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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. Interactions, 34 (1981) 161--171 © Elsevier/North-Holland Scientific Publishers Ltd.
161
METABOLISM OF ZINC AND COPPER IN T H E N E O N A T E : E F F E C T OF CADMIUM A D M I N I S T R A T I O N D U R I N G L A T E G E S T A T I O N IN T H E R AT ON TH E ZINC AND COPPER METABOLISM O F T H E NEWBORN
A. BAKKA*, G.P. S A M A R A W I C K R A M A * *
and M. W E B B t
Toxicology Unit, M.R.C. Laboratories, Woodmansterne S M 5 4 E F (United Kingdom) (Received August 15th, 1980) (Revision received October 16th, 1980) (Accepted October 18th, 1980)
Road, Carshalton,
Surrey,
SUMMARY Rats th at have been treated with Cd (1.0 mg/kg b o d y wt., i.v.) on the 18th day o f gestation give birth t o young, the livers of which are low in Zn, b u t n o t in Cu. With increasing age after birth the hepatic concentrations of total and thionein-bound Zn in these animals increase rapidly to maxima at a b o u t 7 days, approx. 6 days later than in the new born of normal dams, whereas the liver Cu c o n c e n t r a t i o n reaches a higher m a x i m u m at an earlier age than in the control neonate. This rapid uptake o f Cu into the liver of the n e w b o r n o f t he Cd-treated dam is n o t accompanied by a c o n c o m i t a n t increase in the c o n c e n t r a t i o n of soluble thionein-bound Cu. C ad miu m- t r eat m e nt of the dam retards the weight gain of the liver and, at least during the first 6--8 days pos tpart um , the increase in b o d y wt. of the newborn. When the hepatic concentrations of thionein-bound Zn is expressed relative to liver wt. instead of age, there is no significant difference between the n e w b o r n from normal and Cd-treated dams. The Zn concentrations in blood, brain, stomach, duodenum , pancreas, spleen, kidney and muscle of n e w b o r n rats either remain constant, or increase only slightly with age after birth and are not affected significantly by the administration of Cd to the dam in late gestation. This t reat m ent , which p r o b ab ly increases the demand for Zn in the newborn, delays the deposition o f Zn in bone and causes a r e d u c t i o n in the Zn c o n c e n t r a t i o n of the skin. The Cu concentrations in skin and bone, as well as in ot her organs of the n e w b o r n during the first 24 days postpartum, seem to be unaffected by Cdt r e a t m e n t o f the dam. *Fellow of the European Science Exchange Program. Present address: University of Oslo, Akershus Central Hospital, N 1474 Nordbyhagen, Norway. **WHO Fellow. Present address: Department of Community Medicine, Faculty of Medicine, University of Sri Lanka, Peradeniya, Sri Lanka. tTo whom correspondence should be s e n t .
162 It is suggested that hepatic Zn-thionein has an essential function in the Zn metabolism of the liver, b u t is unlikely to control the supply of Zn to other organs in the newborn rat.
INTRODUCTION ~tecent work in this and other laboratories [1---6] has established that the concentration of the metallothionein, Zn-thionein, is high in the livers of late foetal and newborn rats and then falls to the low level, characteristic of the adult, usually within the first 30 days after birth. It has been suggested that this metalloprotein serves as a regulator of hepatic Zn metabolism during the perinatal period [2] and there is some evidence that, in particular, it can provide a source of Zn for the maintenance of the cytosolic nonthionein Zn concentration in the liver at the level essential to sustain metabolic activity [6]. Proof of this regulatory function, however, has y e t to be obtained. In the pregnant rat Cd causes a dose-dependent inhibition of placental Zn-transport [7]. After the administration of Cd (1.0--1.25 mg/kg, i.v.) on, or after the 16th day of gestation, this inhibition initially is severe and persists for the remainder of gestation, although it decreases slowly as the placental Cd-concentration falls with time. In consequence, the accumulation of hepatic Zn-thionein in the foetus is prevented [4,8]. Although foetal uptake of Cd increases with gestational age, even in rats that are treated with ~°gCd (20 ~Ci '°gCd/mg Cd; 1.25 mg Cd/kg b o d y wt.) on the 20th day of gestation, the foetal concentration of Cd is less than 80 ng/g wet wt. tissue at any time before or after birth. Autoradiography of whole b o d y sections of such pregnant animals before delivery shows the presence of '°gCd in the liver, b u t not in any other organ of the foetus; biochemical fractionation reveals that most of this hepatic Cd is incorporated, apparently b y displacement of Zn, in the metallothionein that pre-exists in the 20-day foetal liver [8]. It seemed, therefore, that the small amounts of Cd taken up by the foetuses of the Cd-treated dam and b o u n d predominantly as the hepatic metallothionein, would have little or no effect on their postnatal growth processes. As, however, such newborn would be deficient in Znthionein, comparison of the Zn distribution in postnatal rats from normal and Cd-treated dams might provide information a b o u t the function of the hepatic metallothionein. The results of such comparative studied are summarized in this paper. MATERIALS AND METHODS Animals. Female rats of the Wistar--Porton strain, each of which had had one litter (i.e. of proven fertility) were caged separately with males overnight. The morning that vaginal plugs were found was designated day 0 of pregnancy (see e.g. Ref. 9). Each female then was housed in a stainless
163 steel cage (changed every 3rd day) and maintained on Oxoid Pasteurized I~.at Breeding Diet (Oxoid Ltd., Southwark Bridge Road, London SE1) and tap water. Cadmium (1.0 mg/kg body wt.) as a solution of CdC122~H20 in isotonic saline was administered on the 18th day of gestation; control pregnant females were treated at the same time with the appropriate volume (1.0 ml/kg body wt.) of saline only. In some experiments the Cd solution was supplemented with 1°9Cd (carrier-free; The Radiochemical Centre, Amersham, Bucks) to a specific activity of 20 uCi (740 kBq)/mg Cd. After parturition, pups of both the experimental and control series usually were culled to 8 per litter and kept with their mothers. Foetuses were obtained as described by Samarawickrama [8]. Tissues and tissue fractionation. Each pup was weighed and then killed by aecapitation, samples of blood being collected in preweighed vials. The liver, brain, kidneys, spleen, pancreas, stomach, duodenum anal femurs, together with portions of skin (including hair of the older animals) from the parietal region and muscle from the quadriceps and gluteus groups were separated by dissection. Blood and tissue samples were digested by the method of Thompson and Btanchflower [10] and analysed for Zn and Cu. Liver tissue was homogenized in 10 m=",_" Tris--HC1 buffer (pH 8.0) (2--3 ml/g wet wt. tissue), the metallothionein being separated from the soluble fraction of the homogenate by gel filtration [6]. Analytical methods. Copper and Zn were determine:~ by atomic absorption spectrophotometry. The 1°9Cd isotope was measured as described by Webb and Verschoyle [11]. RESULTS
The results of Table I show that Cd, accumulated by the liver of the TABLE
I
CONCENTRATIONS AND CONTENTS OF NEWBORN RATS AT DIFFERENT Cd T O T H E D A M
OF THIONEIN-BOUND Cd IN T H E L I V E R S AGES AFTER THE ADMINISTRATION OF
The maternal animals were injected intravenously with I°9Cd (10 uCi/mg Cd; 1 m g Cd/kg body wt.) on the 18th day of gestation and were allowed to litter.The pups (12 in each litter) were kept with their mothers from birth to analysis. The results shown below are the means of duplicate analyses, each on the pooled livers from 4 or 2 pups of the same litter. Age (days postpartum)
2 (n = 4) 9 (n = 4) 24 (n = 2)
Thionein-bound Cd Concentration (ng Cd/g wet wt. tissue)
Content (ng Cd in whole liver)
101.7 58.1 14.8
28.5 30.5 30.7
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Fig. 1. Changes in the concentrations o f total Zn ( o , . ) and o f t h i o n e i n - b o u n d Zn (%A) w i t h age in the livers of n e w b o r n rats f r o m normal dams ( ) and dams treated with Cd (1.0 m g / k g b o d y wt., i.v.) on the 18th day of gestation ( - - - - - - ) . Values for total Zn are the means (+S.D.) o f individual analyses o n 8 or 4 livers; t h i o n e i n - b o u n d Zn was measured after f r a c t i o n a t i o n of the pooled tissue (see Materials and Methods).
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165 foetus during late gestation remained b o u n d as the metallothionein after birth. Thus the decrease in the concentration of thionein-bound-Cd with age was due solely to the increase in the weight of the liver, since the c o n t e n t of Cd in this form remained constant (Table I). The presence of this hepatic Cd did not inhibit the synthesis of Zn-thionein in the newborn animals. Thus although the late foetuses of the Cd-treated rats, relative to those of normal dams, had reduced amounts of Zn in their livers (Fig. 1), after birth, which eliminated the block in Zn-transport via the placentae, their hepatic concentrations of thionein-bound and total Zn increased rapidly to reach maxima at a b o u t 7 days of postnatal age; i.e. approx. 6 days later than in the newborn of normal dams (Fig. 1). In contrast with Zn, the hepatic concentration of Cu in the pups of the Cd-treated dams was similar to that in the controls at birth, b u t then increased more rapidly to a maximum (73 pg Cu/g wet wt. tissue), approx. 70% greater and 7 days earlier than that in the controls (42 gg Cu/g wet wt. tissue; Fig. 2). This increase in total liver Cu concentration was not associated with increased uptake of Cu into the metallothionein fraction and, at present, cannot be explained.
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0 I~I821~,T ~4 ~ 8,~121~16,1~20~224 birth Age (dc]ys) Fig. 4. Liver wt. in r e l a t i o n t o age in n e w b o r n rats f r o m n o r m a l d a m s ( - - o - - ) a n d d a m s t r e a t e d w i t h Cd (1.0 m g / k g b o d y w t . , i.v.) o n t h e 1 8 t h day o f g e s t a t i o n ( - - - t - - - - ) . T h e n u m b e r o f m e a s u r e m e n t s at each age is given in brackets.
Cadmium-treatment of the dams which, at 18 days of gestation, is not teratogenic [7,8], did not alter the age at which either the eyes opened or hair-growth was apparent in the newborn. Between 10 and 20 days of age, the rate of growth of the newborn of the Cd-treated dams, as measured by the increase in b o d y wt., appeared to be the same as that of the offspring of normal rats but, during the first 6--8 days, it was retarded (Fig. 3). Thus in terms of b o d y wt. during the period of rapid growth, the former were equivalent to normal animals approx. 2 days younger. The weight gain of the livers
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Fig. 5. Hepatic concentration of thionein-bound Zn in relation to liver wt. in newborn rats from darns (--o--) and darns treated with Cd (1.0 mg/kg body wt., i.v.)on the 18th day of gestation (----e----).
167
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Postnatal age (days) Fig. 6. Z n c o n c e n t r a t i o n s in d i f f e r e n t o r g a n s a n d tissues o f n e w b o r n r a t s f r o m n o r m a l d a m s (---o--) a n d darns t r e a t e d w i t h Cd (1.0 m g / k g b o d y wt., i.v.) o n t h e 1 8 t h day o f g e s t a t i o n ( - - - - ~ - - - - ) , in r e l a t i o n t o age. T h e r e s u l t s f o r each o f t h e ages s h o w n are t h e m e a n s (-+S.D.) o f analyses o n 8 s a m p l e s o f n o r m a l n e w b o r n tissues a n d 4 s a m p l e s of t h e tissues f r o m n e w b o r n o f C d - t r e a t e d dams.
of the newborn was retarded also as a result of Cd-administration to the maternal animal during late gestation and, at any time between the 4th and 24th day after birth, the liver weights of these animals were about the same as those of 2--3 day younger controls (Fig. 4). Thus, as at any age between 10 and 24 days after birth, the whole liver concentrations of Zn in the y o u n g from normal and Cd-treated dams were the same (Fig. 1) whereas the organ weights were different (Fig. 4), it is apparent t h a t the hepatic c o n t e n t of Zn in the former remained below that in the latter t h r o u g h o u t this period. Nevertheless when the hepatic concentration of thionein-bound Zn was expressed as a function of liver wt., there was little difference between the two groups at any time (Fig. 5). In the blood, brain, stomach, d u o d e n u m , pancreas, spleen, kidney and
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Fig. 7. Cu concentrations in the spleen, kidney, bone and skin of newborn rats from normal dams (--~--) and dams treated with Cd (1.0 mg/kg body wt., i.v.) on the 18th day of gestation (----~----), in relation to age. The results are the means (-+S.D.) of analyses on 8 and 4 tissue samples, respectively (see legend to Fig. 6). muscle o f the normal n e w b o r n rat the total Zn concentrations either remained essentially constant, or increased only slightly between 2 an=l 4 ,=lays p o s t p a r t u m . In these tissues and organs the Zn concentrations were n o t affected significantly by prenatal administration of Cd to t he clam (Fig. 6). Such t r e a t m e n t , however, retarded the deposition of Zn in bone of the n ewb o r n , at least until 18 days of age, and caused an abrupt inhibition at 10 days p o s t p a r t u m of the increase in Zn c o n c e n t r a t i o n in the skin (Fig. 6). Since the samples of the latter that were analysed included hair, t he growth o f which was appar e nt at 11 days, this inhibition possibly indicates Zn conservation (i.e. decreased excretion of the metal in hair) in the offspring o f the Cd-treated mothers. A similar conservation mechanism could explain th e decreased Zn c o n c e n t r a t i o n in the bone of these animals. Th e co n ce nt r at i ons of Cu, in contrast with those o f Zn, in bone and skin (Fig. 7) as well as in spleen and kidney (Fig. 7) and in the blood, brain, stomach, d u o d e n u m and muscle {data not shown) of the new born were u n a f f e c t e d by t r e a t m e n t of the dam with Cd on the 18th day of gestation. In all o f these organs and tissues the Cu concentrations remaine,:~ low (usually less than 4 #g/g wet wt.) between the 2nd and 24th day after birth. DISCUSSION
Because o f the block in placental Zn transport t hat results from the administration of an acute dose o f Cd to the pregnant rat [7] the hepatic co n cen tr ations of total Zn and of thionein-bound-Zn in the new born of clams t h a t have been treated with Cd (1.0 mg/kg b o d y wt., i.v.) on the 18th day o f gestation, are m uch less than those of normal pups (Fig. 1). In contrast, placental t r a ns por t of Cu appears to be either unaffected, or less severely affected, since the hepatic concentrations of this metal in the offspring o f the Cd-treated and normal dams are essentially the same at birth (Fig. 2). R educed whole b o d y concentrations of Zn have been founA previously in n e w b o r n rats from mothers who had received Cd in their drinking
169 water (17.2 pg Cd/ml) t h r o u g h o u t gestation [ 1 2 ] . In contrast with the effects of a single acute, parenteral, dose, as reported herein, however, this continual low level oral exposure of the pregnant animal to Cd resulted in significantly lower birth weights and b o d y concentrations of Cu [12]. Although after administration of Cd to the pregnant rat, only small amounts of the metal cross the placentae, the concentration of thioneinb o u n d Cd in the livers of the newborn from such treated dams, as observed in the present work, was somewhat greater (i.e. a b o u t 0.1 pg/g wet wt. tissue; Table I) than that found previously in the foetuses after the injection of a higher dose {1.25 mg Cd/kg b o d y wt.) to the maternal animal on the 20th day of gestation [7,8]. As, however, the presence of Cd-thionein in the liver does not seem to affect the hepatic uptake and thionein binding of Zn in the adult animals [13] it seems reasonable to assume that this concentration of thionein bound Cd in the liver of the newborn is unlikely to influence subsequent Zn metabolism. Also as the content of thionein b o u n d Cd appears to remain essentially constant with increasing age after birth (Table I), further transport of Cd to the newborn via the milk of the treated dam cannot be significant. This, coupled with the rapid increase in hepatic Zn concentration after birth (Fig. 1) suggests that there is no residual antagonism by Cd of Zn metabolism either in the liver of the newborn, or in the mammary gland of the mother at 3 or more days after injection of the toxic metal. Rat pups, suckling females on diets inadequate in Zn, are known to have decreased rates of DNA and protein synthesis in both brain and liver [14] and to exhibit symptoms of acute Zn-deficiency (i.e. scruffy coats, dermatitis of paws and tails; Ref. 15). In the newborn of the Cd-treated dams, however, the Zn-deficiency appears to be confined to the liver. Thus, although the hepatic concentrations of both total Zn and thionein-bound Zn in these animals, relative to the controls, were found to be reJuced by a b o u t 70% (Fig. 1), the Zn concentrations in other organs and tissues, apart from bone, were little affected, at least at 4 days after birth (Fig. 6). These concentrations, other than in bone and skin, however, did not change appreciably with age and thus with b o d y wt. In contrast, the concentration of thioneinbound Zn and, therefore, of total Zn [ 6 ] , in the liver of the normal pup decreaseJ with increase in the liver wt., which is correlated with increase in both b o d y wt. and age [6]. Cadmium-treatment of the dams on the 18th day of gestation retarded the weight gain of the pups during the first 6--8 days after parturition (Fig. 3) and of their livers between the 4th and 24th day postpartum (Fig. 4). Thus, although the hepatic concentrations of thionein-bound Zn in these pups were very different from those of the controls when expressed as functions of age (Fig. 1), they were essentially the same when expressed as functions of liver wt. (Fig. 5). In mice, which are extremely susceptible to Zn deficiency during postnatal development, the growth of the liver is related directly to the level of dietary Zn [16]. It seems possible, therefore, that liver growth in newborn rats may be dependent initially upon the hepatic concentration of
170 Zn a t b i r t h , w h i c h is r e d u c e d b y C d - t r e a t m e n t o f t h e d a m s in late g e s t a t i o n . A f t e r b i r t h , w h e n t h e C d - i n d u c e d b l o c k t o Z n t r a n s p o r t n o longer exists, t h e d e m a n d f o r Z n b y t h e s e d e f i c i e n t p u p s m u s t b e high, b u t t h e i n t a k e o f this m e t a l is l i m i t e d b y its c o n c e n t r a t i o n in m i l k . Relative t o p u p s f r o m n o r m a l litters (n = 8), f o r e x a m p l e , p u p s f r o m large litters (n = 13) o f t h e same age are d e f i c i e n t in h e p a t i c Zn [ 6 ] . T h u s a l t h o u g h t h e u p t a k e o f Z n b y t h e liver of the newborn of the Cd-treated dam occurs rapidly and the concentrations o f t h e m e t a l in various o t h e r organs are m a i n t a i n e d , d e c r e a s e d a m o u n t s a p p e a r t o b e d e p o s i t e d in b o n e a n d p o s s i b l y are e x c r e t e d in t h e hair (Fig. 6). T h e parallel b e t w e e n t h e changes in t h e h e p a t i c c o n c e n t r a t i o n s o f t o t a l a n d t h i o n e i n - b o u n d Zn w i t h age in t h e n e w b o r n o f t h e C d - t r e a t e d d a m s suggests t h a t , as in n o r m a l n e w b o r n r a t s [ 6 ] , t h e m e t a U o t h i o n e i n has an essential f u n c t i o n in h e p a t i c Zn m e t a b o l i s m . A l t h o u g h Cousins [ 1 7 ] c o n siders t h a t , o n a s h o r t - t e r m basis, Z n m e t a b o l i s m in t h e a d u l t is r e g u l a t e d ( b y m e t a l l o t h i o n e i n ) in t h e liver ( a n d intestine) t o a f f e c t t h e p r o c e s s i n g o f d i e t a r y Z n a n d r e d i s t r i b u t i o n o f e n d o g e n o u s Zn, t h e p r e s e n t results p r o v i d e n o e v i d e n c e f o r a c o n t r o l f u n c t i o n o f t h e h e p a t i c m e t a l l o p r o t e i n in t h e s u p p l y o f Z n t o o t h e r organs o f t h e n e w b o r n rat. REFERENCES
1 H. Ohtake, K. Hasegawa and M. Koga, Zinc-binding protein in the livers of neonatal, normal and partially hepatectomized rats, Biochem. J., 174 (1978) 999. 2 J.U. Bell, Native metallothionein levels in rat hepatic cytosol during perinatal development, Toxicol. Appl. Pharmacol., 50 (1979) 101. 3 S.H. Oh and P.D. Whanger, Biological function of metallothionein. VII. Effect of age on its metabolism in rats, Am. J. Physiol., 237 (1979) E18. 4 M. Webb, Functions of hepatic and renal metallothioneins in the control of the metabolism of cadmium and certain other bivalent cations, in: J.H.R. K~gi and M. Nordberg (Eds.), Metallothionein, Birkhauser Verlag, Basel, Boston, Stuttgart, 1979, pp. 313--320. 5 K.-L. Wong and C.D. Klaasen, Isolation and characterization of metallothionein which is highly concentrated in newborn rat liver, J. Biol. Chem., 254 (1979) 12399. 6 R. Mason, A. Bakka, G.P. Samarawickrama and M. Webb, Metabolism of zinc and copper in the neonate: accumulation and function of (Zn, Cu)-metallothionein in the liver of the newborn rat, Brit. J. Nutr. (1980) in press. 7 G.P. Samarawickrama and M. Webb, The effects of cadmium on the pregnant rat and embryo-fetal development, Environ. Health Perspec., 28 (1979) 245. 8 G.P. Samarawickrama, Studies on the toxicity of cadmium in the pregnant rat, Ph.D. Thesis, CNAA, London, 1979. 9 W.S. Webster, Cadmium-induced fetal growth-retardation in the mouse, Arch. Environ. Health, 33 (1978) 36. 10 R.H. Thompson and W.J. Blanchflower, Wet-ashing apparatus to prepare biological materials for atomic absorption spectrophotometry, Lab. Pract., 20 (1971) 859. 11 M. Webb and R.D. Verschoyle, An investigation of the role of metallothionein in protection against the acute toxicity of the cadmium ion, Biochem. Pharmacol., 25 (1976) 673. 12 H. Choudhury, L. Hastings, E. Menden, D. Brockman, G.P. Cooper and H.G. Petering, Effect of low level prenatal cadmium exposure on trace metal body burden and behavior in Sprague--Dawley rats, in: M. Kirchgessner (Ed.), Trace Element Meta-
171
13
14 15 16 17
bolism in Man and Animals -- 3, Institut fiir Ern~rungsphysiologie, Technische Universit~'t Miinchen, Freising°Weihenstephan, 1978, pp. 549--552. G.P. Samarawickrama, Biological effects of cadmium in mammals, in: M. ~Vebb (Ed .), The Chemistry, Biochemistry and Biology of Cadmium, Elsevier/North-Holland, Amsterdam, 1979, pp. 341--421. H.H. Sandstead, D.D. Gillespie and R.N. Brady, Zinc deficiency: effect on brain of suckling rat, Pediat. Res., 6 (1972) 119. P.B. Mutch and L.S. Hurley, Effect of zinc deficiency during lactation on postnatal growth and development of rats, J. Nutr., 104 (1974) 828. R.S. Beach, M.E. Gershwin and L.S. Hurley, Growth and development in postnatally zinc-deprived mice, J. Nutr., 110 (1980) 201. R.J. Cousins, Regulatory aspects of zinc metabolism in liver and intestine, Nutr. Rev., 37 (1979) 97.
161
METABOLISM OF ZINC AND COPPER IN T H E N E O N A T E : E F F E C T OF CADMIUM A D M I N I S T R A T I O N D U R I N G L A T E G E S T A T I O N IN T H E R AT ON TH E ZINC AND COPPER METABOLISM O F T H E NEWBORN
A. BAKKA*, G.P. S A M A R A W I C K R A M A * *
and M. W E B B t
Toxicology Unit, M.R.C. Laboratories, Woodmansterne S M 5 4 E F (United Kingdom) (Received August 15th, 1980) (Revision received October 16th, 1980) (Accepted October 18th, 1980)
Road, Carshalton,
Surrey,
SUMMARY Rats th at have been treated with Cd (1.0 mg/kg b o d y wt., i.v.) on the 18th day o f gestation give birth t o young, the livers of which are low in Zn, b u t n o t in Cu. With increasing age after birth the hepatic concentrations of total and thionein-bound Zn in these animals increase rapidly to maxima at a b o u t 7 days, approx. 6 days later than in the new born of normal dams, whereas the liver Cu c o n c e n t r a t i o n reaches a higher m a x i m u m at an earlier age than in the control neonate. This rapid uptake o f Cu into the liver of the n e w b o r n o f t he Cd-treated dam is n o t accompanied by a c o n c o m i t a n t increase in the c o n c e n t r a t i o n of soluble thionein-bound Cu. C ad miu m- t r eat m e nt of the dam retards the weight gain of the liver and, at least during the first 6--8 days pos tpart um , the increase in b o d y wt. of the newborn. When the hepatic concentrations of thionein-bound Zn is expressed relative to liver wt. instead of age, there is no significant difference between the n e w b o r n from normal and Cd-treated dams. The Zn concentrations in blood, brain, stomach, duodenum , pancreas, spleen, kidney and muscle of n e w b o r n rats either remain constant, or increase only slightly with age after birth and are not affected significantly by the administration of Cd to the dam in late gestation. This t reat m ent , which p r o b ab ly increases the demand for Zn in the newborn, delays the deposition o f Zn in bone and causes a r e d u c t i o n in the Zn c o n c e n t r a t i o n of the skin. The Cu concentrations in skin and bone, as well as in ot her organs of the n e w b o r n during the first 24 days postpartum, seem to be unaffected by Cdt r e a t m e n t o f the dam. *Fellow of the European Science Exchange Program. Present address: University of Oslo, Akershus Central Hospital, N 1474 Nordbyhagen, Norway. **WHO Fellow. Present address: Department of Community Medicine, Faculty of Medicine, University of Sri Lanka, Peradeniya, Sri Lanka. tTo whom correspondence should be s e n t .
162 It is suggested that hepatic Zn-thionein has an essential function in the Zn metabolism of the liver, b u t is unlikely to control the supply of Zn to other organs in the newborn rat.
INTRODUCTION ~tecent work in this and other laboratories [1---6] has established that the concentration of the metallothionein, Zn-thionein, is high in the livers of late foetal and newborn rats and then falls to the low level, characteristic of the adult, usually within the first 30 days after birth. It has been suggested that this metalloprotein serves as a regulator of hepatic Zn metabolism during the perinatal period [2] and there is some evidence that, in particular, it can provide a source of Zn for the maintenance of the cytosolic nonthionein Zn concentration in the liver at the level essential to sustain metabolic activity [6]. Proof of this regulatory function, however, has y e t to be obtained. In the pregnant rat Cd causes a dose-dependent inhibition of placental Zn-transport [7]. After the administration of Cd (1.0--1.25 mg/kg, i.v.) on, or after the 16th day of gestation, this inhibition initially is severe and persists for the remainder of gestation, although it decreases slowly as the placental Cd-concentration falls with time. In consequence, the accumulation of hepatic Zn-thionein in the foetus is prevented [4,8]. Although foetal uptake of Cd increases with gestational age, even in rats that are treated with ~°gCd (20 ~Ci '°gCd/mg Cd; 1.25 mg Cd/kg b o d y wt.) on the 20th day of gestation, the foetal concentration of Cd is less than 80 ng/g wet wt. tissue at any time before or after birth. Autoradiography of whole b o d y sections of such pregnant animals before delivery shows the presence of '°gCd in the liver, b u t not in any other organ of the foetus; biochemical fractionation reveals that most of this hepatic Cd is incorporated, apparently b y displacement of Zn, in the metallothionein that pre-exists in the 20-day foetal liver [8]. It seemed, therefore, that the small amounts of Cd taken up by the foetuses of the Cd-treated dam and b o u n d predominantly as the hepatic metallothionein, would have little or no effect on their postnatal growth processes. As, however, such newborn would be deficient in Znthionein, comparison of the Zn distribution in postnatal rats from normal and Cd-treated dams might provide information a b o u t the function of the hepatic metallothionein. The results of such comparative studied are summarized in this paper. MATERIALS AND METHODS Animals. Female rats of the Wistar--Porton strain, each of which had had one litter (i.e. of proven fertility) were caged separately with males overnight. The morning that vaginal plugs were found was designated day 0 of pregnancy (see e.g. Ref. 9). Each female then was housed in a stainless
163 steel cage (changed every 3rd day) and maintained on Oxoid Pasteurized I~.at Breeding Diet (Oxoid Ltd., Southwark Bridge Road, London SE1) and tap water. Cadmium (1.0 mg/kg body wt.) as a solution of CdC122~H20 in isotonic saline was administered on the 18th day of gestation; control pregnant females were treated at the same time with the appropriate volume (1.0 ml/kg body wt.) of saline only. In some experiments the Cd solution was supplemented with 1°9Cd (carrier-free; The Radiochemical Centre, Amersham, Bucks) to a specific activity of 20 uCi (740 kBq)/mg Cd. After parturition, pups of both the experimental and control series usually were culled to 8 per litter and kept with their mothers. Foetuses were obtained as described by Samarawickrama [8]. Tissues and tissue fractionation. Each pup was weighed and then killed by aecapitation, samples of blood being collected in preweighed vials. The liver, brain, kidneys, spleen, pancreas, stomach, duodenum anal femurs, together with portions of skin (including hair of the older animals) from the parietal region and muscle from the quadriceps and gluteus groups were separated by dissection. Blood and tissue samples were digested by the method of Thompson and Btanchflower [10] and analysed for Zn and Cu. Liver tissue was homogenized in 10 m=",_" Tris--HC1 buffer (pH 8.0) (2--3 ml/g wet wt. tissue), the metallothionein being separated from the soluble fraction of the homogenate by gel filtration [6]. Analytical methods. Copper and Zn were determine:~ by atomic absorption spectrophotometry. The 1°9Cd isotope was measured as described by Webb and Verschoyle [11]. RESULTS
The results of Table I show that Cd, accumulated by the liver of the TABLE
I
CONCENTRATIONS AND CONTENTS OF NEWBORN RATS AT DIFFERENT Cd T O T H E D A M
OF THIONEIN-BOUND Cd IN T H E L I V E R S AGES AFTER THE ADMINISTRATION OF
The maternal animals were injected intravenously with I°9Cd (10 uCi/mg Cd; 1 m g Cd/kg body wt.) on the 18th day of gestation and were allowed to litter.The pups (12 in each litter) were kept with their mothers from birth to analysis. The results shown below are the means of duplicate analyses, each on the pooled livers from 4 or 2 pups of the same litter. Age (days postpartum)
2 (n = 4) 9 (n = 4) 24 (n = 2)
Thionein-bound Cd Concentration (ng Cd/g wet wt. tissue)
Content (ng Cd in whole liver)
101.7 58.1 14.8
28.5 30.5 30.7
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165 foetus during late gestation remained b o u n d as the metallothionein after birth. Thus the decrease in the concentration of thionein-bound-Cd with age was due solely to the increase in the weight of the liver, since the c o n t e n t of Cd in this form remained constant (Table I). The presence of this hepatic Cd did not inhibit the synthesis of Zn-thionein in the newborn animals. Thus although the late foetuses of the Cd-treated rats, relative to those of normal dams, had reduced amounts of Zn in their livers (Fig. 1), after birth, which eliminated the block in Zn-transport via the placentae, their hepatic concentrations of thionein-bound and total Zn increased rapidly to reach maxima at a b o u t 7 days of postnatal age; i.e. approx. 6 days later than in the newborn of normal dams (Fig. 1). In contrast with Zn, the hepatic concentration of Cu in the pups of the Cd-treated dams was similar to that in the controls at birth, b u t then increased more rapidly to a maximum (73 pg Cu/g wet wt. tissue), approx. 70% greater and 7 days earlier than that in the controls (42 gg Cu/g wet wt. tissue; Fig. 2). This increase in total liver Cu concentration was not associated with increased uptake of Cu into the metallothionein fraction and, at present, cannot be explained.
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Cadmium-treatment of the dams which, at 18 days of gestation, is not teratogenic [7,8], did not alter the age at which either the eyes opened or hair-growth was apparent in the newborn. Between 10 and 20 days of age, the rate of growth of the newborn of the Cd-treated dams, as measured by the increase in b o d y wt., appeared to be the same as that of the offspring of normal rats but, during the first 6--8 days, it was retarded (Fig. 3). Thus in terms of b o d y wt. during the period of rapid growth, the former were equivalent to normal animals approx. 2 days younger. The weight gain of the livers
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Postnatal age (days) Fig. 6. Z n c o n c e n t r a t i o n s in d i f f e r e n t o r g a n s a n d tissues o f n e w b o r n r a t s f r o m n o r m a l d a m s (---o--) a n d darns t r e a t e d w i t h Cd (1.0 m g / k g b o d y wt., i.v.) o n t h e 1 8 t h day o f g e s t a t i o n ( - - - - ~ - - - - ) , in r e l a t i o n t o age. T h e r e s u l t s f o r each o f t h e ages s h o w n are t h e m e a n s (-+S.D.) o f analyses o n 8 s a m p l e s o f n o r m a l n e w b o r n tissues a n d 4 s a m p l e s of t h e tissues f r o m n e w b o r n o f C d - t r e a t e d dams.
of the newborn was retarded also as a result of Cd-administration to the maternal animal during late gestation and, at any time between the 4th and 24th day after birth, the liver weights of these animals were about the same as those of 2--3 day younger controls (Fig. 4). Thus, as at any age between 10 and 24 days after birth, the whole liver concentrations of Zn in the y o u n g from normal and Cd-treated dams were the same (Fig. 1) whereas the organ weights were different (Fig. 4), it is apparent t h a t the hepatic c o n t e n t of Zn in the former remained below that in the latter t h r o u g h o u t this period. Nevertheless when the hepatic concentration of thionein-bound Zn was expressed as a function of liver wt., there was little difference between the two groups at any time (Fig. 5). In the blood, brain, stomach, d u o d e n u m , pancreas, spleen, kidney and
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Fig. 7. Cu concentrations in the spleen, kidney, bone and skin of newborn rats from normal dams (--~--) and dams treated with Cd (1.0 mg/kg body wt., i.v.) on the 18th day of gestation (----~----), in relation to age. The results are the means (-+S.D.) of analyses on 8 and 4 tissue samples, respectively (see legend to Fig. 6). muscle o f the normal n e w b o r n rat the total Zn concentrations either remained essentially constant, or increased only slightly between 2 an=l 4 ,=lays p o s t p a r t u m . In these tissues and organs the Zn concentrations were n o t affected significantly by prenatal administration of Cd to t he clam (Fig. 6). Such t r e a t m e n t , however, retarded the deposition of Zn in bone of the n ewb o r n , at least until 18 days of age, and caused an abrupt inhibition at 10 days p o s t p a r t u m of the increase in Zn c o n c e n t r a t i o n in the skin (Fig. 6). Since the samples of the latter that were analysed included hair, t he growth o f which was appar e nt at 11 days, this inhibition possibly indicates Zn conservation (i.e. decreased excretion of the metal in hair) in the offspring o f the Cd-treated mothers. A similar conservation mechanism could explain th e decreased Zn c o n c e n t r a t i o n in the bone of these animals. Th e co n ce nt r at i ons of Cu, in contrast with those o f Zn, in bone and skin (Fig. 7) as well as in spleen and kidney (Fig. 7) and in the blood, brain, stomach, d u o d e n u m and muscle {data not shown) of the new born were u n a f f e c t e d by t r e a t m e n t of the dam with Cd on the 18th day of gestation. In all o f these organs and tissues the Cu concentrations remaine,:~ low (usually less than 4 #g/g wet wt.) between the 2nd and 24th day after birth. DISCUSSION
Because o f the block in placental Zn transport t hat results from the administration of an acute dose o f Cd to the pregnant rat [7] the hepatic co n cen tr ations of total Zn and of thionein-bound-Zn in the new born of clams t h a t have been treated with Cd (1.0 mg/kg b o d y wt., i.v.) on the 18th day o f gestation, are m uch less than those of normal pups (Fig. 1). In contrast, placental t r a ns por t of Cu appears to be either unaffected, or less severely affected, since the hepatic concentrations of this metal in the offspring o f the Cd-treated and normal dams are essentially the same at birth (Fig. 2). R educed whole b o d y concentrations of Zn have been founA previously in n e w b o r n rats from mothers who had received Cd in their drinking
169 water (17.2 pg Cd/ml) t h r o u g h o u t gestation [ 1 2 ] . In contrast with the effects of a single acute, parenteral, dose, as reported herein, however, this continual low level oral exposure of the pregnant animal to Cd resulted in significantly lower birth weights and b o d y concentrations of Cu [12]. Although after administration of Cd to the pregnant rat, only small amounts of the metal cross the placentae, the concentration of thioneinb o u n d Cd in the livers of the newborn from such treated dams, as observed in the present work, was somewhat greater (i.e. a b o u t 0.1 pg/g wet wt. tissue; Table I) than that found previously in the foetuses after the injection of a higher dose {1.25 mg Cd/kg b o d y wt.) to the maternal animal on the 20th day of gestation [7,8]. As, however, the presence of Cd-thionein in the liver does not seem to affect the hepatic uptake and thionein binding of Zn in the adult animals [13] it seems reasonable to assume that this concentration of thionein bound Cd in the liver of the newborn is unlikely to influence subsequent Zn metabolism. Also as the content of thionein b o u n d Cd appears to remain essentially constant with increasing age after birth (Table I), further transport of Cd to the newborn via the milk of the treated dam cannot be significant. This, coupled with the rapid increase in hepatic Zn concentration after birth (Fig. 1) suggests that there is no residual antagonism by Cd of Zn metabolism either in the liver of the newborn, or in the mammary gland of the mother at 3 or more days after injection of the toxic metal. Rat pups, suckling females on diets inadequate in Zn, are known to have decreased rates of DNA and protein synthesis in both brain and liver [14] and to exhibit symptoms of acute Zn-deficiency (i.e. scruffy coats, dermatitis of paws and tails; Ref. 15). In the newborn of the Cd-treated dams, however, the Zn-deficiency appears to be confined to the liver. Thus, although the hepatic concentrations of both total Zn and thionein-bound Zn in these animals, relative to the controls, were found to be reJuced by a b o u t 70% (Fig. 1), the Zn concentrations in other organs and tissues, apart from bone, were little affected, at least at 4 days after birth (Fig. 6). These concentrations, other than in bone and skin, however, did not change appreciably with age and thus with b o d y wt. In contrast, the concentration of thioneinbound Zn and, therefore, of total Zn [ 6 ] , in the liver of the normal pup decreaseJ with increase in the liver wt., which is correlated with increase in both b o d y wt. and age [6]. Cadmium-treatment of the dams on the 18th day of gestation retarded the weight gain of the pups during the first 6--8 days after parturition (Fig. 3) and of their livers between the 4th and 24th day postpartum (Fig. 4). Thus, although the hepatic concentrations of thionein-bound Zn in these pups were very different from those of the controls when expressed as functions of age (Fig. 1), they were essentially the same when expressed as functions of liver wt. (Fig. 5). In mice, which are extremely susceptible to Zn deficiency during postnatal development, the growth of the liver is related directly to the level of dietary Zn [16]. It seems possible, therefore, that liver growth in newborn rats may be dependent initially upon the hepatic concentration of
170 Zn a t b i r t h , w h i c h is r e d u c e d b y C d - t r e a t m e n t o f t h e d a m s in late g e s t a t i o n . A f t e r b i r t h , w h e n t h e C d - i n d u c e d b l o c k t o Z n t r a n s p o r t n o longer exists, t h e d e m a n d f o r Z n b y t h e s e d e f i c i e n t p u p s m u s t b e high, b u t t h e i n t a k e o f this m e t a l is l i m i t e d b y its c o n c e n t r a t i o n in m i l k . Relative t o p u p s f r o m n o r m a l litters (n = 8), f o r e x a m p l e , p u p s f r o m large litters (n = 13) o f t h e same age are d e f i c i e n t in h e p a t i c Zn [ 6 ] . T h u s a l t h o u g h t h e u p t a k e o f Z n b y t h e liver of the newborn of the Cd-treated dam occurs rapidly and the concentrations o f t h e m e t a l in various o t h e r organs are m a i n t a i n e d , d e c r e a s e d a m o u n t s a p p e a r t o b e d e p o s i t e d in b o n e a n d p o s s i b l y are e x c r e t e d in t h e hair (Fig. 6). T h e parallel b e t w e e n t h e changes in t h e h e p a t i c c o n c e n t r a t i o n s o f t o t a l a n d t h i o n e i n - b o u n d Zn w i t h age in t h e n e w b o r n o f t h e C d - t r e a t e d d a m s suggests t h a t , as in n o r m a l n e w b o r n r a t s [ 6 ] , t h e m e t a U o t h i o n e i n has an essential f u n c t i o n in h e p a t i c Zn m e t a b o l i s m . A l t h o u g h Cousins [ 1 7 ] c o n siders t h a t , o n a s h o r t - t e r m basis, Z n m e t a b o l i s m in t h e a d u l t is r e g u l a t e d ( b y m e t a l l o t h i o n e i n ) in t h e liver ( a n d intestine) t o a f f e c t t h e p r o c e s s i n g o f d i e t a r y Z n a n d r e d i s t r i b u t i o n o f e n d o g e n o u s Zn, t h e p r e s e n t results p r o v i d e n o e v i d e n c e f o r a c o n t r o l f u n c t i o n o f t h e h e p a t i c m e t a l l o p r o t e i n in t h e s u p p l y o f Z n t o o t h e r organs o f t h e n e w b o r n rat. REFERENCES
1 H. Ohtake, K. Hasegawa and M. Koga, Zinc-binding protein in the livers of neonatal, normal and partially hepatectomized rats, Biochem. J., 174 (1978) 999. 2 J.U. Bell, Native metallothionein levels in rat hepatic cytosol during perinatal development, Toxicol. Appl. Pharmacol., 50 (1979) 101. 3 S.H. Oh and P.D. Whanger, Biological function of metallothionein. VII. Effect of age on its metabolism in rats, Am. J. Physiol., 237 (1979) E18. 4 M. Webb, Functions of hepatic and renal metallothioneins in the control of the metabolism of cadmium and certain other bivalent cations, in: J.H.R. K~gi and M. Nordberg (Eds.), Metallothionein, Birkhauser Verlag, Basel, Boston, Stuttgart, 1979, pp. 313--320. 5 K.-L. Wong and C.D. Klaasen, Isolation and characterization of metallothionein which is highly concentrated in newborn rat liver, J. Biol. Chem., 254 (1979) 12399. 6 R. Mason, A. Bakka, G.P. Samarawickrama and M. Webb, Metabolism of zinc and copper in the neonate: accumulation and function of (Zn, Cu)-metallothionein in the liver of the newborn rat, Brit. J. Nutr. (1980) in press. 7 G.P. Samarawickrama and M. Webb, The effects of cadmium on the pregnant rat and embryo-fetal development, Environ. Health Perspec., 28 (1979) 245. 8 G.P. Samarawickrama, Studies on the toxicity of cadmium in the pregnant rat, Ph.D. Thesis, CNAA, London, 1979. 9 W.S. Webster, Cadmium-induced fetal growth-retardation in the mouse, Arch. Environ. Health, 33 (1978) 36. 10 R.H. Thompson and W.J. Blanchflower, Wet-ashing apparatus to prepare biological materials for atomic absorption spectrophotometry, Lab. Pract., 20 (1971) 859. 11 M. Webb and R.D. Verschoyle, An investigation of the role of metallothionein in protection against the acute toxicity of the cadmium ion, Biochem. Pharmacol., 25 (1976) 673. 12 H. Choudhury, L. Hastings, E. Menden, D. Brockman, G.P. Cooper and H.G. Petering, Effect of low level prenatal cadmium exposure on trace metal body burden and behavior in Sprague--Dawley rats, in: M. Kirchgessner (Ed.), Trace Element Meta-
171
13
14 15 16 17
bolism in Man and Animals -- 3, Institut fiir Ern~rungsphysiologie, Technische Universit~'t Miinchen, Freising°Weihenstephan, 1978, pp. 549--552. G.P. Samarawickrama, Biological effects of cadmium in mammals, in: M. ~Vebb (Ed .), The Chemistry, Biochemistry and Biology of Cadmium, Elsevier/North-Holland, Amsterdam, 1979, pp. 341--421. H.H. Sandstead, D.D. Gillespie and R.N. Brady, Zinc deficiency: effect on brain of suckling rat, Pediat. Res., 6 (1972) 119. P.B. Mutch and L.S. Hurley, Effect of zinc deficiency during lactation on postnatal growth and development of rats, J. Nutr., 104 (1974) 828. R.S. Beach, M.E. Gershwin and L.S. Hurley, Growth and development in postnatally zinc-deprived mice, J. Nutr., 110 (1980) 201. R.J. Cousins, Regulatory aspects of zinc metabolism in liver and intestine, Nutr. Rev., 37 (1979) 97.