Effect of estrogen administration upon ceruloplasmin and copper concentrations in rat serum

TOXICOLOGYANDAPPLIEDPHARMACOLOGY

Effect

of Estrogen Copper

20,588-598(1971)

Administration Concentrations

upon Ceruloplasmin in Rat Serum1

and

F. WILLIAM SUNDERMAN,JR., SHOZONOMOTO, CONCET~INAG. GILLIES, AND PETERJ. GOLDBLATT Departments of Laboratory Medicine and Pathology, University of Connecticut School of Medicine, Newington, Connecticut 06111 Received April 8, 1971

Effect of Estrogen Administration upon Ceruloplasminand Copper Concentrationsin Rat Serum.SUNDERMAN, F. W., JR.,NOMOTO, S.,GILLIFS, C. G., and GOLDBLATT, P.J. (1971).Toxicol. Appl. Pharmacol. 20,588-598. Administration of estradiol-17fito rats (200 g body wt) in SCdosageof 50 pg/day for 16 days stimulated3-fold increasesin meanconcentrationsof serum ceruloplasmin(CPN) and Cu, without producing ultrastructural alterations of the hepatocytes. A dose-responsestudy showed that increasedconcentrationsof serumCPN and Cu were producedby 16 daily injectionsof estradiol-17/?throughout the doserangefrom 2 to 200pg/day, and that 50pg/day wassufficientfor maximumstimulation.A time-response study showedthat significant increasesin meanconcentrationsof serum CPN and Cu did not occur until after 7 daily injectionsof 50yg of estradiol17/3,and that maximum increasesof serumCPN and CU concentrations wereachievedafter 28 daily injectionsin malerats and after 35 daily injections in ovariectomizedfemalerats. Electron microscopicstudy of livers of rats which received42 daily injectionsof estradiol-17/3 (50 pg/day) revealed accumulationof hyaloplasmiclipid droplets within scatteredhepatocytes. Stimulation of increasedconcentrationsof serumCPN by administration of estradiol-17pprovidesanexperimentalmethodfor the study of hormonal induction of hepatic synthesisof a specificprotein. Turpin et al. (1952) observed that administration of estrogens to rats results in increased concentrations of serum copper, and Meyer et al. (1958) reported that estrogen-

stimulated hypercupremia in rats is a manifestation of increased concentrations of serum ceruloplasmin (CPN). Clinical investigations have shown that estrogen administration produces increased concentrations of serum CPN and Cu in normal persons (Russ and Raymunt, 1956; von Studnitz and Berezin, 1958; Johnson et al., 1959; Musa et al., 1965; Doe et al., 1967) and in some patients with Wilson disease (Russ et al., 1957; German and Bearn, 1961; Plooij et al., 1961). Elevated concentrations of serum CPN and Cu are commonly observed in women who receive estrogen-progestogen combination drugs for oral contraception (Carruthers et al., 1966; Clemetson, 1968; Halsted et al., 1968; O’Leary and Spellancy, 1968; Mondorfet al., 1968; Shokeir, 1968; Tovey and Lathe, 1968; Laurel1 et al., 1967, 1969a, 1970; Methfessel et a/., 1969; Wolf ’ Supportedby U.S. Atomic EnergyCommission Grant No. AT(30-l)-4051and by U.S. Public HealthServiceGrant No. CA-11799. 588

ESTROGEN STIMULATION

OF SERUM CPN AND CU

589

et al., 1969; Mendenhall,

1970), but do not occur in women who are treated with contraceptive progestogens (Laurel1 et al., 1969b; Briggs et al., 1970). The mechanism of estrogen stimulation of increased concentrations of serum CPN and Cu has been partially elucidated by studies of 64Cu metabolism. Owen and Hazelrig (1966) have compared the disposition of 64Cu in isolated, perfused rat livers with that in intact rats, and have demonstrated that CPN is synthesized predominantly, and perhaps exclusively, in the liver. Evans et al. (1970) have performed experiments with 64Cu which indicate that administration of estradiol to rats increases de nova hepatic synthesis of CPN. Evans et al. (1970) have concluded that the estrogen may act as an inducer for synthesis of CPN messenger-RNA, consequently leading to the increased synthesis of CPN protein. These observations suggested to the present authors that estrogen stimulation of increased concentrations of serum CPN might furnish an experimental method for study of hormonal induction of hepatic synthesis of a specific protein. In order to develop such an experimental method, we have investigated the dose-response and timeresponse relationships between administration of estradiol-17/3 and increased concentrations of serum CPN and Cu. Concurrently, we have examined the effects of administration of estradiol-17B upon the ultrastructure of the rat hepatocyte. METHODS The experimental animals were 216 male and 82 female albino rats of the Fischer strain2 which were fed Purina rat chow. The female rats were ovariectomized at age 4 wk (more than 4 wk before the initiation of the experiments). On the day of the first injection of estradiol-17/3, the body wt of the rats averaged 200 g (175-225 g). An aqueous suspension of estradiol-17fi3 was administered by SCinjection daily at 9 AM. For the dose-response study, estradiol-17/3 was injected daily for 16 days in doses of 2,5,10,20,50,100, and 200 pg/day to 7 groups of 8 male rats, and in doses of 5,10,20, 50, 100, and 200 pg/day to 6 groups of 4 female rats. Control rats (16 male, 8 female) received 16 daily SCinjections of 0.1 ml of the aqueous vehicle, which contained NaCl (8.5 mg/ml), polysorbate 80 (15 pg/ml), and phenol (5 mg/ml). For the time-response study, estradiol-17p was injected in doses of 50 pglday for periods of 1, 2, 3,4, 5, 6, 9, 16,21,28,35, and 42 days to 12 groups of 8 male rats, and for periods of 7,13,16,21,28, 35, and 42 days to 7 groups of 4 female rats. Two control rats in each group (i.e., a total of 24 male and 14 female control rats) received daily SCinjections of 0.1 ml of the aqueous vehicle. In the dose-response and time-response studies, the rats were anesthetized with ether, and were exsanguinated by cardiac puncture 24 hr after the last injection. To measure changes in serum CPN and Cu after cessation of estrogen administration, 3 groups of 8 male rats received 21 daily SCinjections of estradiol-17/3 (50 pg/ day), and were exsanguinated on day 8, 15, or 22 after the last injection. Similarly, 2 groups of 4 female rats received 21 daily SCinjections of estradiol-178 (50 pg/day), and were exsanguinated on day 8 or 15 after the last injection. All the rats were fasted during the night before they were killed. Serum CPN was measured by p-phenylenediamine oxidase assay at pH 5.2, as described by Sunderman and Nomoto (1970), and serum Cu was measured by atomic absorption spectrometry, as described by Sunderman and * Charles River Breeding Laboratories, North Wilmington, Massachusetts 01887. 3 “Progynon,” aqueous suspension, Schering Corporation, Bloomfield, New Jersey 07003.

590

SUNDERMAN ET AL.

Roszel (1967). Electron microscopic examinations were performed upon livers from 8 male control rats and from 3 treated male rats in each of 8 selectedexperimental groups, as indicated in Tables 1 and 2. Portions of the left hepatic lobe were rapidly diced in cacodylate-buffered p-formaldehyde at 0-4”C, and postfixed in s-collidinebuffered 2 % 0~0,. Liver sampleswere also fixed in s-collidine-buffered 2 % 0~0, without prior p-formaldehyde fixation. The tissueswere embedded, sectioned, and stained as described by Goldblatt et al. (1970). Sections representative of various zones of the liver were examined by light microscopy and by useof a Phillips 300electron microscope at 60 kV. RESULTS Dose-ResponseStudy In serums of the control rats, the CPN concentrations were (male) 36 i 3 (mean *SD) and (female) 39 -f 7 mg/dl, and the Cu concentrations were (male) 125 f 5 and (female) 136 i 25 pg/dl. Following 16 daily SCinjections of estradiol-17/I, increased mean concentrations of serum CPN and Cu were observed in both male and female rats at all the dosestested (Table 1, Fig. 1). Maximum stimulation of serum CPN and Cu TABLE 1 DOSE-RESPONSE STUDY”

Relative meanconcentrations(uscontrols = 1.0) Dosageof estradiol-17P (p&W

M

F

M

F

0 2 5 10 20 50* 100 200

1.0 1.3” 1.4’ I .6” 2.2’ 3.1’ 2.9” 3.1’

1.0 1.5’ 1.6* 1.8’ 2.8” 2.4’ 2.3’

1.0 1.3” 1.4’ 1.8”

1.0 1.4* 1.5*

2.4’ 3.2” 3.1’ 3.3’

1.8’ 2.9” 2.5” 2.3’

SerumCPN

SerumCu

a Estradiol-17P was injected SCat the indicated dose for 16 consecutive days, and blood was collected 24 hr after the last injection. The control group contained 16 male and 8 female rats ; the test groups contained 8 male and 4 female rats. M, male; F, female. b Liver sections of male rats were examined by electron microscopy. c Probability c 0.05 (based upon null hypothesis for differences between values for test and control groups, computed by Student two-sided t test). d Probability c 0.01. e Probability -C0.001.

concentrations was achieved with daily injections of 50 pg of estradiol-17/3. At this dose, the CPN concentrations were (male) I13 + 11 and (female) 108f 8 mg/dl, and the Cu concentrations were (male) 398 f 42 and (female) 390 & 36 pg/dl. No significant changes in serum concentrations of CPN or Cu were observed when the dosage of estradiol-17/3 was increased from 50 pg/day to 100 or 200 pg/day for 16 days.

ESTROGEN

STIMULATION

OF SERUM

CPN

AND

CU

FIG. 1. Dose-response curves for the effect of estradioLl7p upon ceruloplasmin and copper concentrations in rat serums. Open and filled symbols indicate the means & SE in female and male rats, respectively. Estradiol-17,8 was injected SCfor 16 consecutive days at the designated dose, and the rats were killed on day 17.

Time-Response Study In serums of the control rats, the CPN concentrations were (male) 35 + 4 and (female) 37 i 6 mg/dl, and the Cu concentrations were (male) 120 IL 16 and (female) 131 f 23 pg/dl. After SCinjections of estradiol-17/I in doses of 50 ,ug/day, no significant increases in concentrations of serum CPN and Cu were observed until after the seventh daily injection (Table 2, Fig. 2). Maximum stimulation of serum CPN and Cu concentrations was accomplished when the daily injections of 50 ,ug of estradiol-I 7/3were continued for 28 days in male rats (CPN = 122 f 5 mg/dl; Cu = 427 + 17 pg/dl); and when the injections were continued for 35 days in female rats (CPN = 117 f 13 mg/dl; Cu = 414 i 45 pg/dl). Prolongation of the daily injections of 50 pg of estradiol-17)3 until 42 days did not produce any further alterations in serum CPN or Cu concentrations in either male or female rats. Response after Cessation of Estrogen Administration Following the termination of 21 daily injections of estradiol-l7/3 in doses of 50 lug/day, the serum concentrations of CPN and Cu slowly diminished toward the normal levels (Table 2, Fig. 2). In serums of male rats which were collected on day 22 after cessation of estradiol-17/I, the CPN concentration was 46 of 4 mg/dl and the Cu concentration was

592

SUNDERMAN ET AL.

161i 7 pg/dl. In serumsof female rats which were collected on day I5 after cessationof estradiol-17/3, the CPN concentration was 49 i 6 mg/dl and the Cu concentration was 176i 23 pg/dl. TABLE 2 TIME-RESPONSE STUDY~

Number of injections of estradiol-17fi

Relative meanconcentrations(UScontrols = 1.O) __ Serum CPN SerumCu

Ob lb

M

--

1.0 1.0 0.9 1.0

F

M

F

1.0 -

1.0 -

-

1.0 1.o 0.9 1.0

-

1.0 1.0 1.1

2 3 4 9 6 7 9b 13 16b 21b 28b 35 42b 21 (+8 days)

1.2 1.3d 3.2’ 3.4” 3.5’ 3.4’ 3.4” 2.8’

1.9’ 2.9’ 3.0” 3.1” 3.0’ 2.0’

3.3’ 3.4 3.6’ 3.5” 3.4’ 2.8”

21 (+15 days) 21 (+22 days)

2.1” 1.3d

1.3d -

1.9” 1.3d

1.0 1.2

1.44 -

1.4d -

1.4” 1.9” 2.9” 3.0’ 3.2’ 3.1’ 1.9’ 1.3” -

(1Estradiol-178 was injected SC50 ,ug/day for the indicated nnmber of consecutive days. Blood was collected 24 hr after the last injection, except where it is specified that the blood was collected on the Sth, 15th or 22nd day after the last injection. The control group contained 24 male and 14 female rats. The test groups contained 8 male and/or 4 female rats. M, male; F, female. b Liver sections of male rats were examined by electron microscopy. c Probability c 0.05. d Probability <: 0.01. e Probability c 0.001.

Copper: CeruloplastninRatios The concentration of Cu in serumsof the male control rats averaged 0.349 f 0.018 “/L of the serum CPN concentration. In the rats which received estradiol-17P in dosageof 50 pg/day, significant diminutions in the ratios of serum Cu to serum CPN were observed after the secondto sixth injections (Table 3). No significant alterations in the ratios of serumCu to serumCPN were observedin any of the other experimental groups. Electron Microscopic Observations Despite specific attention to nucleolar ultrastructure, no consistent nucleolar alterations were observed in the hepatocytes of male rats which received estradiol-17/I, such

ESTROGEN

STIMULATION

OFSERUM

CPN

AND

CU

593

FIG. 2. Time-response curves for the effect of estradiol-17/l upon ceruloplasmin and copper concentrations in rat serums. Open and closed symbols indicate the means + SE in female and male rats, respectively. Estradiol-17/3 was injected SC,50 pg/day, for the designated number of days, and the rats were kiIled24hr later(exceptformeasurementsmarked byanasterisk).Theasterisksindicate that the rats received estradiol-17/3 injections, 50 pg/day, for 21 days, but were not killed until 8, 15, or 22 days later. TABLE RATIOS

OF MEAN

CONCENTRATIONSOF

Number of injections of estradiol-17p 0 1 2 3 4

5 6 9 16 21 28 42

3 SERUM

COPPER

AND CERULOPLASMIN'

Serum Cu: CPN ratios (pg Cu/mg CPN, mean i SD) 3.49 3.38 3.20 3.02 3.17 2.96 3.27 3.50 3.52 3.49 3.52 3.53

+0.18

f 0.38 &0.24' ~0.20" kO.27" f0.17' + 0.24b i0.24 +I.05 + 0.04 iO.04 Lko.04

a Estradiol-178 was administered to male rats SC,50 pg/day, for the indicated number of consecutive days. Blood was collected 24 hr after the last injection. The control group contained 24 rats, and the test groups contained 8 rats. b Probability < 0.05. ’ Probability < 0.01. 22

594

SUNDERMAN

ET

AL.

as have been previously reported in uterine myocytes of estrogen-treated ovariectomized rats (Laguens, 1964). Several nucleoli exhibited small dense plaques, but these were also seen in hepatocyte nucleoli of one control rat. Dilatation of endoplasmic reticulum was frequently noted, but was also present in hepatocytes of control rats. No mitochondrial abnormalities were detected, such as have been found in hepatocytes of women who receive oral contraceptives (Perez et al., 1969). Glycogen was present in hepatocytes of all the rats which were examined. A consistent and unequivocal abnormality was the accumulation of numerous hyaloplasmic lipid droplets within scattered hepatocytes of rats which received 42 daily injections of estradiol-17/3 in doses of

FIG. 3. Electron micrograph of hepatic parenchymal cell of a male rat which had received 42 daily SC injections of estradiol-17/3 (50 pg/day). Note the marked accumulation of hyaloplasmic lipid droplets (L.). Uranyl acetate-lead citrate stain. x6800.

50 pg!day (Fig. 3). The fat-laden cells were evident on both light and electron microscopic examination, and they appeared to be randomly distributed throughout all zones of the liver. Cells which contained abundant fat droplets were usually surrounded by hepatocytes which were devoid of such droplets. Such accumulations of hyaloplasmic lipid droplets were not seen within hepatocytes of any of the control rats, nor were they found in hepatocytesVof rats which received 1, 5, 9, 16, 21, or 28 daily injections of estradiol-17/3 (50 pg/day). DISCUSSION

Insofar as the authors can ascertain, the only previous investigation in rats of the dose-response and time-response relationships between estrogen therapy and increased concentrations of serum CPN or Cu was the study by Turpin et al. (1952). They found

ESTROGEN

STIMULATION

OF SERUM

CPN

AND

CU

595

that daily administration of an estrogen (diethylstilbestrol, 10 pg/kg/day) for 5 days was insufficient to produce hypercupremia in rats, but that continuation of the injections for a total of 14 days resulted in 53% increase in the mean concentration of serum Cu. Turpin et al. (1952) noted that relatively low dosages of estrogen were most effective in stimulating hypercupremia, and that administration of diethylstilbestrol for 14 days in doses of 100-1000 pg/kg/day did not produce hypercupremia. The present study confirms the report by Turpin et al. (1952) that 5 days of estrogen therapy is insufficient to produce hypercupremia in rats, but the present study appears to contradict their finding of suppression of hypercupremia by high doses of estrogen. It is possible that this difference may be attributed to use of estradiol-17P in the present study, rather than diethylstilbestrol. According to Magdoff-Fairchild et al. (1969), the molecular weight of human CPN is 132,000, and each molecule of CPN contains 6 atoms of Cu. Therefore, the Cu content of CPN should comprise 0.288 % of the weight of the CPN molecule. These computations are in substantial agreement with measurements by Morel1 et al. (1969) which indicate that Cu comprises 0.275 f 0.009 ‘A of the weight of purified human CPN. No comparable measurements are available for the copper content of rat CPN. However, ultracentrifugal analyses reported by Holzman and Gaumnitz (1970) indicate that the molecular weight of rat CPN does not differ significantly from that of human CPN. On this basis, it may be inferred that the Cu content of rat CPN is essentially the same as that of human CPN. The present finding that the concentration of total Cu in serums of control rats averages 0.349% of the serum CPN concentration is consistent with the expected partition of serum Cu between CPN-bound and albumin-bound fractions (Sternlieb et al., 1969). For comparison, Warren et al. (1969) reported that the concentration of total copper in 51 human serums averaged 0.355 % of the serum ceruloplasmin concentration. Our observation that the serum Cu:CPN ratio was significantly decreased after the second to sixth injections of estradiol-17/I in doses of 50 pg/day suggests that depletion of serum albumin-bound Cu may occur during this phase of estrogen stimulation of ceruloplasmin synthesis. Laguens (1964) reported that nucleoli of uterine myocytes of ovariectomized rats were enlarged 6 hr after a single injection of estradiol of 8 pg/lOO g body weight. At 24 hr after the injection the nucleoli became increased in number, and at 48 and 72 hr there were numerous large nucleoli which adhered to the nuclear membrane. Despite careful search in the present study, such nucleolar alterations were not seen in the rat hepatocytes. Perez et al. (1969) found a high incidence of intramitochondrial crystalline arrays in hepatocytes of women on sequential oral contraceptive therapy. Such mitochondrial abnormalities were not detected in the present study. The striking increase in hyaloplasmic lipid droplets which was seen in the present study in scattered hepatocytes of rats which had received 42 daily injections of estradiol-17p has not been reported previously. However, Aftergood and Alfin-Slater (1969) observed marked increase in hepatic cholesterol content of rats which received repeated po administration of a combination of norethynodrel and mestranol. The possibility that estrogens and androgens might exert antagonistic effects upon lipid deposition in hepatocytes has been suggested by reports that castration increases the susceptibility of male rats to ethionine-induced fatty liver (Farber et al., 1951; Schlunk et al., 1968). Although the liver is not commonly regarded as a target organ for estradiol-176,

596

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recent studies have indicated that estradiol-17/3 can influence the control of genetic transcription and translation in the hepatocyte. Thus, Hamilton (1968) has observed substantial binding of estradiol-17j3 to hepatic chromatin, and a small but repeatable stimulatory effect of estradiol-178 upon the template activity of hepatic chromatin. Church and McCarthy (1970) have reported that administration of estradiol-17j3 stimulates the synthesisof hepatic RNA, attended by (a) activation of hepatic DNAdependent RNA polymerase, (b) derepressionof portions of the hepatic genome, and (c) augmentation of transport of RNA from the hepatocyte nucleus to the cytoplasm. All thesemechanismsmight be involved in the stimulation of hepatic synthesisof CPN which has been shown to occur following administration of estradiol-17j3(Evans et al., 1970). To date, no evidence is available concerning the possibleeffects of estradiol-17p upon the stability of hepatic messenger-RNA templates for CPN, nor upon the rates of turnover and catabolism of CPN. Investigations of the mechanismsof estrogen stimulation of hypercupremia and hyperceruloplasminemia are currently in progress in our laboratory. Basedupon the results of the present study, administration of estradiol-17/3 to male rats (200 g body wt) in dosesof 50 pg/day for 16 days has been selectedas the basic regimen for these experiments, inasmuch as this doseschedulestimulates greater than 3-fold increasesin the mean concentrations of serum CPN and Cu, without producing pathological alterations of the hepatocytes. REFERENCES APTERGOOD, L., and ALFIN-SLATER, R. B. (1969).Effect of an oral contraceptivesteroid mixture on someaspectsof lipid metabolismin the rat. In: Metabolic Effects of Gonadal Hormones and Contraceptive Steroids (H. A. Salhanick,D. M. Kipnis, and R. L. Van de Wiele,

eds.),pp. 265-274.PlenumPress,New York. BRIGGS, M., AUSTIN, J., and STANIFORD, M. (1970).Oral contraceptivesand copper metabolism.Nature (London) 225, 81.

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CPN

AiiD

CU

597

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SUNDERMAN, F. W., JR., and ROSZEL, N. 0. (1967). Measurements of copper in biological materials by atomic absorption spectrometry. Amer. J. Clin. Puthol. 48, 286-294. TOVEY, L. A. D., and LATHE, G. H. (1968). Caeruloplasmin and green plasma in women taking oral contraceptives, in pregnant women, and in patients with rheumatoid arthritis. Lancet 2,596-600. TURPIN, R., JEROME, H., and SCHMITT-JUBEAU, H. (1952). Action de doses progressives de diethylstilboestrol sur la cupremie chez le rat. C. R. Sot. Biol. 146, 1703-1706. VON STUDNITZ, W., and BEREZIN, D. (1958). Studies on serum copper during pregnancy, during the menstrual cycle, and after the administration of oestrogens. Acta Endocrinol. 27,245-252. WAKREN, R. L., JELLIFFE, A. M., WATSON, J. V., and HOBBS, C. B. (1969). Prolonged observations on variations in the serum copper in Hodgkin’s disease. Clin. Radiol. 20,247-256. WOLF, P., ENLANDER, D., DALZIEL, J., and SWANSON, J. (1969). Green plasma in blood donors. N. Engl. J. Med. 281,205.