Liver glucocorticoid receptor and heat shock protein 70 levels in rats exposed to different stress models.

The aim of the present study was to define the stress-induced pattern of cytosolic glucocorticoid receptor (GR) and Hsp70 protein in the liver of male Wistar rats exposed to different stress models: acute (2 h/day) immobilization or cold (4 degrees C); chronic (21 days) isolation, crowding, swimming or isolation plus swimming and combined (chronic plus acute stress). Changes in plasma levels of corticosterone were studied by radioimmunoassay (RIA). The results obtained by Western immunoblotting showed that both acute stressors led to a significant decrease in cytosolic GR and Hsp70 levels. Compared to acute stress effects, only a weak decrease in the levels of GR and Hsp70 was demonstrated in chronic stress models. Chronically stressed rats, which were subsequently exposed to novel acute stressors (immobilization or cold), showed a lower extent of GR down-regulation when compared to acute stress. The exception was swimming, which partially restores this down-regulation. The observed changes in the levels of these major stress-related cellular proteins in liver cytosol lead to the conclusion that chronic stressors compromise intracellular GR down-regulation in the liver.

Inhibition of rat brain ecto-atpase activity by various drugs.

The in vitro effect of digoxin, verapamil, propranolol, carbamazepine, diazepam and promethazine were investigated on the ecto-ATPase activity of synaptosomal plasma membranes from the rat brain. ATP hydrolyzing activities of the enzyme were not affected by digoxin while the use of all other drugs resulted in significant and dose-dependent ihibition in ATP hydrolysis. According to values of IC(50) and K(iapp), the order of inhibitory potency of the drugs applied was: diazepam > promethazine > verapamil > propranolol >> carbamazepine. Kinetic analysis of the nature of the ATPase inhibition revealed that it resulted from a direct action of drugs on the enzyme protein. The aim of the present study was to determine the potential neuromodulatory side effects of the drugs investigated. The results achieved indicated that all investigated drugs, except digoxin, may modulate neuronal activities via the purinergic receptors P2 by increasing extracellular concentrations of ATP as a consequence of inhibition of the ecto-ATPase activity. Our findings indicate that it may be useful to take into consideration the possible side effects of the investigated drugs, when they are used in treatment of different pathologies, particularly in the treatment of epilepsy by carbamazepine and diazepam.

Selective inhibition of brain Na,K-ATPase by drugs.

The effect of drugs from the class of cardiac (methyldigoxin, verapamil, propranolol), antiepileptic (carbamazepine), sedative (diazepam) and antihistaminic (promethazine) drugs on Na,K-ATPase activity of plasma membranes was studied in rat brain synaptosomes. Methyldigoxin in a concentration of 0.1 mmol/l inhibits enzyme activity by 80 %. Verapamil, propranolol and promethazine in concentrations of 20, 20 and 2 mmol/l respectively, entirely inhibit the ATPase activity. Carbamazepine and diazepam in concentrations of 0.02-60 mmol/l have no effect on the activity of this enzyme. According to the drug concentrations that inhibit 50 % of enzyme activity (IC(50)), the potency can be listed in the following order: methyldigoxin promethazine verapamil ? propranolol. From the inhibition of commercially available purified Na,K-ATPase isolated from porcine cerebral cortex in the presence of chosen drugs, as well as from kinetic studies on synaptosomal plasma membranes, it may be concluded that the drugs inhibit enzyme activity, partly by acting directly on the enzyme proteins. Propranolol, verapamil and promethazine inhibitions acted in an uncompetitive manner. The results suggest that these three drugs may contribute to neurological dysfunctions and indicate the necessity to take into consideration the side effects of the investigated drugs during the treatment of various pathological conditions.

Localization of recognition site between transforming growth factor-beta1 (TGF-beta1) and TGF beta receptor type II: possible implications in breast cancer.

Although overexpression of TGF-beta1 protein has been demonstrated in advanced breast cancer (BC) patients, as well as in other solid tumours, the molecular mechanism of this process remains obscure. This paper proposes that a genetic/epigenetic alteration might occur in the TGF-beta1 gene, within the region coding for the recognition site with TGFbeta receptor type II, leading to a disruption of the ligand-receptor interaction and triggering the TGF-beta1 cascade-related BC progression. To establish the operational framework for this hypothesis, in the present study, this recognition site was identified by the Informational Spectrum Method (ISM) to comprise two TGF-beta1 peptides (positions 47-66 aa and 83-112 aa) and one receptor peptide at positions 112-151 aa of the extracellular domain of the receptor (TbetaRIIM). The TbetaRIIM locus was further evaluated by ISM-derived deletion analysis of the TbetaRII sequences. To provide experimental support for the proposed model, a pilot study of plasma TGF-beta1 analysis was performed in advanced BC patients (n = 8). Two commercial ELISA assays, one with specific alphaTGF-beta1 MAb (MAb) and other with TbetaRIIM as the immobilized phase, revealed pronounced differences in the pattern of plasma TGF-beta1 elevation. In MAb-profile, the TGF-beta1 increase was detected in 7 of 8 patients, whereas analogous TbetaRIIM-profile revealed the elevation in 3 of 8 patients, taking a 50% of maximal elevation as the cut-off value. These findings are consistent with the proposed aberration of TGF-beta1 ligand within the TbetaRII recognition site. Summarizing, this model system is a good starting point for further genetic studies, particularly on genetic/epigenetic alterations of sequences involved in TGF-beta1 and TbetaRIIM interaction, with putative prognostic value for breast cancer.

Plasma TGF-beta1-related survival of postmenopausal metastatic breast cancer patients.

A pilot study was conducted to assess whether plasma levels of transforming growth factor-beta1 (TGF-beta1) might facilitate biological subgrouping of postmenopausal metastatic breast cancer patients, and, accordingly, its applicability in clinical oncology. This study included 29 postmenopausal metastatic breast cancer patients. Plasma TGF-beta1 levels were detected by enzyme-linked immunosorbent assay (ELISA). Estrogen and progesterone receptors were assayed by radioligand binding, in accordance with the recommendation of the EORTC. Concentrations of 17-beta estradiol were determined by using ELISA-microwell method (DIALAB). Overall survival was followed for 24 months for each individual patient. Stratification of the patients by ER/PR status showed that 14 patients with estrogen receptor-negative, progesterone receptor-negative carcinomas displayed a statistically significant increase in plasma TGF-beta1 levels when compared to plasma TGF-beta1 levels of 6 patients with ER-positive, PR-positive carcinomas (P=0.04). In this study, 7 out of 14 patients with negative receptors' status had no plasma TGF-beta1 values overlapping with patients having positive receptors' status. The TGF-beta1 cut-off value was defined as the highest plasma TGF-beta1 level of ER-positive, PR-positive patients: 3.28 ng/ml. This plasma TGF-beta1 cut-off value defined low-risk subgroup of 19 patients (< or = 3.28 ng/ml) and high-risk subgroup of 10 patients (> 3.28 ng/ml) (P=0.047). Plasma TGF-beta1-related survival was independent of the classical prognostic factors of metastatic breast cancer. Accordingly, a clinical significance of elevated plasma TGF-beta1 levels may be suggested.

Total monoamine oxidase activity in the hypothalamus, ovary and uterus of rats with an extreme number of ovarian corpora lutea.

The activity of total monoamine oxidase (MAO) in the rat ovary and uterus fluctuates significantly under various physiological conditions. We analyzed total MAO activity in the hypothalamus, uterus and ovary in adult rats, having an extreme number of corpora lutea (hyperluteinized ovaries) resulting from the mechanical lesions in the posterior hypothalamic region of neonatal rats. Total MAO activity in the hypothalamus (30.21 +/- 1.53 pmol/mg tissue/min) and uterus (3.16 +/- 0.61 pmol/mg tissue/min) of rats with hyperluteinized ovaries did not show a significant difference as compared to that of intact controls (31.09 +/- 1.72 and 2.90 +/- 0.40 pmol/mg tissue/min, respectively). In contrast, in the ovaries of hyperluteinized rats, total MAO activity (21.16 +/- 1.70 pmol/mg tissue/min) was significantly higher (p<0.01) when compared to that of intact controls (13.61 +/- 1.30 pmol/mg tissue/min). The increased MAO activity in the hyperluteinized ovaries may be attributed to the increased number of transformed and accumulated corpora lutes as a consequence of diminished luteolysis.

Integrated natural radiation exposure studies in stable Yugoslav rural communities.

The results of field investigations of natural radiation exposures of the general population in two stable rural communities in Yugoslavia are presented. The principal emphasis was on exposures to contemporary indoor radon, but measurements of external penetrating radiation absorbed dose rates in air were carried out in the majority of cases. In addition, in a limited number of dwellings, measurements of thoron gas concentrations were made. By means of making a series of sequential 3-month radon measurements, both seasonal variations and annual average radon levels in the dwellings were determined. Using passive alpha track detectors, individual radon and thoron indoor concentrations as high as 9591 Bq m(-3) and 709 Bq m(-3), respectively, were detected while absorbed dose rates in air in the dwellings as high as 430 nGy h(-1) were recorded. On the basis of these different types of measurements, assessments could be made of the integrated natural radiation exposures being received by the populations. In addition to contemporary radon measurements, retrospective radon exposure assessments in most of the dwellings were made on the basis of measurements of 210Po concentrations in both surface (glass) traps and in volume (porous materials) traps. A description is given of the sampling strategies and protocols used in this field work. It is shown that at least one stable rural community receiving high natural radiation exposures, has been clearly identified and plans for future health investigations of the population there are outlined.

Selenium intake as a modulator of responsiveness to oxidative stress.

A deficiency in important components of the endogenous antioxidative defense system (AODS) against the production of reactive oxygen species, including free radicals, results in the accumulation of oxidative damage, inducing oxidative stress. A dietary deficiency in selenium (Se), an important part of AODS, can increase the sensitivity of a living system to oxidative stress. We investigated the effects of Se supplementation, in the form of Se-enriched yeast, on the AODS resistance of red blood cells (RBC) to experimentally induced oxidative stress. We analyzed the alterations in main components of the AODS, such as the amount of reduced (GSH) oxidized glutathione (GSSG), Se-dependent glutathione peroxidase (GSH-Px), Se content, catalase (CAT), and thiobarbituric acid reactive substances (TBARS), in RBC of male Wistar rats exposed to gamma rays and supplemented with Se-enriched yeast (SeY) in drinking water. The results suggested that the increased Se level generally exhibited a protective effect against whole body irradiation, reducing the expenditure of the AODS components in defense. These reductions differed depending on the time observed and the parameter investigated but, generally, SeY supplementation induced a faster restoration of the AODS after this kind of oxidative stress.

Effect of X-rays on selenium content in rat jaws.

It is known that X-radiation of the growing jaws results in impaired dental development. We investigated the role of selenium (Se) in radiation-induced dental growth retardation. The heads of 8-day-old female rats were irradiated with a single dose of 9.6 Gy of X-rays. Another group of animals was irradiated under the same conditions but, for radioprotective purposes, were also in deep hypothermia during radiation exposure. Nonirradiated animals served as controls. The Se content in the upper and lower jaws was analyzed by hydride generation atomic absorption spectrophotometry. In the upper jaws of the animals exposed to irradiation, the Se concentration was significantly lower than in those irradiated under conditions of hypothermia (p < 0.05), although both groups showed no significant difference in Se concentration when compared to nonirradiated controls. In the lower jaws, the concentration of Se was significantly reduced in the irradiated group when compared with controls and the rats irradiated under conditions of hypothermia (p < 0.05). There was no significant difference in Se concentration between rats irradiated under conditions of hypothermia and nonirradiated controls. There was no significant change in Se concentration in the jaws of the rats protected by hypothermia during radiation exposure compared with nonirradiated controls. Although Se loss in the irradiated bone could be prevented by hypothermia, we could not prove that Se loss per se is the cause of growth retardation nor that its retention has a radioprotective effect.

Regulation of ovarian hyperluteinization.

Adult female rats with neonatally damaged posterior hypothalamus, made by a transversal cut, were investigated. Plasma levels of prolactin (PRL), gonadotropic hormones (GTH) and female gonadal steroids (GS) were determined by radioimmunoassay. The animals were sacrificed, at the ages of 4 and 6 months and their hypothalamus, pituitary gland, ovary and uterus were examined using light microscopy. The results can be summarized as follows: body mass of animals, with damaged posterior hypothalamus, was significantly reduced. Masses of luteinized ovaries were increased and uterine tissues decreased. Serum levels of PRL were significantly increased and luteinizing hormone (LH) decreased. Ultrastructural changes in the corpora lutea (CL), previously described, showed clear signs of their reduced capacities to produce GS, both estradiol (Oe) and progesterone (Pg) per total ovarian mass. However, prostaglandin 2 alfa (PGF2alpha) known as a luteolytic factor, was also diminished in the evidently retarded endometrium. As a result of decreased plasma values of LH, Pg and PGF2 alpha, luteolysis of CL in hyperluteinized ovaries did not occur, and their new generations were accumulated during subsequent cycles. The character of interruption and recovery of aminergic and peptidergic neurons, involved in regulation of hypothalamic-pituitary gonadal axis and feed-back effects of steroid hormones, require further studies.

The effects of continuous light and darkness on the activity of monoamine oxidase A and B in the hypothalamus, ovaries and uterus of rats.

For assessing monoamine oxidase (MAO-A and -B) activities in the hypothalamus, ovaries and uterus, mature female rats were exposed to either continuous light or dark over 6 weeks. Confirming previous studies, continuous light induced constant estrus in all animals. The majority of animals kept under continuous dark during the six weeks remained mostly in diestrus with estrus appearing sporadically. The endocrinological function of the ovaries was disturbed by continuous light, which resulted in the development of polycystic ovaries, their morphological appearance being not significantly affected by continuous dark. Hypothalamic MAO-A activity was markedly reduced under the influence of both light (p < 0.05) and dark (p < 0.01). The activity of hypothalamic MAO-B was reduced only under the influence of dark (p < 0.01). In the ovaries, no significant differences were detected in either MAO-A or -B activity as a result of these environmental manipulations. In the uterus of rats exposed to continuous light which developed polycystic ovaries, MAO-A activity was significantly lower (p < 0.05) in comparison to either intact controls or rats exposed to continuous dark. These results demonstrate that chronic changes in photoperiodicity may considerably influence MAO-A activity and to a lesser extent MAO-B activity dependent on the tissue studied.

Basal and stress induced concentrations of adrenal gland catecholamines and plasma ACTH during aging.

Basal and stress levels of catecholamines (CA) in the adrenal glands, and circulatory levels of adrenocorticotropic hormone (ACTH) were examined in female Wistar rats aged 1, 3, 10 and 24 months. Our data showed reduction in basal dopamine (DA) concentration in adrenal glands and an increase in this catecholamine in response to stress at all ages (1, 3, 10, 24 months). The greatest levels of basal norepinephrine (NE) and epinephrine (E) concentrations in the adrenal glands were noted in intact rats at the age of 24 months. On the other hand, the stress response of NE and DA had a tendency to fall, reaching basal values at the age of 10 and 24 months of age. Basal circulatory levels of ACTH showed a reduction with age. The stress response of ACTH was reduced in animals aged 10 and 24 months. Reduced basal values of adrenal DA and increased NE and E values, suggest that there is increased adrenomedullar activity at the age of 24 months. On the other hand, the reduced or even absent stress response of NE and E observed in the adrenals, in 10 and 24 months old rats, may be of interest in considering the ability of these animals for adaptation. Basal and stress values of plasma ACTH are significantly reduced with the onset of senescence in female rats.

Rat pinealocyte reactive response to a long-term stress inducement.

The pineal gland-reactive response to long-term multifactor stress inducement performed by 18 h immobilization was found in the occurrence of different pinealocyte forms--cells of the basal activity, functionally animated cells and cells assumed to be threatened by an irreversible injury. Functionally animated pinealocytes were recognized as entities of neuroendocrine-like and ependymal-like activities displaying an episode of their initially increased secretory activity determined by Golgi apparatus and succeeded by a period determined by the storage of compounds arising from the cisterns of the granular reticulum. The domination of the pinealocytes with neuroendocrine-like activity was considered to evince a stimulated, peptidergic-mediated pineal gland activity. The adrenocorticotropic hormone-reactive secretion, employed as the evaluation parameter in morphofunctional observations, corroborated the morphologically estimated increased pineal gland activity in long-term stress inducement.

The effect of constant light on the concentration of catecholamines of the hypothalamus and adrenal glands, circulatory hadrenocorticotropin hormone and progesterone.

The participation of adrenocorticotropic hormone (ACTH) in the control of adrenal glands and the regulation of ACTH secretion in female rats exposed to constant light for six weeks were studied. A significant increase of plasma ACTH (p < 0.05) in rats exposed to constant light is in correlation with an increase of epinephrine (E) synthesis in adrenal gland (p < 0.05) when compared to intact controls. On the other hand, ACTH secretion is in inverse relation with a significantly reduced concentration of dopamine (DA) and norepinephrine (NE) in hypothalamus (p < 0.05). In addition, higher concentration of plasma ACTH in rats exposed to constant light through its effect on the adrenal cortex is responsible for the appearance of polycystic ovaries. As a contribution to this assumption is the result showing an increased concentration of serum progesterone in rats exposed to constant light when compared to corresponding controls which is probably of adrenal origin. These results indicate an inverse relationship between plasma ACTH concentrations and DA and NE concentrations in hypothalamus and that the hypothalamo-pituitary-adrenal axis has a significant role in the regulation of ovarian function.

Catecholamines in hypothalamus, ovaries and uteri of rats with precocious puberty.

The concentration of catecholamines in the hypothalamus and reproductive organs of rats with hypothalamic lesions placed on the day of birth was investigated. In an attempt to contribute to the discussion concerning the role of catecholamines in regulation of puberty, the aim of this study was to examine the content of hypothalamic, ovarian and uterine catecholamines in female rats with precocious vaginal opening. The animals were sacrificed on the day of precocious puberty, which occurred at the age of 25.71 +/- 0.98 days. The data obtained were compared with intact (pubertal) controls obtained on the day of vaginal opening (40.13 +/- 1.35 days) as well as in infantile intact controls of corresponding age of the lesioned animals. In the ovaries of both animals (i.e. lesioned and sacrificed on day of vaginal opening and the infantile controls of corresponding age), small and medium sized follicles were present. Ruptured follicles were evident only in the pubertal controls. The mass of ovaries and uteri from lesioned rats was also significantly lower than in the pubertal controls. Concentrations of norepinephrine in both ovaries and uteri on the day of vaginal opening of the animals with posterior hypothalamic lesions and of infantile controls, was significantly higher than in pubertal controls on the day of vaginal opening. Epinephrine was significantly higher in the uterine tissues of lesioned and infantile controls as compared to pubertal animals. The levels of dopamine and norepinephrine in the hypothalamus of lesioned and pubertal controls on the day of vaginal opening were significantly higher than in infantile controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat pineal gland suppresses the injection stress-reactive ACTH outflow.

The involvement of the pineal gland in the regulation of the initial ACTH reactive outflow to short-term stress inducement was studied. The stress trial, consisting of the subcutaneous saline injection in the animal hind leg, elicited ACTH release, the peak appearing in the 5th min followed by a rapid downturn to the prestress level in the 15th min. The higher ACTH level in the pinealectomized rats was monitored in the 5th min. The results obtained show the implication of the pineal gland in restricting the magnitude of the initial stress-reactive ACTH outflow.

Selenium content and distribution in rat tissues irradiated with gamma rays.

The effects of supplementation with selenous yeast and ionizing radiation on selenium (Se) content and distribution were evaluated in rat tissues (liver, kidney, spleen, heart, muscle, blood, front brain, hind brain, hypothalamus, pituitary, adrenal glands, testes, and hair). This study had 16 Se-supplemented (0.5 micrograms Se/d) and 16 placebo adult male Wistar rats. One half of the animals (eight Se-supplemented and eight placebos) were irradiated with a single dose of 4.2 Gy from a Co-60 source and sacrificed 7 d after irradiation along with nonirradiated animals and analyzed for Se content determination. The data obtained showed that selenous yeast supplementation increased Se levels in rat tissues (highest increases in hypothalamus, 161%; hind brain, 126%; spleen, 110%; and adrenal gland, 105%). Ionizing radiation induced significant changes in Se content and distribution (decrease in liver, blood, hair, femoral muscle, spleen, and hypothalamus; increase in kidney, testes, adrenal glands, and brain of placebo group). Supplementation with selenous yeast reduces changes in Se content and distribution after irradiation. It seems that the animal tissue susceptibility to oxidative damage may be correlated to their ability to retain Se in tissues.

Characteristics of a prompt morphodynamic response of the pineal gland to an acute ACTH injection.

The morphodynamic response of the pineal gland in the period 5-30 min after a single ACTH injection was studied. In 5 min a saline injection, functioning as a control, elicited a prompt release of the pineal active compounds advocated to be contained in clear vesicles and structures traditionally labeled lipid droplets. In the subsequent time period, 15 min, a rise in the relative volume of the granular endoplasmic reticulum (GER) and the Golgi apparatus, as well as prosecretory granules, was interpreted as a sign of an increased pinealocyte activity which leads to the restoration of the pineal gland endocrine potential. A decline in the increased mass of this membranous elaborative compartment in 30 min could be understood as an illustration of the stabilisation of the pinealocyte stress-evoked response. The ACTH injection did not apparently change the pattern of the morphodynamic pinealocyte response promoted by the saline injection in 5 min. However, starting from 15 min, a significant increase of the mass of GER and the Golgi apparatus was found in both pinealocyte populations. The augmented presence of prosecretory granules and lipid droplets, in comparison with the saline-injected animals in the respective time period, as well as the absence of a clearly observable evidence of the pinealocyte secretory activity pointed out that the ACTH stimulative action on the pinealocyte membranous elaborative compartment rather facilitated the pinealocyte endocrine recovery than supported their current secretory activity. From the functional viewpoint, the results obtained suggest that a stress-induced ACTH burst could modulate the post-secretory restitution of the endocrine potential of previously stress-activated pinealocytes.

The effects of X-ray irradiation of the head region of eight-day-old rats on the development of molar and incisor teeth.

The effects of irradiation on the development of molars and incisors up to the age of 57 days was studied applying locally a single dose of 9.6 Gy of X-rays to the head region of eight-day-old female rats. Radiography of jaws revealed the absence of the third molar in all irradiated rats, as a result of germ stunting which was, at the moment of irradiation, in the phase of histodifferentiation. In the case of the first and second molar, being at the time of irradiation in the phase of root formation, changes were noted in the development and morphological features of the roots. In animals sacrificed 18 days after irradiation, the radiographs of jaws showed a break in the continuity of the incisors located under the mesial root of the first molar. In all animals the break in continuity of the incisors became macroscopically visible between the 28th and 42nd day after irradiation. 15 days after the loss of incisors, the incisival functional occlusion was restored in all animals.

Morphodynamic response of the pineal gland to initial stress attack.

The influence of an initial stress attack (a subcutaneous saline injection plus rough handling) on the morphofunctional behaviour of the pineal gland was studied. Both light and electron microscopy pointed to an enhanced endocrine activity on the gland parenchyma. The occurrence of the clusters of highly activated light pinealocytes, as well as the appearance of two functionally different types of these cells, revealed that the introduction of the pineal gland to a new stress-induced steady state was based on the gradual promotion of a number of pinealocytes to the level of a high activity. Dark pinealocytes were less numerous and rather engaged in the synthesis than in the secretion. The ultrastructural characteristics of both pinealocyte populations show that the pineal gland meets the secretory demands upon an initial stress attack by a striking discharge of its active compounds and a successive activation of a new elaborative cycle. The morphodynamic conclusion about an enhanced pineal gland secretory activity is fully evaluated in the change of the rate of prolactin surge. A possible impact of the morphodynamic reactivity of the endocrine parenchyma of the pineal gland upon the functional interpretation of the morphological properties of pinealocytes in case of some manipulative procedures with animals was discussed.