1 Department of Psychobiology of the School of Psychology, University of La Laguna, Tenerife, and Laboratory of Neurobiology and Experimental Neurology; Departments of 2 Physiology and 3 Anatomy and Pathology of the School of Medicine and 4 University Hospital of Canary Islands, Canary Islands, Spain
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Abstract |
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Key words: adenoma/prolactin/prolactinoma/serotonin/tryptophan
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Introduction |
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Materials and methods |
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L-Tryptophan (200 mg/kg; Sigma, St Louis, MO, USA) was given daily by stomach intubation to young pregnant rats (150200 g body weight before gestation). The amino acid or its saline vehicle was administered during the light cycle from day 15 to day 21 of gestation. Timing of pregnancies was determined by daily vaginal washings checking for spermatozoa; the day on which spermatozoa were found was regarded as day 0. Only the rats that were born on day 21 of gestation were included in the study. Immediately after weaning, 180 female rats were divided into two equal groups, the tryptophan-mother and saline vehicle-mother groups. In order to prevent a litter effect, only two female offspring per rat were used. At 240, 380, 517, 566, 593 and 665 days after birth, the rats were killed by decapitation at the beginning of the dark period of the second day of dioestrous (determined by analysing vaginal smears for at least eight consecutive days). Different samples were obtained for biochemical studies of serum and brain tissue, and for histological studies of pituitary and mammary tissue.
Blood was collected from the trunk and allowed to clot at room temperature. Serum was separated by centrifugation (2000 g for 15 min), divided into aliquots, and stored at 40°C until assay. Serum luteinizing hormone (LH) and prolactin radioimmunoassays were performed using materials provided by the National Hormone and Pituitary Program (Rockville, MD, USA), and the results were expressed in terms of the respective standard hormone preparation according to previously validated procedures (Mas et al., 1984). Progesterone and oestradiol were quantified by radioimmunoassay (Coat-a-count kit; Diagnostic Products Corporation, Los Angeles, CA, USA). The intra- and inter-assay variation coefficient was <8% in all the radioimmunoassays, as calculated at the concentration of the 50% displacement.
Immediately after extraction, the brain was laid on its dorsal surface and the hypothalamus and medial preoptic area (MPOA) dissected from the forebrain structures according to a previously described procedure (Gonzalez et al., 1986). The brain pieces were quickly removed and weighed in conical 5 ml test-tubes. A 2 ml aliquot of perchloric acid (PCA) (0.1 mol/l) containing 4x105 mol/l sodium metabisulphite was pipetted into the tubes to avoid the metabolism or oxidation of monoamines. Thus, at 2 min after killing of the rat the brain monoamines were protected in a stable solution. The mixture was then sonicated at 100 W for about 12 s on ice, and the homogenate centrifuged for 15 min at 15 000 g. The supernatant and pellet were kept in separate tubes. All samples were stored at 70°C to avoid deterioration until biochemical measurements were carried out (within 2 months of brain dissection). Using this procedure, the concentrations of compounds studied were not significantly modified before their measurement (Arevalo et al., 1991
).
Concentrations of tryptophan, serotonin (5-hydroxytryptamine; 5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in brain tissue were measured using liquid chromatography with electrochemical detection, according to previously validated procedures (Afonso et al., 1990; Arevalo et al., 1991
; Santana et al., 1994
). An aliquot of the supernatant was injected into the chromatographic column (300x3.9 mm stainless steel column packed with Nova-Pack c18, 4 µm particle size; Waters, Milford, MA, USA). The mobile phase consisted of 0.1 mol/l NaH2PO4H2O, 0.5 mmol/l EDTA, 1 g/l sodium heptanesulphonate (PIC B7) and 6% acetonitrile. The final solution (pH 4.35) was filtered (0.45 µm Millipore filter) before use. Standards of serotonin and 5-HIAA (Sigma) were dissolved in PCA containing 4x105 mol/l sodium metabisulphite and kept as stock solutions at 20°C. They were diluted with ice-cold PCA/sodium metabisulphite shortly before chromatographic injection. All separations were performed isocratically at a flow rate of 1.0 ml/min at room temperature. The electrochemical detector used was a Waters Model 460 (Waters, Milford, MA, USA). The detector potential was 0.82 V. Quantifications were performed from standard curves of peak height.
Mammary and pituitary tissues were dissected and processed for histological studies with light microscopy. Two processing methods were used, one for epoxy resin-embedded tissue and the other for paraffin-embedded tissue. In the first case, samples were fixed with 2.5% glutaraldehyde in Millonig buffer (monosodic phosphate 1.69%, sodium hydroxide 0.387%, glucose 0.488% and calcium chloride 0.0045% in distilled water), post-fixed with osmium tetroxide, dehydrated in a graded ethanol series and embedded in epoxy resin (EPON®; Tousimis Research Corp., Rockville, MD, USA). Semithin sections (1 µm) were cut and stained with toluidine blue. In the second case, samples were fixed in 10% formaldehyde, embedded in paraffin with an automatic processor (Autotechnicon®; Technicon Instruments Corp., Tarrytown, NY 10591, USA), sectioned at 5 µm and stained with haematoxylin and eosin. Selected slices of paraffin-embedded tissue were used for immunohistochemical detection of prolactin in both pituitary and mammary tissues. Cells with prolactin were detected according to a published procedure (Bratthauer and Adams, 1994) and using the HistostainTM SP Kit (Zymed Lab. Inc., San Francisco, CA, USA). Briefly, after deparaffinization, hydration and blocking endogenous oxidation with 3% hydrogen peroxide (10 min), tissues were incubated overnight in 10% non-inmune goat serum at room temperature. Three drops of the primary prolactin antibody provided by the NHPP (National Hormone and Pituitary Program) (1:100 000) were added to sections. After 1 h, and after washing the tissue with phosphate-buffered saline and goat serum, three drops of the biotinylated secondary antibody were added for 45 min. Finally, slices were washed, covered with horseradish peroxidasestreptavidin for 45 min, developed with diaminobenzidine (Sigma FastTM, 3-3' Diamino benzidine tablet sets, product number D-4293), counterstained with Mayer haematoxylin (1 min), dehydrated in an ethanol series, rinsed in xylene, and covered with Eukitt (O.Kindler GmbH & Co., Freiburg, Germany).
The statistical analyses were performed using analysis of variance (ANOVA) followed by Scheffé post hoc test, Student's t-test and 2 test (Statistica; Statsoft, Tulsa, OK, USA). Differences were considered to be significant when associated with a probability of 5% or less. Values were expressed as the mean ± SEM.
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Results |
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Discussion |
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We have previously reported that tryptophan administered to pregnant rats crosses the placental barrier and induces a dose-related increase in tryptophan concentration in the placenta, body and brain of the fetus (Arevalo et al., 1991) and a parallel increase in 5-HT and 5-HIAA levels in the fetal brain (Arevalo et al., 1991
). In the present study it was found that, in addition to this global action on the serotonergic system, maternal intake of tryptophan induced a permanent facilitation of serotonergic neurotransmission in the hypothalamus of female offspring (Table I?). Serotonergic neurones differentiate during early ontogenesis and their fibres reach the hypothalamus in the last days of prenatal life (Hyyppä 1972
; Zeisel et al., 1981
; Wallace and Lauder, 1983
; Arevalo et al., 1991
). However, the final termination density and the formation of precise termination patterns are not reached until adulthood (Jacobs and Azmitia, 1992
). Thus, present data show that during early neurogenesis and before synapsis formation, the central availability of tryptophan can modulate serotonergic cell differentiation, inducing permanent functional consequences in the hypothalamic serotonergic system (Arevalo et al., 1987
; Mattson, 1988
; Santana et al., 1994
).
During adulthood, serotonin is involved in the neural regulation of prolactin release by the anterior pituitary (Kamberi et al., 1971; Kordon et al., 1973
; Lu and Meites, 1973
; Chen and Meites, 1975
; Woolf and Lee, 1977
; van der Kar et al., 1980
). The peripheral administration of 5-hydroxytryptophan (Jacobs and Azmitia, 1992
), the intravenous administration of 5-HT (Pilotte and Porter, 1979
) or the administration of serotonergic agonists (Jacobs and Azmitia, 1992
) facilitate prolactin release to plasma. The drug-blockade of serotonergic neurotransmission decreases the serum prolactin concentration (Gid-Ad et al., 1976
). Thus, it has been proposed that hypothalamic 5-HT activates prolactin release from the anterior pituitary gland (Simpkins et al., 1977
; Meites, 1980
, 1982
). We observed an increase in both the serum prolactin and the serotonergic activity of the hypothalamus in tryptophan-mother offspring who developed a mammary adenoma. These data suggest that the prenatal administration of tryptophan induces not only a long-lasting activation of serotonergic neurotransmission but also a persistent enhancement of the facilitatory activity of 5-HT on prolactin release in a significant number of rats.
It has been reported in rats that treatments resulting in increased prolactin secretion (medial eminence lesion, reserpine administration) enhance the development of spontaneous mammary tumours. On the other hand, treatments that reduce prolactin secretion inhibit the development of spontaneous mammary tumours (Simpkins et al., 1977; Welsch and Nagasawa, 1977
; Meites et al., 1978
; Meites, 1980
, 1982
). In the present study, a 400% increase in plasma prolactin was found in rats that developed a mammary adenoma. Thus, it is possible that the increase in incidence of mammary adenoma found in the offspring of tryptophan-mothers could be induced by an enhancement in the plasma prolactin concentrations produced by a permanent facilitation of the hypothalamic serotonergic cells. Despite the fact that this hypothesis is supported by the presence of lactorrhoea in most mammary tumours, the epithelial component of adenomas is not necessarily induced by the chronic hyperprolactinaemia. In addition to the high prolactin concentrations, the female offspring of tryptophan-mothers that developed mammary adenomas showed a high concentration of plasma progesterone. As progesterone causes growth of the lobules and proliferation of alveoli cells, this hormone may also be involved in the high incidence of mammary adenomas observed here.
Pituitary adenomas have been found in 825% of human autopsies, a percentage that increases with age (Post et al., 1980). This high age-related incidence of prolactinomas has also been found in rats and mice (Post et al., 1980
; Meites, 1982
). A high age-related incidence of spontaneous mammary adenomas has also been reported in women and female rats or mice (Welsch and Nagasawa, 1977
). As far as we know, and despite the fact that mammary adenoma has been associated with a high plasma concentration of prolactin (Brown et al., 1982
), the relationship between mammary adenomas and pituitary prolactinomas (particularly in the case of prolactin-secreting microadenomas) has not been established. In addition, we have not found any information about the aetiology of spontaneous pituitary prolactinomas or the possible action of the long-lasting activation of hypothalamic serotonergic systems on lactotroph proliferation. Present data suggest that a persistent increase in hypothalamic serotonin due to age (Meites et al., 1978
) or other factors (high levels of tryptophan during prenatal life) may cause both pituitary prolactinomas and, in a second stage, mammary adenomas in rats. However, the morphological difference between the mammary adenomas found here in rats and the mammary fibroadenomas reported in the human species suggests that the result presented in this study must not be directly extrapolated to women.
In conclusion, the present study shows that excessive ingestion of one amino acid, tryptophan, during gestation could have important consequences on the brain development of the offspring. It has been reported that in humans transient tyrosinaemia during neonatal life can induce permanent intellectual impairment 810 years later (Menkes et al., 1972; Mamunes et al., 1976
). Further studies are warranted to evaluate the functional consequences of the neonatal increase in serum tryptophan on human development. Present data show that prenatal administration of tryptophan induces a permanent facilitation of central serotonergic neurotransmission, disrupting the serotonergic regulation of pituitary prolactin release and increasing the incidence of pituitary prolactinomas and mammary adenomas. We used a tryptophan dose 1020 times higher than the mean requirement for this amino acid in humans (Lazaris-Brunner et al., 1998
). However, tablets with a high dose of this amino acid can be obtained in most countries without medical authorization. This study suggests that it is necessary to act cautiously when tryptophan is prescribed as a dietary supplement, particularly in the case of pregnant women or low-weight newborns. Finally, and according to the hypothesis previously proposed for tyrosine and the catecholaminergic systems (Arevalo et al., 1987
; Garabal et al., 1988
; Mattson, 1988
; Rodriguez et al., 1994
; Santana et al., 1994
), present data support the idea that serotonin may have a neurotrophic role before the ontogenic start of neurotransmission.
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Acknowledgments |
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Notes |
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References |
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Submitted on January 25, 1999; accepted on May 12, 1999.