ANTIESTROGEN-INDUCED ALTERATIONS OF HYPOTHALAMIC DOPAMINE AND NOREPINEPHRINE

LEVELS IN THE FEMALE RAT

SAMARENDRA N. BAKSI, THERESA E. REDINGTON and MAYSZE J. HUGHES Department of Physiology. Texas Tech. University Health Sciences Center, School of Medicine.

Lubbock, TX 79430. U.S.A.

(Accepted 20 June 1981)

Summary-The influence of antiestrogen (tamoxifen) administration in ciao on the steady state concen- tration of dopamine (DA) and norepinephrine (NE) in the hypotha~mus-median eminence area of mature female rats was investigated. Rats were treated with tamoxifen at 3 different dose levels (0.f. 1.0 and 10.0 mg/kg, s.c.) daily for 7 or 21 days. Control groups were injected with vehicle only (107: ethanol; l.Oml/kg). In a separate experiment, mature female rats were ovariectomized or sham operated and killed either 7 or 21 days after operation. Dopamine and NE were estimated by high pressure liquid chromatography with electro-chemical detection. Seven days of tamoxifen treatment at 3 different dose levels did not alter DA and NE concentrations compared to the control. The chronic (21 days) tamoxi- fen treatment in the smallest dose (0.1 mg) significantly increased the DA concentration. The concen- tration of NE was reduced only at the l.Omg/kg dose. There was no significant difference in the NE concentration 7 or 21 days aRer ovariectomy or sham operation. but the DA concentration was sign% cantly increased 21 days after ovariectomy. The uterine weights were reduced significantly after 21 days of treatment with tamoxifen in the two Iarger doses, but were increased with the smallest dose of tamoxifen. Uterine weight after ovariectomy both after 7 and 21 days was significantly reduced. These results suggest that chronic low-dose antiestrogen treatment may affect the hypothaiamic-median emin- ence DA and NE concentrations. The data also suggests that antiestrogenic action (uterine growth inhibition) of tamoxifen is not related to the alterations of DA and NE in the hypothalamus-median eminence.

Experimental studies indicate that sex hormones may alter central catecholamine levels (Greengrass and Tonge, 1971; Gudelsky. Annuziato and Moore. 1977) and modify behavioral and biochemical activity as- sociated with dopaminergic mechanisms (Nausieda, Keller. Weiner and Klawans. 1979; Koller, Weiner. Ktawans and Nausieda, 1980; Hruska and Silbergeld, 198Oa). Dopamine is presumed to have a role in the pathophysioIogy of choreatic movement and affective disorder (Klawans and Weiner, 1976) as well as in pituitary prolactin release (Ben-Jonathan, 1980). Ster- oidal oral contraceptives have also been reported to induce chorea (Koller. Weiner, Klawans and Nau- sieda. 1979). To the present authors’ knowledge, the effect of antiestrogens on central catecholamine levels have not been reported, except for the work of Luine and McEwen (1977) in which the authors studied the effect of antiestrogens on monoamine oxidase activity in the corticomedial amygdaia.

One group of antiestrogens of the triphenyl ethyl- ene type, such as tamoxifen (Harper and Walpole, 1967). is being used as a therapeutic agent in certain types of breast cancer (Ward, 1973: Legha, Davis and Muggia, 1978). In the present study, an investigation was made of the influence of tamoxifen treatment on the steady-state concentration of hypothaIamic cate-

Key words: antiestrogen, tamoxifen, hypothalamus. dopamine. norepinephrine. ovariectomy, rat.

cholamines in mature female rats. A preliminary report of this work has appeared elsewhere (Baksi, Red&ton and Hughes, 1980).

METHODS

Mature female Sprague-Dawley rats were obtained from King Animal Laboratories, Oregon, WI. when they were 60 days old, weighing 15@175 g. The rats were housed 3 per cage, with free access to food (Ral- ston Purina Rat Pellets, St Louis, MO) and tap water. The room temperature was 25°C and the light/dark cycle was controlled with 14 hr of light (on at 03:oO hr). The animals were acclimatized for one week before the start of the experiments. Tamoxifen citrate (ICI 46,474; p-fi-dimethylaminoethoxyphe- nyl-1,2 diphenyl but-1-ene, Lot No. BX 79jNXA/45) used in this study was a gift from ICI Americas (Dr D. H. McCurdy). The drug was dissolved in absolute alcohol and diluted with distilled water to the required volume and injected subcutaneously daily for either 7 or 21 days at three different dose levels (0.1, 1.0 and 10.0 mg/kg). The control groups received only vehicle (loo/, ethanol; l.Oml/kg). In a separate experiment under identical husbandry conditions, female rats of similar age and body weight were ovar- iectomized under light ether anesthesia. A group of sham-operated rats served as control. Twenty-four hours after the last drug or vehicle treatment and 7

1163

1164 SAMARFNDKA N. BAKSI PI al

and 21 days after ovariectomy or sham operation each rat was decapitated, the brain was quickly re- moved and its hypothalamus-median eminence area dissected on a chilled glass plate on ice, according to the procedure described by Glowinski and Iversen (1966). All rats were decapitated between 09:OO and 11:OO hr. The excised brain part was quickly frozen on dry ice and stored in sealed plastic tubes at - 20°C for the estimation of catecholamines. The uterus was removed. cleaned of fatty tissue and weighed.

The catecholamines in the brain parts were esti- mated by high pressure liquid chromatography with electrochemical detection as described by Felice, Felice and Kissinger (1978) with modifications.

A Waters Associate Liquid Chromatography Sys- tem (Model 6000A; Waters Associate, Milford, MA) with a 30 cm x 3.9 mm id. reverse phase C, 8 column (Waters Associate) was equipped with a carbon paste amperometric detector (TL-3: Model LC-4. Bioanaly- tical System, West Lafayette, IN). The mobile phase was composed of 21.6g KH,PO,; 3.3 ml SSp; H,PO,; 0.186g Octyl Sodium Sulfate: 2.0ml 0.1 M EDTA Na, and 20.0ml methanol adjusted to a volume of 2.01. The detector potential was set at +0.72 V vs Ag/AgCl reference electrode. The mobile phase (pH 2.8) was pumped at 1.0 mlimin. The injec- tion volume was 25 jtl.

Chemicals and their sources were as follows: 3.4-dihydroxybenzylamine-HBr (Aldrich Chemical Co.. Milwaukee. WI): norepinephrine bitartate. epi- nephrine bitartate and dooamine-HCl (Sigma Chemi- cal Co.. St. Louis. MO) and Sodium Octyl Sulfate and acid washed aluminum oxide (Bioanalytical System). Catecholamine working standards consisted of long/PI in 0.05 M HCIO, with 0.1 mM NaHS03 pre- pared every 2 weeks and stored at 4-C.

Three milliliters of chilled 0.4 N HC104 (with 0.01 M NaHSO, and 0.0015 M EDTA Na,) and 150 ng internal standard (dihydroxybenzylamine. 10 ng/pl) were added to tissue samples (up to 125 mg). The mixture was homogenized with a Brinkman Polytron homogenizer (Brinkman. Westbury, NY) for 1.5 set under an ice jacket and centrifuged at 15,OOOg for 15min in a refrigerated centrifuge. The superna- tant was transferred to a conical screw cap vial which contained 1.5 ml pH 8.6 Tris buffer (3.0M) and IOOmg of aluminum oxide. After the addition of the su~rnatant. the vials were immediately shaken for 15min in a Dubnoff shaking incubator which was cooled to 15’C with crushed ice. After the alumina settled. the supernatant was discarded by aspiration. The alumina was washed once with 2.0ml of Tris buffer (pH 8.6, 6mM) and twice with 2.0ml distilled water. After l.Oml of 0.05 N HCLOI with 0.1 mM

NaHSO, was added to the alumina, the vials were shaken for 15 min. The supernatant was removed with a pasture pipet and filtered through 0.45pm filter paper and a Swinny stainless steel filter assembly unit (Milipore, Bedford, MA) into 1.5 ml polypropylene eppendorf micro test tubes (Allied Container Corp., Dedham, MA). Each sample was capped and stored at 4-C until used for chromatography within 24 hr. A preliminary study in this laboratory indicates that samples stored in this way retained 929; of norepine- phrine and 729; of dopamine activity up to 50 days with virtually no loss during the first week.

To calculate the concentration of catecholamines, two additional brain tissue samples, one blank and one spiked were prepared for calibration according to the method described by Felice et cd. (1978). The amount of catecholamine was expressed as rig/g tissue. The percentage extraction of each catechol- amine was also estimated from the peak height of a known amount of working standard solution. The percentage extraction in all the experiments was between 70 and 80”;.

Statistics

Where appropriate, the data are presented as mean values accompanied by the standard errors of the mean (SEMI. Comparisons were made using Student’s f-test and Multiple Range Test (Duncan, 1955) and Analysis of Variance for significance.

REWLTS

Seven days treatment with tamoxifen at 3 different dose levels in female rats produced no significant change in steady-state concentrations of dopamine (DA) and norepinephrine (NE) in the hypothalamus compared with those in the vehicle-treated control group (Table 1). Epinephrine was not detectable in any sample. Treatment with the largest dose (10.0 mg/kg) of tamoxifen decreased (P < 0.05) the body weight gain (data not shown).

In contrast. treatment with tamoxifen for 21 days altered the hypothalamic concentration of DA and NE in some groups. The concentration of DA was not significantly different in groups which were treated with 10.0 and I .O mg/kg of tamoxifen. although, at the l.Omg/kg dose, DA concentrations were slightly, but not significantly. reduced. Concentrations of DA in- creased significantly (P < 0.01) in the group treated with the smallest dose (0.1 mg/kg) of tamoxifen. Con- centrations of NE were reduced significantly (P < 0.05) at the 1.0 mg/kg dose of tamoxifen (Fig. 1). Epinephrine was not detectable in any group. Body weights were reduced significantly (P < 0.01) in all groups trea;ed with tamoxifen. Uterine weights were reduced significantly (P < 0.01) after 21 days treat- ment with tamoxifen in the two larger doses (10.0 and

Antiestrogen and catecholamines 1165

Table 1. Concentrations of dopamine (DA) and norepinephrine (NE) in the hypothalamus-median eminence of mature female rats after 7 days of treatment with tamoxifen (TAM) at 3 dose levels or vehicle (1(X/, ethanol: I.0 mg/kg) and 7 days after ovariectomy or sham oper- ation. Mean f SEM of 6 rats in each group. There was no significant

difference due to TAM treatment or ovariectomy

Catecholamines rig/g tissue DA NE

Tamoxifen treated (7 days) Control (vehicle) 0.1 mg/kg TAM 1.0 mg/kg TAM

10.0 mg/kg TAM Ovariectomy (7 days)

Control (sham operated) Ovariectomized

1144 k 288 1549 + 236 1377 a 314 1788 + 330 980 + 150 1661 + 354

1463 h 262 1775 + 365

1498 k 173 898 + 77 1446 + 152 1053 + 104

1.0 mg/kg) but were increased significantly (P < 0.01) with the smallest (0.1 mg/kg) dose (Table 2).

In rats ovariectomized for 7 days there was no change in hypothalamic DA and NE concentrations

compared to the sham-operated control (Table 1). But hypothalamic DA concentrations significantly (P < 0.001) increased in rats ovariectomized for 21 days. There was no change in NE concentrations

(Fig. 2). Epinephrine was not detectable in any group. The uterine weights were reduced significantly (P < 0.001) 7 and 21 days after ovariectomy (Table 2).

1

1400

1200 1

o,b I**

DA

I000 - DA

1600 -

0

.L b

I

I H NE

f a

d

E 1200 -

P

5 900. -

600 -

300-

d

.L

Ll - NE

c cd I_

- : - .L .-

CONTROL

10.0 I.0 u.1

TAMOXFEN mg /kg

Fig. 1. Concentration of dopamine (DA) and norepine- phrine (NE) in the hypothalamus-median eminence of mature female rats after 21 daily treatments with 3 different doses of tamoxifen or vehicle control (IO’,, ethanol: l.Oml/kg. SC.). The height of each column represents the mean concentration of DA (upper panel) and NE (lower panel) and the vertical line represents +SEM of 6 animals. * P < 0.05 significantly different: ** P < 0.001: from con- trol values. Means bearing the same letters are significantly (P i 0.05) different by the multiple range test (Duncan.

Shorn Ovormctomized Operated

Fig. 2. Concentration of Dopamine (DA) and Norepine- phrine (NE) in the hypothalamus-median eminence of mature female rats 21 days after ovariectomy or sham op- eration. The height of each column represents the mean concentration of DA (upper panel) and NE (lower panel) and the vertical line represents SEM of 6 rats. ** P < 0.001 significantly different from sham-operated control by Stu-

1955). dent’s t-test.

1166 SAMARENDRA N. BAKSI et al.

Table 2. Uterine weight of mature female rats after 21 days of treatment with tamoxifen (TAM) at 3 dose levels or vehicle as well as 7 and 21 days after ovariec-

tomy or sham operation. Mean + SEM of 6 rats in each group

Uterine weight (mg/lOO g body wt)

Tamoxifen treated (21 days) Control (vehicle)

0.1 mg/kg TAM 1 .O mg/kg TAM

10.0 mg/kg TAM Ovariectomy experiment

Control (sham operated) Ovariectomized (7 days) Ovaricetomized (21 days)

244* 13 361 + 10*“,h 118 7 4**” 98 k 4**b

360 + 4 129 + 5**’ 83 + 19**’

* P < 0.01: ** P -c 0.001. Significantly different from control values. Means bear- ing the same superscripts are significantly (P < 0.01) different by multiple range test (Duncan, 1955)

DISCUSSION

This investigation indicates that chronic (21 days) treatment with antiestrogen at the lowest dose level tested (0.1 mg/kg) increased the hypothalamic-median eminence steady-state concentrations of DA in rats. The NE concentrations, on the other hand, were reduced at the l.Omg/kg dose level. The antiestroge- nit activity of tamoxifen at 10.0 and l.Omg/kg doses caused a marked reduction (60 and 52% respectively) of uterine weights but the smallest dose (0.1 mg/kg) of tamoxifen behaved as a partial estrogen agonist (68% increase in uterine weight). It is interesting to note that, at this dose, the concentration of DA in the hypothalamus-median eminence area also increased significantly. The antiestrogen treatment at any dose for 7 days had no significant effect on DA and NE concentrations. Although no record was made of the uterine weights after 7 days treatment with tamoxifen, other investigators have reported changes in uterine weights after a comparable dose-level of tamoxifen (Bichon and Bayard. 1980). Gudelsky et al. (1977) have shown that long-term ovariectomy in rats in- creases median eminence DA concentrations which could be reversed by estrogen replacement. Estrogen treatment reduces the hypothalamic prolactin-inhib- iting factor (Ajika, Krulich, Fawcett and McCann,

1972) which is presumed to be DA (Gibbs and Neill, 1978). If the small dose of tamoxifen acted as an estro- gen agonist, then it should decrease hypothalamic DA, but in the present study, an increase of DA con- centration in the hypothalamus-median eminence area was found. Euvrard, Oberlander and Boissier (1980) have also demonstrated an antidopaminergic effect of estrogens at the striata: level in rats. The larger dose of tamoxifen which acted as an estrogen antagonist (reduced uterine weight) in the present study, did not alter the DA concentration in the hypothalamus-median eminence. The reason for this is not clear at present. It is, however, possible that tamoxifen itself or one of its metabolites (Borgna and

Rochefort, 1981) had a direct or indirect effect on the dopaminergic system unrelated to its estrogen- antagonizing property. Nagy, Valdenegro and McLeod (1980) have recently reported that tamoxifen administration in rats caused a 45% reduction in pro- lactin synthesis and release in thv. These authors also showed no interaction between antiestrogen and the dopaminergic system.

The interpretation of these results is difficult because steady-state concentrations of DA and NE do not represent dynamic measures of neural activity. There are also diurnal variations in the DA levels of rat brain areas (Owasoyo, Walker and Whitworth. 1979) as well as in DA receptor binding in the rat striatum (Naber, Wirz-Justice, Kafka and Wehr, 1980). However, in this study, all the rats were sacri- ficed between 09:OO and 1l:OO hr. so that any changes due to diurnal rhythms did not affect the DA concen- trations.

The functional significance of the antiestrogen- induced increase in hypothalamic DA concentration remains to be determined, since long-term treatment with the drug at a comparable dose level is being used for certain types of human breast cancer (Ward, 1973; Legha et al.. 1978). What, if any, effect these treat- ments cause in the alterations of the central dopamin- ergic system is not known. The known effects of estro- gens on the DA response (Keller et a[., 1980) and the stimulation of DA receptor activity (Hruska and Sil- bergeld, 1980a, b; Hruska. Ludmer and Silbergeld, 1980) as well as on oral contraceptive-induced chorea (Koller et al., 1980) warrant a more detailed study of the effect of antiestrogen on the dopaminergic system.

Acknolrledyement-This work was supported in part by a grant from the National Institutes of Health. HL 16240.

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