Vitamin D-neutralizing CYP24A1 expression, oncogenic mutation states and histological findings of human papillary thyroid cancer.

OBJECTIVE: The aims of the present study were to examine gene and protein expression of the vitamin D-inactivating 24-hyroxylase (CYP24A1) and the activating 1-alpha-hydroxylase (CYP27B1) enzyme in human papillary thyroid cancer (PTC), furthermore, to investigate the association between CYP24A1 expression and numerous clinical, histological parameters and somatic oncogene mutation status of thyroid tumor tissues. MATERIALS AND METHODS: Gene expression analysis was carried out in 100 Hungarian thyroid samples, both normal and papillary tumor tissue sections of the same patient. The specific mRNA to the selected genes was analyzed by TaqMan probe-based quantitative real-time RT-PCR. The somatic oncogene mutation states of BRAF, NRAS, HRAS and KRAS were also tested. RESULTS: CYP24A1 mRNA expression was markedly increased in 52 cases (52%) of the examined papillary cancers compared with that of normal thyroid tissue. There was a tendency toward difference in the distribution of high-level CYP24A1 in the PTC accompanied with somatic oncogene mutation. Positive correlation was seen between increased CYP24A1 expression rate and a group of variables reflecting tumor malignity (mainly vascular invasion, lymph node metastasis, tumor size, hypothyreosis) by principal components analysis. No significant alteration was seen in CYP27B1 gene expression between neoplastic and normal tissues. CONCLUSIONS: A definite alteration was seen in vitamin D3-inactivating CYP24A1 gene activity in PTC compared to their normal tissues on a relatively large patient population. Our findings raise the possibility that CYP24A1 may also directly be involved in thyroid carcinogenesis.

The effects of hypokalaemia on the hormone exocytosis in adenohypophysis and prolactinoma cell culture model systems.

The extracellular ion milieu determines the exocytosis mechanism that is coupled to spontaneous electrical activity. The K(+) ion plays crucial role in this mechanism: as the potassium current is associated with membrane hyperpolarization and hormone release through protein cascade activation. The primary aim of this study was to investigate the response mechanisms of normal adenohypophysis and adenohypophyseal prolactinoma cell populations at different extracellular K(+) levels with an otherwise isoionic milieu of all other essential ions. We focused on prolactin (PRL) and adrenocorticotrophic hormone (ACTH) release.In our experimental study, female Wistar rats (n=20) were treated with estrone-acetate (150 µg/kg b.w./week) for 6 months to induce prolactinomas in the adenohypophysis. Primary, monolayer cell cultures were prepared by enzymatic and mechanical digestion. PRL and ACTH hormone presence was measured by radioimmunoassay or immuno-chemiluminescence assay. Immunocytochemistry was used to assess the apoptotic cells.Differences between the effects of hypokalaemia on normal adenohypophysis cultures and prolactinoma cell populations were investigated. Significant alteration (p<0.001, n=10) in hormone exocytosis was detected in K(+) treated adenohypophyseal and prolactinoma cell cultures compared to untreated groups. Immunocyto-chemistry showed that Bcl-2 expression was reduced under hypokalaemic conditions.The decrease in hormone exocytosis was tightly correlated to the extracellular K(+) in both cell types, leading to the conclusion that external K(+) may be the major factor for the inhibition of hormone release. The significant increase in hormone content in supernatant media suggests that hypokalaemia may play important role in apoptosis.

Behavioral and endocrine effects of chronic exposure to low doses of chlorobenzenes in Wistar rats.

Chlorobenzenes have often been applied to study persistent organic pollutants with endocrine disruptor effects (POP/EDCs), but with the focus mainly on physiological aspects. Few data exist on the effects of chlorobenzenes and most POP/EDCs on anxiety or other arginine-vasopressin (AVP)- and oxytocin (OXT)-mediated behavior, albeit exposure to POP/EDCs or their ambient mixtures, even in low doses, may pose health risks for subjects living in contaminated areas and/or consuming polluted food. Our primary aim was therefore to demonstrate behavioral effects of longterm exposure to a discrete dose of a chlorobenzene mixture, and to draw attention to the results of subtoxic oral exposure on anxiety-related elements and the possible underlying endocrine processes. Adult male Wistar rats were treated daily with a mixture (ClB) of 1 µg/kg each of hexachlorobenzene and 1,2,4-trichlorobenzene via a gastric tube for 30, 60 or 90 days. After exposure, anxiety-related behavioral elements were determined in open-field and elevated plus maze tests. At euthanasia, the plasma levels of AVP, OXT and adrenocorticotrophic hormone (ACTH) were measured. Simultaneously, pituicytes from subjects were cultured to study the levels of basal and serotonin- or norepinephrinestimulated AVP and OXT secretion. Various anxiety-related behavioral elements were observed to be increased in both tests. The plasma AVP, OXT and ACTH concentrations were increased, to extents depending on the duration of exposure. The basal and monoamine-stimulated levels of AVP and OXT secretion of pituicytes prepared from the ClB-exposed rats were also elevated. Thus, certain anxietyrelated behavioral and endocrine elements were modulated by long-term exposure to ClB. As adult subjects were involved, which are generally less susceptible to toxic agents, it may be concluded that discrete doses of POP/EDC chlorobenzenes that are low enough to fall below the range of legal regulation may exert anxiogenic effects, which suggests that certain anxiogenic disorders may be induced environmentally in exposed human populations.

Further analysis of behavioral and endocrine consequences of chronic exposure of male Wistar rats to subtoxic doses of endocrine disruptor chlorobenzenes.

Many chemicals utilized by humans are present as environmental pollutants and may influence homeostasis from neurological, immunological, endocrinological and/or behavioral aspects. Such agents, acting alone or in ambient mixtures, may be biologically active even at extremely low doses, and it may be postulated that stable, bioaccumulative, reactive endocrine disruptors may affect central and/or peripheral secretion of arginine-vasopressin (AVP) and oxytocin (OXT) and thereby related physiological and behavioral functions, potentially leading to disorders in exposed subjects. The primary aim of this study was to demonstrate effects of chronic exposure to a low dose of an orally administered chlorobenzene mixture on anxiety-related and aggressive behavior mediated largely by AVP and OXT. Chlorobenzenes were applied to model ambient mixtures of endocrine disruptors. Adult, male Wistar rats were exposed daily to 0.1 µg/kg of 1,2,4-trichlorobenzene and hexachlorobenzene via a stomach tube for 30, 60 or 90 days, after which anxiety-related and aggressive behavioral elements were examined in open-field, elevated plus maze and resident-intruder tests. The plasma levels of AVP, OXT and adrenocorticotrophic hormone at the endpoints were measured by radioimmunoassay or immunochemiluminescence assay. The levels of basal and serotonin- or norepinephrine-stimulated AVP and OXT secretion in pituicyte cultures prepared from the posterior lobe of the pituitaries were also measured. The hormone levels proved to be increased to extents depending on the duration of exposure to the chlorobenzenes. Several anxiety-related and aggressive behavioral elements were also enhanced following chlorobenzene exposure, while certain explorative and locomotive elements of the animals were decreased. As both physiological and behavioral elements were modulated by chronic, subtoxic doses of chlorobenzenes, it is concluded that doses of such environmental pollutants low enough to fall outside the range of legal regulation may pose potential risks of anxiogenic and/or aggressive consequences in exposed subjects, including humans.

Segregation of the V804L mutation and S836S polymorphism of exon 14 of the RET gene in an extended kindred with familial medullary thyroid cancer.

In multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid cancer (FMTC), the majority of germline mutations are restricted to specific positions in exons 10 and 11 of the RET gene. However, germline mutations may very occasionally occur in other exons, including exon 14 of the RET gene. Interestingly, an increased frequency of a rare germline sequence variant of the RET exon 14, S836S, has been detected in patients with sporadic medullary thyroid cancer (MTC), and this variant has been proposed to play a role in the genesis of MTC and, perhaps, FMTC. In this study we report the segregation of a germline V804L mutation and a germline sequence variant S836S in exon 14 of the RET gene in an extended Hungarian FMTC kindred comprising 80 individuals of four generations. Molecular analysis of the RET gene was performed by direct DNA sequencing in 23 family members, of whom 12 had the V804L mutation, three had the V804L mutation and S836S polymorphism in separate alleles, and six had the S836S polymorphism, all in heterozygous forms. Two of the family members had neither mutation nor polymorphism of the RET gene. Three of the family members who had the V804L mutation and one member who could not be tested for mutation were operated for non-metastatic MTC, while one member with MTC who had the V804L mutation refused surgery. In all patients affected with MTC, the disease developed relatively late in life and never caused death. None of the other family members carrying the V804L mutation and/or the S836S polymorphism had clinical or biochemical evidence of MTC. These observations suggest that the co-existence of the V804L mutation and S836S polymorphism in separate alleles does not seem to aggravate the relatively low-risk disease phenotype characteristic in most patients with codon 804 mutations of the RET exon 14.

Primary mono-layer cell cultures as model system for studying of environmental toxic agents: organochlorine compounds.

Organic pollution of water and soil has various harmful effects on biological systems (1). Chlorine substituted benzol compounds are one these xenobiotic substances, which are toxic to the environment (2). They can also accumulate in plant and animal tissues (3), which provides ample reason to study the effects of sublethal doses of chloro-benzols on various cell cultures. In this study the toxic effects of chloro-benzols were investigated on avian fibroblast and mammalian hepatocyte cultures. The fibroblast cultures were prepared from eggs preadapted to chloro-benzol during a fourteen-day-long incubation period. The Wistar rat hepatocyte monolayer cultures were exposed to a direct treatment of 1,2,4-tri-chloro-benzol (0.01 microgram/ml-1 microgram/ml) for 3 hours. Following the treatment with chloro-benzol, the viability of the cells was measured, together with lactic dehydrogenase activity, in both kinds of cultures. The effect of tri-chloro-benzol treatment on chicken eggs was not significant. The cells of chicken embryos were not damaged after the 1,2,4-tri-chloro-benzol treatment. The hepatocyte cultures showed the toxic effects of 1,2,4-tri-chloro-benzol after the direct and acute treatment. The cell viability decreased and the LDH activity increased significantly. These results show that the primary cell cultures are suitable for studying the effects of organochlorine compounds.

Functional membrane changes due to tumor induction in rat pituitary cell cultures.

Membrane functions in tumorous cells are different from those in healthy cells. The aim of the present study was to investigate the changes in pituitary cell membrane functions and hormone secretion after tumor induction in vivo and in vitro. Prolactinomas were induced in vivo in female Wistar rats with estrone acetate. Normal anterior pituitaries and prolactinomas of female Wistar rats were dissociated enzymatically and mechanically, then cultured on collagen-treated plastic dishes. Some normal anterior pituitary cultures were treated with benz(c)acridines as tumorigenic agents in vitro. Intracellular 3',5'-cyclic-adenosine monophosphate (cAMP) levels were determined by a competitive binding technique, membrane fluidity was assayed by fluorescence anisotropy, and ATP-ase activities were estimated via ATP loss. The results indicated decreased membrane fluidity in tumorous cell cultures. However, in vitro benz(c)acridine treatment exerted more pronounced effects than those observed after in vivo estrone treatment. The ATP-ase activities were highly increased in benz(c)acridine-treated cells and in estrogen-induced prolactinoma cells, more strongly so in the former ones. The intracellular cAMP levels were higher than normal in both of them. The results concerning the ACTH, alpha-MSH, PRL and GH levels of normal and tumorous cell cultures were published in our previous study. Our findings show that the tumorous transformation of pituitary cells can cause significant changes in functional membrane parameters and hormone secretion. Decreased membrane fluidity was accompanied by an increased exocytosis (hormone release) and adenylate cyclase activity in tumorous cells.

[Experience with ambulatory radioiodine therapy of hyperthyroidism]. / Ambuláns radiojód kezeléssel szerzett tapasztalatok a hyperthyreosis gyógyításában.

Since 1993, outpatient radioiodine therapy has been available in Hungary. The reported study evaluated the efficacy of outpatient radioiodine treatment in subjects with hyperthyroidism. The data on 118 patients with Graves' disease and 36 patients with thyroid autonomy were analysed retrospectively. All patients were treated within the period 1994-1997. The activities of radioiodine were individually calculated. The applied dose in Graves' disease was 150 Gy, and in thyroid autonomy 150 Gy or 300 Gy. The efficacy of the treatment were evaluated 3, 6, and 12 months after radioiodine therapy. In patients with persistent hyperthyroidism repeated therapies were performed. Overall the radioiodine therapy was successful in 85% of the Graves' disease patients. The first 150 Gy treatment was effective in 70% of the patients with Graves' disease and in 43% of the patients with increased radioiodine turnover. In thyroid autonomy, the treatment with 150 Gy was successful in 71%, with 300 Gy in 89% of the patients. The efficacy of radioiodine treatment was similar to the results of one dose application. It was concluded, that radioiodine therapy with 150 Gy absorbed dose in Graves' disease and with 300 Gy absorbed dose in thyroid autonomy proved successful by the method of the authors.

[15-year echocardiographic follow-up of acromegalic patients]. / Acromegaliás betegek 15 éves echocardiographiás követése.

An echocardiographic follow-up of 25 patients (pts) was performed, after the first examinations in 1978-1979. 15 pts have died in the last 15 years (Group 1), while 10 still living (Group 2). In Group 1 either the impaired global left ventricular at the time of the first echocardiography predicted a high mortality i.e. there were only two pts with normal echocardiographic findings. The other 6 acromegalic pts with a "normal heart" have survived. The left ventricular hypertrophy of pts in Group 2 was observed to increase during the 15-year follow-up, because of the development of systemic hypertension. In conclusion, the echocardiographic findings are of a good predictive value in estimations of the survival rate in acromegalic pts.

The effects of oral clonidine on the growth hormone level in acromegalic patients.

The effects of the noradrenergic agent clonidine on GH secretion of the adenohypophysis were studied in 10 healthy volunteers and 11 acromegalic patients. Orally administered clonidine led to a considerable serum human growth hormone (GH) level increase in the healthy individuals. In the acromegalic patients the clonidine resulted in a slight, but not significant GH increase. These results support the findings that the mechanism of noradrenergic regulation of serum GH secretion is impaired in acromegaly.

Lack of effect of desglycinamide-arginine-vasopressin (DGAVP) on memory in patients with korsakoff's syndrome.

The effect of desglycinamide9-[Arg8]-vasopressin (DGAVP) on memory processes was studied in patients with Korsakoff's syndrome. Intranasal treatment with DGAVP for 7 days affected neither attention nor short- and long-term memories. It is suggested that treatment with DGAVP is not indicated for all types of memory disorders, and that the beneficial effect of this treatment may depend on the integrity of certain brain structures.

Effects of desglycinamide-arginine-vasopressin (DG-AVP) on memory processes in diabetes insipidus patients and non-diabetic subjects.

The effects of desglycinamide9-arginine8-vasopressin (DG-AVP) on memory processes have been studied in patients with central diabetes insipidus (DI) and in non-diabetic control patients. Acute im injection of DG-AVP improved some aspects of short-term memory. Subchronic intranasal administration of DG-AVP facilitated short-term memory more consistently and in addition improved long-term memory. DG-AVP increased the attention, but only in the non-diabetic subjects. The effects of DG-AVP on memory processes persisted after discontinuation of treatment. DG-AVP did not affect the parameters for water and electrolyte metabolism, blood pressure and pulse rate neither in DI nor in the control patients. Thus, the memory effects of DG-AVP are probably mediated by a direct action on the central nervous system.

Effects of lysine-vasopressin and 1-deamino-8-D-arginine-vasopressin on memory in healthy individuals and diabetes insipidus patients.

Central diabetes insipidus (DI) patients showed impairments in short- and long-term memory functions, but not in attention and concentration, as compared to healthy individuals. A single i.m. injection or sub-chronic intranasal administration of either lysine-vasopressin (LVP) or 1-deamino-8-D-arginine-vasopressin (DDAVP) normalized the disturbed memory functions in DI patients. These peptides also improved memory functions in healthy individuals.