, 1979; Bakka and Webb, 1981; Lui, 1987; Klein et al., 1991). The developmental pattern of MT concentration in the perinatal liver in most cases parallels that of Zn and/or Cu, which prompted a suggestion that the protein may (1) regulate Zn and/or Cu metabolism during development or (2) supply the essential metals for various metabolic processes during periods of rapid growth and development (Cousins, 1985; Bremner, 1987; Richards, 1989). There are, however, significant species differences in the developmental profile of hepatic Zn, Cu and MT, as well as in a ratio of the metals bound to the protein in the cytoplasm. For example, elevated
concentration of Cu-rich MT are found in bovine (Hartman and Weser, 1977), Syrian hamster (Bakka and Webb, 1981) and guinea-pig (Lui, 1987) perinatal liver, while in other species such as the rat (Mason et al., 1980), Chinese hamster and mouse (Bakka and Webb, 1981), sheep (Bremner et al., 1978) and humans (Riordan and Richards, 1980; Klein er al., 1991) more Zn than Cu is associated with the protein. The reason for these species differences in perinatal Zn and Cu metabolism has not yet been satisfactorily recognized. In recent years, immunohistochemical methods have provided new interesting information on MT intracellular distribution in the perinatal liver (Panemangalore et al., 1983; Templeton et al., 1985; Nartey et al., 1987). These studies revealed, for example, in rats that there is a gradual increase in levels of cytoplasmatic MT during gestation, but at day 20, MT is mainly localized in the nuclei and remains so for several days after birth; thereafter, the intranuclear localization of MT decreased with age. Unfortunately, it is not known what is the role of MT in nuclei, as well as whether the intranuclear MT is present as a Zn- and/or Cu-containing protein. To obtain answers to these questions, at least in part, the present work was designed to examine postnatal changes in the subcellular concentrations of Cu, Zn and MT in the liver of bank voles during the first 30 days of their life. Previously, it has been found that in normal adult voles hepatic MT is induced principally by small amounts of cadmium (Cd) (Wlostowski, 1992a), but the protein binds Cu in the cytoplasm and subsequently the complex is taken up by lysosomes and to some extent by nuclei. It is of interest, whether or not similar relationships also exist in the neonates of this species.
285
286
T. MATERIALS
WLOSTOWSKI
AND METHODS
The bank voles, from our own laboratory stock, were used throughout the study. All animals were housed in stainless-steel cages in a room maintained at 1%20°C on a 16-hr light/S-hr dark cycle. The bank voles were fed on a diet that was made on the basis of data taken from literature concerning bank vole’s food maintaining the same percentage of components as in the natural food, that is, seeds, green feed and animal food (Krasowska, 1989). Thus, the diet induded seeds (wheat), 65%; dried green feed (clover, alfalfa, grasses), 22% as we11as meat and bone meal, 13%. The diet was also enriched by vitamins, Polfamix (I g~m/kiIogram diet). The food and tap water were provided ad libitum. The neonates were raised with their dam until they were studied on days 1, 3, 5, 10, 15, 20 and 30 after birth. Three litters with a litter-size of 4-5 were examined at each time point. The livers of 4-5 pups (male and female) from each litter were pooled and used as one experimental sample. The bank vole pups were anaesthetized with ether and the livers were removed and weighed. Immediately a 0.5 g liver sample pooled from pups of the same litter was transferred to 4.5 ml of 0.25 M sucrose solution (O’C) and homogenized with a motor-driven Teflon pestle in a PotterElvehjem glass homogenizer. The obtained homogenate was then subjected to differential centrifugation at 4°C. The crude nuclear fraction (nuclei and celhtlar debris) was obtained by centrifuging the homogenate at 13OOg for Smin. The resulting supematant was then centrifuged at 20,ooOg for 20min, yielding the pelleted mitochondrial-lysosomal fraction and supematant-the post-mitochondrial fraction (cytosol+ microsomes). Aliquots (IOOyl) of the supematant were removed for MT assay. The remaining supematant and the nuclear and mitochondrial-lysosomal fraction were digested with the mixed nitric and perchloric acids (Wlostowski et al., 1988). The concentrations of Cu and Zn were determined by atomic absorption spectrophotometry in air-acetylene flame, whereas Cd analyses were carried out by electrothermal atomic absorption spectrophotometry using AAS 3 Carl Zeiss Jena instrument with an EA 3 furnace attachment. MT con~ntration in the ~st-mit~hondrial fraction was determined by a Cd-hem method ~ostowski, I992a) which was based on the works of Onosaka and Cherian (1982) and Heiimaier and Summer (1985). In some cases (l- and 3day-old pups) MT was also measured in the nuclear and mitochondrial-lysosomal fractions. The pooled liver (0.5 g) was processed as described above. The nuclear and mitochondrial-lysosomal fractions were further treated according to Mehra and Bremner (1984) by resuspending in 1.5 ml of 1% (v/v) 2-mercaptoethanol in H,O. After freezing and thawing three times, as well as leaving at 4°C overnight, the homogenates were then centrifuged at 20,000 g for 20 min. In the obtained supematants MT concentration was determined by using both the Cd-hem method which only determines Zn (Cd)-thioneins and the tetrathiomolybdateCd-saturation method (Klein ef al., 1990) which is also suitable to quantify Cu~ontaining MT. In the two cases a stable cadmium was used. MT was calculated according to
the molecular weight of 6600 and the definite molar ratio of 7 moles of Cd per mole of MT (Winge and Miklossy, 1982). The Duncan’s multiple range test and the Student’s t-test were used for the determination of the statistical significance of differences between means.
RESULTS The postnatal changes in total and subcellular concentrations of Cu in the liver of bank voles are shown in Fig. fA. The total Cu concentration increased rapidly from approximately 15 pg/g wet wt in the 1 day-old pups to a peak level (43 pg/g wet wt)
II
1
3
5
I
” 10
20
20
ARC (dags)
Fig. 1. Changes in (A) the concentrations of total hepatic copper (-a---), nuclear copper (-0-), post-mitochondrial copper (-a--) and mitochondrial-lysosomal copper (--A-) and (B) the liver weight of bank voles during postnatal development. Values represent the mean (SE. are presented only for the mean total copper concentrations and tiver weight).
by day 5 postpartum. Thereafter, the hepatic Cu level declined by approximately 50% in the 10 day-old voles and the value remained relatively unchanged until 20 day postpartum. The Cu concentration approached adult level (7 fig/g wet wt) by the 30th day after birth. As can be seen from Fig. 1A, the nuclear Cu followed closely that of the total metal concentration. From day 3 to 20 postpartum, the percentage contribution of the nuclear fraction to the total tissue Cu concentration reached 62-71%. In the 1 and 30 day-old voles this contribution amounted to 40%, which was assumed to be characteristic for adult bank voles (Wtostowski, 1992a). The age-dependent changes of Cu concentration in the mitochondriallysosomal fraction were similar to some degree to those observed in the nuclear fraction, but in the former one there was no peak concentration at day 5 following parturition. The contribution of the mitochondrial-lysosomal fraction to the total Cu concentration ranged from 9% at day 5 to 19% at day 3. Unlike the nuclear and mitochondrial-lysosomal Cu, the concentration of this metal in the post-mitochondrial (cytoplasm) fraction declined abruptly from 7 pg/g wet liver in the 1 day-old pups to 2.5 pg/g wet liver in the 3 day-old ones. This decrease was regained
Cu, Zn and metallothionein
Age (da#s)
Fig. 2. Changes in (A) the concentrations of total hepatic zinc (-•-), nuclear zinc (-_O-), post-mitochondrial zinc (-_O-) and mitochondrial-lysosomal zinc (-A-) and (B) the concentration of metallothionein (MT) in the post-mitochondrial fraction of the liver in bank voles during postnatal development. Values represent the mean (S.E. are presented only for the mean total zinc and MT concentrations). by day 5 postpartum and thereafter the concentration of Cu in the post-mitochondrial fraction appeared to decrease gradually over the next 10 days, approaching adult level by the 15th day following birth (Fig. 1A). The contribution of the post-mitochondrial fraction to the total tissue Cu from day 3 to 20 amounted only to 12-22%. This value increased to approximately 45% in the 1 and 30 day-old voles. The total and subcellular concentrations of Zn also underwent dramatic changes during the course of postnatal development (Fig. 2A). The total Zn concentration decreased rapidly from 28 pg/g wet wt in the 1 day-old pups to approximately 20 pg/g wet wt in the 3 and 5 day-old voles. Then it appeared to increase gradually over the next 25 days, attaining adult value by the 30th day following parturition. The sharp decline in the total Zn concentration between 1 and 3 days after birth was accompanied by a simultaneous
drop (by approximately 9pg/g wet wt) in the level of Zn in the post-mitochondrial fraction (Fig. 2A). At the same time the nuclear Zn remained unchanged and the mitochondrial-lysosomal Zn increased insignificantly from 1.5 to 2.5 fig/g wet wt. While the post-mitochondrial Zn decrease regained by day 5 postpartum, the nuclear and mitochondrial-lysosomal Zn declined significantly at this time, suggesting an intracellular redistribution of the metal. Thereafter the nuclear Zn abruptly rose in parallel with the total Zn concentration and reached adult value in the 10 day-old voles. The concentration of Cd in the postnatal liver of bank voles could not be detected until the 30th day following parturition when it amounted to 0.01 pg/g wet wt. The maximum concentration of MT (approximately 130 pg/g wet wt) in the post-mitochondrial fraction occurred in the 1 day-old voles (Fig. 2B). The value decreased rapidly to 30 pg/g wet liver in the 3 day-old age group and to 10 pg/g in the 5 day-old voles. Thereafter, the concentration of MT approached adult level (about 5 pg/g) at 10 days of age. Since the estimation of MT in the post-mitochondrial fraction was performed by using a Cd-hem method which only determines Zn (Cd)-thioneins (Scheuhammer and Cherian, 1986), and there was no Cd in the liver between 1 and 20 days following birth, it may thus be assumed that the changes in the protein concentration reflected primarily those of Zn-MT. In addition, there was a simultaneous sharp decline in the concentrations of the total hepatic Zn (by 8.5 pg/g), post-mitochondrial Zn (by 9.5 pg/g), as well as postmitochondrial MT (by 100 pg/g or 7 pg Zn in MT/g) in the period between 1 and 3 days after birth. These data might suggest a removal of the complex Zn-MT from the liver. However, at the same time there was also an abrupt decrease (by 4.5 pg/g) in the postmitochondrial Cu and a concomitant sharp increase particularly in the nuclear Cu concentration. These data together with those for Zn and MT might suggest, on the other hand, that in the period between 1 and 3 days postpartum the cytosolic Cu displaced Zn ions already bound to MT and subsequently the complex Cu-MT was translocated from the cytoplasm to the nuclei. To test this possibility MT concentration was also determined in the nuclear and mitochondrial-lysosomal fractions of the 1 and 3 day-old pups. The data of this experiment (Table 1) confirmed the above assumption as most of the cytosolic MT in the 1 day-old pups was recovered in the nuclei of the 3 day-old age group. In addition, by using simultaneously the th&molybdate-Cd-satur&ion method and
Table I. Subcellular concentrations of metailothionein (MT) in the liver of
Age
Nuclear Cu
Nuclear MT
Id 3d
6.0 i 0.3 15.2 + 0.q
15k3(1.7) 90 f 13t (10.5)
281
Lysosomal MT 5.0 + 0.3 (0.57) 10.0+0.5$(1.15)
I and 3 dav-old bank voles* Post-mitochondrial
MT
140 * 15 40 + 5t
‘Values (pg/g wet wt) represent the mean f SE. of 3 determinations. MT assay was performed by using a Cd-hem method (Wlostowski, 1992a) which determines only Zn (Cd)-containing thioneins (data not shown) and a thiomolybdate-Cd-saturation method (Klein ef (II., 1990) which also measures Co-containing thioneins (data presented above). There were only traces of the nuclear and lysosomal MT when assayed by a Cd-hem method but similar amounts of the post-mitochondrial MT were detected by using the two methods. In parentheses the amount of Cu bound to MT is shown, assuming that a mole of MT (m. wt 6600) binds 12 moles of Cu (Bremner, 1987). tP < 0.01, $I’ < 0.05 as compared with the 1 day-old age group (Student’s r-test).
T.
288
WLOSTOWSKI
the Cd-hem method, it was possible to explore that nearly 100% of metal-binding sites in MT present in the nuclei and lysosomes were occupied by Cu, whereas those of MT localized in the cytoplasm by Zn. DISCUSSION
The present paper demonstrated that the postnatal liver of bank vole exhibits dramatic changes in the intracellular distribution of Cu, Zn and MT. In addition, these changes are probably interdependent. For instance, the cytosolic MT present in the highest amounts in the 1 day-old pups contained predominantly Zn, but at the 3rd day MT was localized mainly in the nuclei and contained primarily Cu (Fig. 2, Table 1). The changes in the intracellular localization and metal composition of MT may be explained by a substitution process involving the pool of Zn-MT already present in the cytoplasm (Fig. 2) and the increasing concentration of Cu during that period (Fig. 1). Because Cu has a higher binding affinity for MT than does Zn (Day et al., 1981; Klgi and SchXer, 1988), it cannot be ruled out that Cu ions displaced the Zn bound to MT and then a complex Cu-MT was translocated to the nucleus and to some extent to lysosomes. Simultaneously, the liberated from the protein Zn was probably removed from the liver since there was a corresponding decrease in its total tissue concentration (Fig. 2). Thus, the data obtained strongly suggest that there is a metal composition exchange in the protein, from Zn-MT to Cu-MT, and subsequent translocation of Cu-MT from the cytoplasm to nuclei in the neonatal bank vole liver. The fact that MT is present in high amounts in the nuclei of the postnatal bank vole liver remains in a good agreement with recent immunolocalization studies carried out with other species such as rat and human fetuses and neonates (Panemangalore et al., 1983; Templeton et al., 1985; Nartey et al., 1987). The protein was also demonstrated biochemically in the nuclei of fetal deer liver (Leighton, 1987, cited in Bremner and Beattie, 1990). Interestingly, MT was also found immunohistochemically in the nuclei of leptotene spermatocytes (Nishimura et al., 1990), as well as in the nuclei of primary cultured adult rat hepatocytes stimulated by a growth factor (Tsujikawa et al., 1991). Furthermore, in the partially hepatectomized rat liver, which was used as a model for actively growing tissues, MT was localized predominantly in the nuclei, whereas MT was found only in the cytoplasm of the laparotomized rat liver (Nishimura et al., 1989). These studies strongly suggested a close association of MT with cell proliferation, especially with the early stages of this process. It is interesting to note that also in the present paper an increase in MT, as well as in Cu concentrations in the nuclei preceded a rapidly growing liver (Figs 1 and 2, Table 1). This could suggest that both MT and Cu are involved in the hepatocyte proliferation. Although the exact role of MT and Cu in the nuclei of dividing cells remains to be determined, one may conclude, on the basis of the data obtained in this work, that MT may function (1) as a Cu transfer protein from cytoplasm to nucleus or (2) as a Cu detoxifying factor in the cell nuclei. Also, it cannot
be excluded that the complex Cu-MT, by itself, is involved in some way in the initiation of nuclear DNA synthesis. The latter assumption is supported, at least in part, by a recent study (Tsujikawa et al., 1991) which revealed that MT was present in the cytoplasm of hepatocytes in the G, phase, but was localized mainly in the cell nuclei in the early S phase. Obviously further studies with dividing cells are needed to obtain an exact answer to the question whether or not the translocation of MT from cytoplasm to nucleus is associated with a concomitant transport of Cu. These studies would aid to explore the precise role of high levels of Cu in the perinatal liver of various mammalian species (Terao and Owen, 1977; Bakka and Webb, 1981; Lui, 1987; Klein et al., 1991), as well as of those being accumulated to a great extent by cells of malignant tumors (Nederbragt et al., 1989). The present work revealed also that the synthesis of MT in the newborn bank vole liver was not related to small amounts of Cd as it had been recently found for adult bank voles or rats under normal physiological conditions (Wlostowski, 1992a, b). It is also unlikely that Zn and Cu ions induced the synthesis of MT since the Zn concentration was probably too low and the nuclear Cu concentration was rather inversely than positively correlated with the cytosolic MT level (Figs 1 and 2). In agreement, it has been reported that perinatal liver Zn and Cu do not appear to be important for the regulation of hepatic MT mRNA in the rat (Mercer and Grimes, 1986). Although the synthesis of perinatal liver MT is assumed to be regulated mainly by some hormones (Quaife et al., 1986), Zn ions appear to be important in maintaining high levels of MT in the cytoplasm as newborn liver MT content is severely decreased as a result of maternal Zn deficiency (Gallant and Cherian, 1986). This indicates that either Zn in the perinatal liver plays an important role in the stabilization of the protein as it has been previously suggested for adult liver MT (Bremner et al., 1978; Cain and Holt, 1979; Wlostowski, 1992b) or MT acts as a Zn storage protein during development. But even if the latter possibility is true, a question arises as to why the concentration of Zn in the liver of newborn voles declined between 1 and 3 days postpartum, just before the moment when the liver started to rapidly grow and Zn ions are known to be required in higher amounts for this process (unless the removed Zn was urgently needed to be utilized by some extrahepatic tissues-but this aspect of Zn metabolism is as yet unknown). Nevertheless, the present results as well as those obtained by Lui (1987) for the perinatal guineapig suggest a limited role of MT as a Zn storage protein during development. Still, it may be assumed that Zn ions are involved in the stabilization of thionein molecules during development of various mammalian species. It may be supposed further that at the definite time of development Zn ions bound to MT are displaced by Cu and then a complex Cu-MT is translocated to nuclei or lysosomes. Although this assumption requires further verification, there is some circumstantial evidence in the literature, indicating that it may be also the case in rats. For example, an abrupt decline in the cytosolic Zn-MT at 7-14 days postpartum observed by Wong and Klaassen (1979)
Cu, Zn and metallothionein coincides with a sharp increase in the total and nuclear Cu concentrations (Evans et al., 1970; Terao and Owen, 1977). In addition, at the 13th day following birth the cytosolic MT contains greater amounts of Cu than Zn (Holt et al., 1987), suggesting an involvement of the substitution process. In summary, the present paper demonstrated interdependent changes in the intracellular distribution of Cu, Zn and MT in the newborn bank vole liver. In contrast to the adult bank voles, the synthesis of MT in the liver of newborns is not induced by small amounts of Cd, but presumably some hormones regulate hepatic MT level during development. It is also hypothesized that MT and Cu are involved in some way in the hepatocyte proliferation since in the period directly preceding a rapidly growing liver the protein already present in the cytoplasm as Zn-MT is translocated to the nuclei as Cu-MT. The precise role of MT and high levels of Cu in the nuclei still remains to be elucidated.
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0306~4492/92 $5.00+ 0.00 0 1992Pergamon Press Ltd
POSTNATAL CHANGES IN SUBCELLULAR DISTRIBUTION OF COPPER, ZINC AND METALLOTHIONEIN IN THE LIVER OF BANK VOLE (CLETHRIONOMYS GLAREOLUS): A POSSIBLE INVOLVEMENT OF METALLOTHIONEIN AND COPPER IN CELL PROLIFERATION T. WLOSTOWSKI Institute of Biology, Bialystok Branch of Warsaw University, Swierkowa 20B, 15-950 Bialystok, Poland (Received 28 April 1992; accepted for publication 5 June 1992)
Abstract-l. Dramatic interdependent changes in the intracellular concentrations of copper (Cu), zinc (Zn) and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were observed. 2. The post-mitochondrial Cu, Zn and MT (Zn-MT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (C&MT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex Cu-MT was translocated to the nuclei. 3. The nuclear Cu concentration reached the highest level (62-7 1% of the total tissue Cu) in the period
from day 3 to day 20 post-partum, just prior to and during a rapidly growing liver. 4. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.
INTRODUCTION
Essential elements such as zinc (Zn) and copper (Cu) are indispensable for normal growth, development and differentiation (Walravens, 1980; Prasad, 1984; Cousins, 1985; Prohaska, 1987). The perinatal liver of most mammalian species thus far studied contains higher concentrations of these metals than do extrahepatic tissues (Shiraishi et al., 1991). The concentration of the two metals in the liver undergoes marked age-related changes during development and the peak levels of hepatic Zn and Cu in this period are several-fold higher than those in the adult liver (Terao and Owen, 1977; Riordan and Richards, 1980; Bakka and Webb, 1981; Klein et al., 1991). However, the precise role for these high levels of Zn and Cu during fetal and postnatal development remains to be determined. It has been shown that appreciable amounts of Zn and/or Cu concentrated in the cytosol of the perinatal liver are bound to metallothionein (MT), a lowmolecular-weight sulfhydryl-rich protein with a high affinity for divalent metals (Wang and Klaassen, 1979; Bakka and Webb, 1981; Lui, 1987; Klein et al., 1991). The developmental pattern of MT concentration in the perinatal liver in most cases parallels that of Zn and/or Cu, which prompted a suggestion that the protein may (1) regulate Zn and/or Cu metabolism during development or (2) supply the essential metals for various metabolic processes during periods of rapid growth and development (Cousins, 1985; Bremner, 1987; Richards, 1989). There are, however, significant species differences in the developmental profile of hepatic Zn, Cu and MT, as well as in a ratio of the metals bound to the protein in the cytoplasm. For example, elevated
concentration of Cu-rich MT are found in bovine (Hartman and Weser, 1977), Syrian hamster (Bakka and Webb, 1981) and guinea-pig (Lui, 1987) perinatal liver, while in other species such as the rat (Mason et al., 1980), Chinese hamster and mouse (Bakka and Webb, 1981), sheep (Bremner et al., 1978) and humans (Riordan and Richards, 1980; Klein er al., 1991) more Zn than Cu is associated with the protein. The reason for these species differences in perinatal Zn and Cu metabolism has not yet been satisfactorily recognized. In recent years, immunohistochemical methods have provided new interesting information on MT intracellular distribution in the perinatal liver (Panemangalore et al., 1983; Templeton et al., 1985; Nartey et al., 1987). These studies revealed, for example, in rats that there is a gradual increase in levels of cytoplasmatic MT during gestation, but at day 20, MT is mainly localized in the nuclei and remains so for several days after birth; thereafter, the intranuclear localization of MT decreased with age. Unfortunately, it is not known what is the role of MT in nuclei, as well as whether the intranuclear MT is present as a Zn- and/or Cu-containing protein. To obtain answers to these questions, at least in part, the present work was designed to examine postnatal changes in the subcellular concentrations of Cu, Zn and MT in the liver of bank voles during the first 30 days of their life. Previously, it has been found that in normal adult voles hepatic MT is induced principally by small amounts of cadmium (Cd) (Wlostowski, 1992a), but the protein binds Cu in the cytoplasm and subsequently the complex is taken up by lysosomes and to some extent by nuclei. It is of interest, whether or not similar relationships also exist in the neonates of this species.
285
286
T. MATERIALS
WLOSTOWSKI
AND METHODS
The bank voles, from our own laboratory stock, were used throughout the study. All animals were housed in stainless-steel cages in a room maintained at 1%20°C on a 16-hr light/S-hr dark cycle. The bank voles were fed on a diet that was made on the basis of data taken from literature concerning bank vole’s food maintaining the same percentage of components as in the natural food, that is, seeds, green feed and animal food (Krasowska, 1989). Thus, the diet induded seeds (wheat), 65%; dried green feed (clover, alfalfa, grasses), 22% as we11as meat and bone meal, 13%. The diet was also enriched by vitamins, Polfamix (I g~m/kiIogram diet). The food and tap water were provided ad libitum. The neonates were raised with their dam until they were studied on days 1, 3, 5, 10, 15, 20 and 30 after birth. Three litters with a litter-size of 4-5 were examined at each time point. The livers of 4-5 pups (male and female) from each litter were pooled and used as one experimental sample. The bank vole pups were anaesthetized with ether and the livers were removed and weighed. Immediately a 0.5 g liver sample pooled from pups of the same litter was transferred to 4.5 ml of 0.25 M sucrose solution (O’C) and homogenized with a motor-driven Teflon pestle in a PotterElvehjem glass homogenizer. The obtained homogenate was then subjected to differential centrifugation at 4°C. The crude nuclear fraction (nuclei and celhtlar debris) was obtained by centrifuging the homogenate at 13OOg for Smin. The resulting supematant was then centrifuged at 20,ooOg for 20min, yielding the pelleted mitochondrial-lysosomal fraction and supematant-the post-mitochondrial fraction (cytosol+ microsomes). Aliquots (IOOyl) of the supematant were removed for MT assay. The remaining supematant and the nuclear and mitochondrial-lysosomal fraction were digested with the mixed nitric and perchloric acids (Wlostowski et al., 1988). The concentrations of Cu and Zn were determined by atomic absorption spectrophotometry in air-acetylene flame, whereas Cd analyses were carried out by electrothermal atomic absorption spectrophotometry using AAS 3 Carl Zeiss Jena instrument with an EA 3 furnace attachment. MT con~ntration in the ~st-mit~hondrial fraction was determined by a Cd-hem method ~ostowski, I992a) which was based on the works of Onosaka and Cherian (1982) and Heiimaier and Summer (1985). In some cases (l- and 3day-old pups) MT was also measured in the nuclear and mitochondrial-lysosomal fractions. The pooled liver (0.5 g) was processed as described above. The nuclear and mitochondrial-lysosomal fractions were further treated according to Mehra and Bremner (1984) by resuspending in 1.5 ml of 1% (v/v) 2-mercaptoethanol in H,O. After freezing and thawing three times, as well as leaving at 4°C overnight, the homogenates were then centrifuged at 20,000 g for 20 min. In the obtained supematants MT concentration was determined by using both the Cd-hem method which only determines Zn (Cd)-thioneins and the tetrathiomolybdateCd-saturation method (Klein ef al., 1990) which is also suitable to quantify Cu~ontaining MT. In the two cases a stable cadmium was used. MT was calculated according to
the molecular weight of 6600 and the definite molar ratio of 7 moles of Cd per mole of MT (Winge and Miklossy, 1982). The Duncan’s multiple range test and the Student’s t-test were used for the determination of the statistical significance of differences between means.
RESULTS The postnatal changes in total and subcellular concentrations of Cu in the liver of bank voles are shown in Fig. fA. The total Cu concentration increased rapidly from approximately 15 pg/g wet wt in the 1 day-old pups to a peak level (43 pg/g wet wt)
II
1
3
5
I
” 10
20
20
ARC (dags)
Fig. 1. Changes in (A) the concentrations of total hepatic copper (-a---), nuclear copper (-0-), post-mitochondrial copper (-a--) and mitochondrial-lysosomal copper (--A-) and (B) the liver weight of bank voles during postnatal development. Values represent the mean (SE. are presented only for the mean total copper concentrations and tiver weight).
by day 5 postpartum. Thereafter, the hepatic Cu level declined by approximately 50% in the 10 day-old voles and the value remained relatively unchanged until 20 day postpartum. The Cu concentration approached adult level (7 fig/g wet wt) by the 30th day after birth. As can be seen from Fig. 1A, the nuclear Cu followed closely that of the total metal concentration. From day 3 to 20 postpartum, the percentage contribution of the nuclear fraction to the total tissue Cu concentration reached 62-71%. In the 1 and 30 day-old voles this contribution amounted to 40%, which was assumed to be characteristic for adult bank voles (Wtostowski, 1992a). The age-dependent changes of Cu concentration in the mitochondriallysosomal fraction were similar to some degree to those observed in the nuclear fraction, but in the former one there was no peak concentration at day 5 following parturition. The contribution of the mitochondrial-lysosomal fraction to the total Cu concentration ranged from 9% at day 5 to 19% at day 3. Unlike the nuclear and mitochondrial-lysosomal Cu, the concentration of this metal in the post-mitochondrial (cytoplasm) fraction declined abruptly from 7 pg/g wet liver in the 1 day-old pups to 2.5 pg/g wet liver in the 3 day-old ones. This decrease was regained
Cu, Zn and metallothionein
Age (da#s)
Fig. 2. Changes in (A) the concentrations of total hepatic zinc (-•-), nuclear zinc (-_O-), post-mitochondrial zinc (-_O-) and mitochondrial-lysosomal zinc (-A-) and (B) the concentration of metallothionein (MT) in the post-mitochondrial fraction of the liver in bank voles during postnatal development. Values represent the mean (S.E. are presented only for the mean total zinc and MT concentrations). by day 5 postpartum and thereafter the concentration of Cu in the post-mitochondrial fraction appeared to decrease gradually over the next 10 days, approaching adult level by the 15th day following birth (Fig. 1A). The contribution of the post-mitochondrial fraction to the total tissue Cu from day 3 to 20 amounted only to 12-22%. This value increased to approximately 45% in the 1 and 30 day-old voles. The total and subcellular concentrations of Zn also underwent dramatic changes during the course of postnatal development (Fig. 2A). The total Zn concentration decreased rapidly from 28 pg/g wet wt in the 1 day-old pups to approximately 20 pg/g wet wt in the 3 and 5 day-old voles. Then it appeared to increase gradually over the next 25 days, attaining adult value by the 30th day following parturition. The sharp decline in the total Zn concentration between 1 and 3 days after birth was accompanied by a simultaneous
drop (by approximately 9pg/g wet wt) in the level of Zn in the post-mitochondrial fraction (Fig. 2A). At the same time the nuclear Zn remained unchanged and the mitochondrial-lysosomal Zn increased insignificantly from 1.5 to 2.5 fig/g wet wt. While the post-mitochondrial Zn decrease regained by day 5 postpartum, the nuclear and mitochondrial-lysosomal Zn declined significantly at this time, suggesting an intracellular redistribution of the metal. Thereafter the nuclear Zn abruptly rose in parallel with the total Zn concentration and reached adult value in the 10 day-old voles. The concentration of Cd in the postnatal liver of bank voles could not be detected until the 30th day following parturition when it amounted to 0.01 pg/g wet wt. The maximum concentration of MT (approximately 130 pg/g wet wt) in the post-mitochondrial fraction occurred in the 1 day-old voles (Fig. 2B). The value decreased rapidly to 30 pg/g wet liver in the 3 day-old age group and to 10 pg/g in the 5 day-old voles. Thereafter, the concentration of MT approached adult level (about 5 pg/g) at 10 days of age. Since the estimation of MT in the post-mitochondrial fraction was performed by using a Cd-hem method which only determines Zn (Cd)-thioneins (Scheuhammer and Cherian, 1986), and there was no Cd in the liver between 1 and 20 days following birth, it may thus be assumed that the changes in the protein concentration reflected primarily those of Zn-MT. In addition, there was a simultaneous sharp decline in the concentrations of the total hepatic Zn (by 8.5 pg/g), post-mitochondrial Zn (by 9.5 pg/g), as well as postmitochondrial MT (by 100 pg/g or 7 pg Zn in MT/g) in the period between 1 and 3 days after birth. These data might suggest a removal of the complex Zn-MT from the liver. However, at the same time there was also an abrupt decrease (by 4.5 pg/g) in the postmitochondrial Cu and a concomitant sharp increase particularly in the nuclear Cu concentration. These data together with those for Zn and MT might suggest, on the other hand, that in the period between 1 and 3 days postpartum the cytosolic Cu displaced Zn ions already bound to MT and subsequently the complex Cu-MT was translocated from the cytoplasm to the nuclei. To test this possibility MT concentration was also determined in the nuclear and mitochondrial-lysosomal fractions of the 1 and 3 day-old pups. The data of this experiment (Table 1) confirmed the above assumption as most of the cytosolic MT in the 1 day-old pups was recovered in the nuclei of the 3 day-old age group. In addition, by using simultaneously the th&molybdate-Cd-satur&ion method and
Table I. Subcellular concentrations of metailothionein (MT) in the liver of
Age
Nuclear Cu
Nuclear MT
Id 3d
6.0 i 0.3 15.2 + 0.q
15k3(1.7) 90 f 13t (10.5)
281
Lysosomal MT 5.0 + 0.3 (0.57) 10.0+0.5$(1.15)
I and 3 dav-old bank voles* Post-mitochondrial
MT
140 * 15 40 + 5t
‘Values (pg/g wet wt) represent the mean f SE. of 3 determinations. MT assay was performed by using a Cd-hem method (Wlostowski, 1992a) which determines only Zn (Cd)-containing thioneins (data not shown) and a thiomolybdate-Cd-saturation method (Klein ef (II., 1990) which also measures Co-containing thioneins (data presented above). There were only traces of the nuclear and lysosomal MT when assayed by a Cd-hem method but similar amounts of the post-mitochondrial MT were detected by using the two methods. In parentheses the amount of Cu bound to MT is shown, assuming that a mole of MT (m. wt 6600) binds 12 moles of Cu (Bremner, 1987). tP < 0.01, $I’ < 0.05 as compared with the 1 day-old age group (Student’s r-test).
T.
288
WLOSTOWSKI
the Cd-hem method, it was possible to explore that nearly 100% of metal-binding sites in MT present in the nuclei and lysosomes were occupied by Cu, whereas those of MT localized in the cytoplasm by Zn. DISCUSSION
The present paper demonstrated that the postnatal liver of bank vole exhibits dramatic changes in the intracellular distribution of Cu, Zn and MT. In addition, these changes are probably interdependent. For instance, the cytosolic MT present in the highest amounts in the 1 day-old pups contained predominantly Zn, but at the 3rd day MT was localized mainly in the nuclei and contained primarily Cu (Fig. 2, Table 1). The changes in the intracellular localization and metal composition of MT may be explained by a substitution process involving the pool of Zn-MT already present in the cytoplasm (Fig. 2) and the increasing concentration of Cu during that period (Fig. 1). Because Cu has a higher binding affinity for MT than does Zn (Day et al., 1981; Klgi and SchXer, 1988), it cannot be ruled out that Cu ions displaced the Zn bound to MT and then a complex Cu-MT was translocated to the nucleus and to some extent to lysosomes. Simultaneously, the liberated from the protein Zn was probably removed from the liver since there was a corresponding decrease in its total tissue concentration (Fig. 2). Thus, the data obtained strongly suggest that there is a metal composition exchange in the protein, from Zn-MT to Cu-MT, and subsequent translocation of Cu-MT from the cytoplasm to nuclei in the neonatal bank vole liver. The fact that MT is present in high amounts in the nuclei of the postnatal bank vole liver remains in a good agreement with recent immunolocalization studies carried out with other species such as rat and human fetuses and neonates (Panemangalore et al., 1983; Templeton et al., 1985; Nartey et al., 1987). The protein was also demonstrated biochemically in the nuclei of fetal deer liver (Leighton, 1987, cited in Bremner and Beattie, 1990). Interestingly, MT was also found immunohistochemically in the nuclei of leptotene spermatocytes (Nishimura et al., 1990), as well as in the nuclei of primary cultured adult rat hepatocytes stimulated by a growth factor (Tsujikawa et al., 1991). Furthermore, in the partially hepatectomized rat liver, which was used as a model for actively growing tissues, MT was localized predominantly in the nuclei, whereas MT was found only in the cytoplasm of the laparotomized rat liver (Nishimura et al., 1989). These studies strongly suggested a close association of MT with cell proliferation, especially with the early stages of this process. It is interesting to note that also in the present paper an increase in MT, as well as in Cu concentrations in the nuclei preceded a rapidly growing liver (Figs 1 and 2, Table 1). This could suggest that both MT and Cu are involved in the hepatocyte proliferation. Although the exact role of MT and Cu in the nuclei of dividing cells remains to be determined, one may conclude, on the basis of the data obtained in this work, that MT may function (1) as a Cu transfer protein from cytoplasm to nucleus or (2) as a Cu detoxifying factor in the cell nuclei. Also, it cannot
be excluded that the complex Cu-MT, by itself, is involved in some way in the initiation of nuclear DNA synthesis. The latter assumption is supported, at least in part, by a recent study (Tsujikawa et al., 1991) which revealed that MT was present in the cytoplasm of hepatocytes in the G, phase, but was localized mainly in the cell nuclei in the early S phase. Obviously further studies with dividing cells are needed to obtain an exact answer to the question whether or not the translocation of MT from cytoplasm to nucleus is associated with a concomitant transport of Cu. These studies would aid to explore the precise role of high levels of Cu in the perinatal liver of various mammalian species (Terao and Owen, 1977; Bakka and Webb, 1981; Lui, 1987; Klein et al., 1991), as well as of those being accumulated to a great extent by cells of malignant tumors (Nederbragt et al., 1989). The present work revealed also that the synthesis of MT in the newborn bank vole liver was not related to small amounts of Cd as it had been recently found for adult bank voles or rats under normal physiological conditions (Wlostowski, 1992a, b). It is also unlikely that Zn and Cu ions induced the synthesis of MT since the Zn concentration was probably too low and the nuclear Cu concentration was rather inversely than positively correlated with the cytosolic MT level (Figs 1 and 2). In agreement, it has been reported that perinatal liver Zn and Cu do not appear to be important for the regulation of hepatic MT mRNA in the rat (Mercer and Grimes, 1986). Although the synthesis of perinatal liver MT is assumed to be regulated mainly by some hormones (Quaife et al., 1986), Zn ions appear to be important in maintaining high levels of MT in the cytoplasm as newborn liver MT content is severely decreased as a result of maternal Zn deficiency (Gallant and Cherian, 1986). This indicates that either Zn in the perinatal liver plays an important role in the stabilization of the protein as it has been previously suggested for adult liver MT (Bremner et al., 1978; Cain and Holt, 1979; Wlostowski, 1992b) or MT acts as a Zn storage protein during development. But even if the latter possibility is true, a question arises as to why the concentration of Zn in the liver of newborn voles declined between 1 and 3 days postpartum, just before the moment when the liver started to rapidly grow and Zn ions are known to be required in higher amounts for this process (unless the removed Zn was urgently needed to be utilized by some extrahepatic tissues-but this aspect of Zn metabolism is as yet unknown). Nevertheless, the present results as well as those obtained by Lui (1987) for the perinatal guineapig suggest a limited role of MT as a Zn storage protein during development. Still, it may be assumed that Zn ions are involved in the stabilization of thionein molecules during development of various mammalian species. It may be supposed further that at the definite time of development Zn ions bound to MT are displaced by Cu and then a complex Cu-MT is translocated to nuclei or lysosomes. Although this assumption requires further verification, there is some circumstantial evidence in the literature, indicating that it may be also the case in rats. For example, an abrupt decline in the cytosolic Zn-MT at 7-14 days postpartum observed by Wong and Klaassen (1979)
Cu, Zn and metallothionein coincides with a sharp increase in the total and nuclear Cu concentrations (Evans et al., 1970; Terao and Owen, 1977). In addition, at the 13th day following birth the cytosolic MT contains greater amounts of Cu than Zn (Holt et al., 1987), suggesting an involvement of the substitution process. In summary, the present paper demonstrated interdependent changes in the intracellular distribution of Cu, Zn and MT in the newborn bank vole liver. In contrast to the adult bank voles, the synthesis of MT in the liver of newborns is not induced by small amounts of Cd, but presumably some hormones regulate hepatic MT level during development. It is also hypothesized that MT and Cu are involved in some way in the hepatocyte proliferation since in the period directly preceding a rapidly growing liver the protein already present in the cytoplasm as Zn-MT is translocated to the nuclei as Cu-MT. The precise role of MT and high levels of Cu in the nuclei still remains to be elucidated.
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v
I_