Effect of combined neonatal imprinting by vitamin A, vitamin D3, benzpyrene and allylestrenol on adult rat thymus glucocorticoid and uterine estrogen receptors

ISSN 0306-3623/97 $17.00 + .00 PII S0306-3623(97)00019-0 All rights reserved

Gen. Pharmac. Vol. 29, No. 5, pp. 779-781, 1997 Copyright @ 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER

Effect of Combined Neonatal Imprinting by Vitamin A, Vitamin D3, Benzpyrene and Allylestrenol on Adult Rat Thymus Glucocorticoid and Uterine Estrogen Receptors G. Csaba* and/~. Inczefi-Gonda DEPARTMENTOF BIOLOGY, SEMMELWEISUNIVERSITYOF MEDICINE, H-1445 BUDAPEST,P.O.B. 370, HUNGARY

ABSTRACT. 1. Combined neonatal imprinting with allylestrenol, vitamins A and D3 and benzpyrene significantly increased thymic glucocorticoid receptor capacity in male and female animals and decreased receptors affinity in adult females only. 2. Uterine estrogen receptor affinity or density was not influenced. 3. Considering that perinatal treatment with allylestrenol or vitamin D3 decreased glucocorticoid receptor capacity, the dominance of the positive effect of retinol should be surmised. 4. The experiments call attention to the interrelation of different materials acting simultaneously in the perinatal period. GEN PHAm~AC 29;5:779--781, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Hormonal imprinting, neonatal imprinting, allylestrenol, vitamin A, vitamin D, benzo(a)pyrene, hormone receptors INTRODUCTION Hormone receptors develop parallel with the differentiation of cells (Hubbert and Miller, 1974). After birth, when the maturing receptor meets the appropriate hormone develops the hormonal imprinting, which is needed for finishing receptor maturation (Csaba, 1980, 1981). Without imprinting the receptors do not reach the binding capacity characteristic of adult age (Csaba and Nagy, 1985). However, in the perinatal critical period of development, the discriminating ability of receptors is not complete, and thus molecules similar to the appropriate hormone can be bound by them (Csaba, 1984, 1986) . These molecules are members of the same hormone family, synthetic hormone analogues, or molecules in the food, air or water, having structures similar to hormones. In their presence false imprinting takes place, and changes in hormone binding become the lifelong consequences (Csaba, 1991). Because not only hormones but their receptors are similar, there is an overlap between the false imprinting effects, causing disturbances not only for their own but also for foreign receptors (Inczefi-Gonda et al., 1986). Perinatal influences at the receptorial level also provoke disturbances at the biochemical, morphological and functional levels (Bern et al., 1973, 1987; Gibson et al., 1991; Gray-Nelson et al., 1994; Iguchi, 1992; Iguchi et al., 1987; Levine and Mullins, 1964; McLahlan et al., 1980; Sato et al., 1994; Tchernitchin and Tchernitchin, 1992). Every type of hormone (amino acid, peptide or steroid) can provoke imprinting and molecules similar to them can cause misimprinting (Csaba, 1986, 1991). However, the significance of steroid misimprintings seems to be more important. In earlier experiments single neonatal diethylstilbestrol (DES) or allylestrenol treatment (both have been used for protecting endangered human pregnancies) has been shown to reduce the estrogen receptor number in rat uterus to one third and one half, respectively (Csaba et al., 1986), *To whom all correspondence should be addressed. Received 10 September 1996; revised 7 January 1997; accepted 27 January 1997.

and reduce the binding capacity of thymic glucocorticoid receptors, also (Csaba et al., 1986). Perinatal imprinting with the environmental pollutant benzpyrene (having a steroid-like structure) also reduced the binding capacity of these receptors (Csaba and InczefiGonda, 1984) and, in addition, dramatically reduced the sexual activity of the animals studied (Csaba et al., 1993). Perinatal treatment (imprinting) with the steroid-like vitamin D3 also profoundly decreased the sexual activity of adult male and female rats (Mirzahosseini et al., 1996) and this was also done by vitamin A (retinol) completed with influences to receptors and sexual hormone level (Csaba and Gafil, 1996; Ga~I and Csaba, 1996). This is especially interesting as retinol has a structure entirely different from steroids, nevertheless, its receptors belong to the same receptor superfamily as steroid hormones (Mangelsdorf, 1994). In normal conditions, the aforementioned effects are not found separately. In cases of endangered pregnancy the fetus receives allylestrenol and vitamin D3 from the mother's circulation, obtains retinol for prevention and benzpyrene is present in its circulation. This makes it reasonable to study the combined effects of these molecules in steroid hormone receptor development.

MATERIALS A N D METHODS Newborn rats of our closed-bred Wistar strain were treated with a combination of 20 ~g allylestrenol (Richter, Budapest, Hungary), 20 jxg benzpyrene (Fluka, Buchs, Switzerland), 10,000 IU retinol (vitamin A, Egis, Budapest) and 250 ~g vitamin D3 (Sigma) before 24 h after birth, subcutaneously, in 0.2 ml of sunflower seed oil. Controls were treated with the oil only. Thymi of 6-week-old male and female animals were studied for glucocorticoid receptors. Uteri of 3-months-old females were studied for estrogen receptors 8 days after ovariectomy. Three thymi or four uteri were homogenized together for obtaining preparations for measurement. All assays were performed in duplicate. Data (means) were obtained from measurements of four to seven different homogenates in each group.

780

R e c e p t o r assays Preparation ofcytosol fraction. All procedures were performed at icewater temperature. Tissues (1.5 ml/g wet weight) were cut into pieces and homogenized in Tris-HC1 buffer containing 1.5 mM EDTA (pH 7.4), freshly supplemented with 20 mM molybdate and 2 mM dithiotreitol, with a motor-driven glass-teflon Potter homogenizer. Homogenates were centrifuged at 100,000g for 60 rain at 4°C and the supernatants were used for receptor assays. Protein content was estimated by the Coomassie blue method. Glucocorticoid receptor--thymus cytosol. Five hundred micrograms of protein was incubated with 10, 5, 2.5, 1.25, 0.6, 0.3 and 0.15 nM 3H-dexamethasone acetate (Amersham, Buckinghamshire, UK; specific activity 1.8 TBq/mmol) in the absence or presence of 1000-fold molar excess of unlabeled ligand (Sigma) in a total volume of 100 t*1 at 0°C for 18 hr. Bound glucocorticoid was separated by the charcoal method and counted in OptiPhase, HiSafe (Pharmacia, Lund, Sweden, 35% efficiency). Radioactivity measured in the presence of 1000 nM dexamethasone was regarded as nonspecific binding. Estrogen receptor--uterus cytosol, three hundred micrograms of cytosolic protein was incubated with 5, 2.5, 1.25, 0.62, 0.31, 0.15 and 0.07 nM of 2,4,6,7-3H-estradiol (Izinta, Budapest; 3.2 TBq/mmol specific activity) in the absence or presence of 1000-fold molar excess of unlabeled ligand (Organon, Oss, Holland). Conditions of incubation, termination of the reaction and counting were identical to those for the receptor assay on thymus cytosol. Statistical analysis Analysis of the results was carried out by the computer programs EBDA and LIGAND (McPherson, 1983, 1985). EBDA was used to process raw data. LIGAND (nonlinear curve-fitting program) was used to obtain final parameter estimates. Statistical analysis of the final parameters was calculated by the computer program DATAANALYSIS, v.l.0 (analysis of variance, simple F-test comparison). RESULTS A N D DISCUSSION Steroid hormones and steroid structures can influence each other's effects in adult age even at a receptorial level. Estrogens influence glucocorticoid receptor (Burgess and Handa, 1992), and there is a cross inhibition by estrogen receptor and glucocorticoid receptor activation (Meyer et al., 1989). Of the aromatic hydrocarbons, dioxin (TCDD) modulates receptors for glucocorticoids and estrogen (Ryen et al., 1989; Umbreit and Gallo, 1988), and methylcholantrene decreases nuclear estrogen receptor number (Chaloupka et al., 1992). Retinoids modulate the binding capacity of glucocorticoid receptor (Audoin-Chevallier et al., 1992, 1993). The overlapping effects are more intense in the perinatal critical period (Csaba, 1986, 1991). In the present experiments, the effect of combined treatment was manifested in the thymus. In females there was a significant increase (of about 50%) of glucocorticoid receptor density and a significant decrease in receptor affinity (Table 1). In males, only the receptor capacity (Bmax) was elevated significantly, and the receptor affinity was not changed. There were no changes in binding capacity of uterine estrogen receptors (Table 2). The combined treatment was done with substances causing disturbances employed in the perinatal critical period, at the receptorial level. Three of them (allylestrenol and vitamins A and I)3) are bound by the members of the steroid receptor superfamily; however, only two of them have a steroid structure (allylestrenol and vitamin D3), and the third (vitamin A) has a structure different from steroids. The fourth substance (benzpyrene) has a steroid-like struc-

G. Csaba and/k. Inczefi-Gonda TABLE 1. Effect of combined neonatal imprinting on the dexamethasone binding of thymus glucocorticoid receptors (values in 10 -9 M -+ SD) Group

n

Gender

Ka

Bm~

Control Imprinted

6 6

F F

2.45 + 1.43 4.14 -+ 0.57 a

2.30 -+ 1.17 3.85 -+ 1.08 a

Control Imprinted

5 7

M M

4.71 -+ 0.78 3.68 -+ 0.89

2.71 _+ 0.40 4.11 -+ 0.49 b

P < 0.05; b p < 0.005.

ture; however, it is bound by a separate helix-loop-helix receptor (Hankinson, 1994; Okey et al., 1994). Nevertheless, this receptor also has many similarities to the members of the steroid receptor superfamily, first of all to the glucocorticoid receptor (Dennis et al., 1988; Hapgood et al., 1989; Wilhelmsson et al., 1986). Allylestrenol is a synthetic gestagene, vitamins A and D3 have hormone-like effects (Laudet et al., 1992; Pemrick et al., 1994) and benzpyrene is a polycyclic hydrocarbon and, as such, has estrogenic or antiestrogenic capacity bound to estrogen receptor (Bimbaum, 1994; Chaloupka et al., 1992; White et al., 1995; Zacharewski et al., 1994). Neonatal treatment with each material decreases glucocorticoid and estrogen receptor density alone (Csaba, 1986, 1991; Csaba et al., 1986; Inczefi-Gonda et al., 1986), except retinol, which increases glucocorticoid receptor density of the thymus and increases uterine estrogen receptor affinity for life (Ga~il and Csaba, 1997). Considering the present results the dominance of retinol effect on the thymus glucocorticoid receptor seems obvious. The otherwise receptor density decreasing effect of the three other materials has been suppressed in the presence of retinol and its density elevating effect was manifested. This is interrelated with the fact that retinol is a "master regulator"of hormonal signaling (Mangelsdorf, 1994), whose receptor is in the center of heterodimerization with the others (Mangelsdoff and Evans, 1995)--or with its influence on the binding properties of glucocorticoid receptor (Audoin-Chevallier et al., 1993, 1995). It is worthy of mention that the affinity-decreasing effect of the combined treatment manifested only in females. This could be explained by the higher sensitivity of the female organism to the treatment (McCormick et al., 1995) as well as with the differences in the velocity of maturation of the two genders, perinatally. Neonatal hormonal (steroid) imprinting always influences receptor density for life (Csaba, 1991). Receptor affinity seemed to be strictly programmed at the gene level. In the present case, the glucocorticoid receptors affinity strongly decreased. As none of the materials used influenced glucocorticoid receptor affinity alone (Csaba, 1986, 1991; Csaba and Inczefi-Gonda, 1984; Inczefi-Gonda et al., 1986), this demonstrates the profound effect of combined treatment on the thymic glucocorticoid receptors. The combined treatment was ineffective in the uterine estrogen receptor's binding capacity. Neither density nor affinity changed at all. Because in earlier experiments (Csaba and Inczefi-Gonda, 1984; Gafil and Csaba, 1996; Inczefi-Gonda et al., 1986) retinol increased

TABLE 2. Effect of combined neonatal imprinting on the estradiol binding of uterine estrogen receptors (values in 10 -9 M +- SD)

Group

n

Ka

Bm~x

Control Imprinted

4 5

2.17 -+ 1.15 2.51 +_ 1.38

1.33 --- 0.33 1.46 +-- 0.28

Hormonal Imprinting by Multiple Treatments estrogen receptor's affinity, and the others diminished receptor density, some equilibration of receptor binding capacity could be supposed. T h e results obtained by combined imprinting compared to the resuits of individual treatments call our a t t e n t i o n to the differences caused by the interrelations of substances acting in the same period. Assuming that perinatal imprinting would be similar in humans, and taking into consideration the possible differences in doses and the quantity of possible acting agents, such effects may be unforeseeable.

This work was supported by the National Research Fund (OTKA-TO17775 ) and by the Scientific Research Council, Ministry of Welfare (T11-708), Hungary.

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