NITRIC OXIDE SYNTHASE ACTIVITY AND ITS CONCENTRATION IN THE TISSUES OF HUMAN THYROID CARCINOMAS.

The study of the activity of the constitutive form of nitric oxide synthase (cNOS) revealed that in the papillary thyroid carcinomas it corresponded to that detected in unchanged extratumoral tissue, while the enzyme activity in follicular carcinoma was half lesser. At the same time, the activity of inducible nitric oxide synthase (іNOS) was higher in the papillary and follicular carcinomas. Such changes in the enzyme activity were associated with an increase in its level in papillary carcinomas, and with minor changes in follicular carcinomas. In medullary carcinomas the parameters under study corresponded to those in unchanged tissue, and in the papillary carcinoma metastases without changes in enzyme activity of nitric oxide formation, the level of the latter was much higher. Elevated levels of nitric oxide and іNOS activity in papillary thyroid carcinomas did not depend significantly on the aggression characteristics of the latter, being however absent in tumors of T4 category on a background of reduced cNOS activity and less expressed in tumors surrounded by the tissue in the presence of a chronic thyroiditis. Furthermore, in the papillary carcinomas of papillary or follicular structure nitric oxide level did not differ from the normal range, being slightly higher in tumors of solid or heterogeneous structure with presence of solid areas, whereas in carcinomas of papillary-follicular structure it was twice, and in tissue of solidinsular structure three times higher. іNOS hyperactivity was observed in the carcinomas of different structure, except for tumors of solid structure, in the tumor of which enzyme activity was within the normal range, and in tumor of solid-insular structure where it was significantly higher (as well as cNOS activity) compared with tumors of other structure. Nitric oxide generating system is involved in the transformation of thyroid cells and progression of tumor growth, including through apoptosis regulation, as shown by the results of an analysis of data obtained both in the present study and previously. The nature of such involvement in papillary thyroid carcinomas with different histological structure is different. Key words: nitric oxide; constitutive and inducible nitric oxide synthase; thyroid carcinoma; apoptosis.

[Changes in active cysteine cathepsins in lysosomes from tissues thyroid papillary carcinomas with various biological characteristics].

To clarify possible role of cysteine cathepsin H, B and L in the proteolytic processes that contribute to the progression of tumor growth in the thyroid, we studied their activity in lysosomes isolated from the tissue of papillary carcinomas. It was shown that for these enzymes there is a dependence of the changes in their activity on a number of biological characteristics of the tumors. Thus, the sharp increase in the activity ofcathepsin H observed in lysosomes of tissue carcinomas category T2 and T3, with intra-and ekstrathyroid and lymphatic invasion of tumor cells. An increase in the activity of cathepsin B is set in the lysosomes of tissue heterogeneous follicular structure, especially in the presence of solid areas, in comparison with typical papillary tumors and in the lysosomes of tissue carcinomas in intrathyroid and cathepsin L-at extrathyroid invasion. A common feature of the enzymes is to increase the activity of cathepsins in lysosomes of tissue nonencapsulated papillary carcinomas. These enzymes probably do not take part in the invasion of tumor cells into blood vessels and in the mechanisms of tumor metastasis to regional lymph nodes. The latter shows no changes in the activity of cathepsins in lysosomes of tissue carcinomas category N1. The results indicate the different role of cathepsin H, B and L in thyroid carcinogenesis, where each enzyme has its specific function.

Abnormalities of apoptosis of the thyroid gland cells from extratumoral microfollicular tissue.

AIM: The aim of this study was to evaluate the frequency of existence of thyroid extratumoral normo- and microfollicular tissue in patients with thyroid carcinoma and peculiarities of apoptosis in mentioned tissue. MATERIALS AND METHODS: Using samples of normo- and microfollicular thyroid tissue it was determined the content of fragmented DNA and intensity of stimulated internucleosomal DNA fragmentation; activities of caspase-3 and cysteine lysosomal cathepsins. RESULTS: It was found that normofollicular tissue is observed more often in patients with nodal euthyroid goiter but microfollicular tissue is more common for patients with carcinoma. Extratumoral microfollicular tissue was found in the thyroid of patients above 50 years old mostly, and more rarely in young ones. The fragmented DNA concentration in microfollicular tissue was lower by a factor of 3.5 and intensity of stimulated internucleosomal DNA fragmentation was also decreased. Activity both of cathepsin B in lisosomes and caspase-3 in lysates of such tissue was also decreased. CONCLUSIONS: The decrease of intensity of spontaneous apoptosis and the absence of its modulation/induction following proapoptotic factors in extratumoral microfollicular thyroid tissue may be considered as a respond of the thyroid gland tissue to an existence of carcinoma.

[B-, H- and L-cathepsin-like activity in blood plasma of patients with diseases of the thyroid, parathyroid and adrenal glands].

B-, H- and L-catepsine-like activity regarding Na-benzoyl-D,L-arginine-4-nitroanilide, L-leucine-4-nitroanilide and azocasein was studied in the blood plasma of patients with different diseases of thyroid, parathyroid and adrenal glands. It has been shown that the high H- catepsine-like activity confirming the activation of blood catepsin H secretion accompanied by the tissue growth under any pathology took place in the blood plasma of patients with all the above diseases. High B- catepsine-like activity in the blood plasma of patients with thyroid diseases was exceptionally fixed under the nodular forms of thyroid pathology, while the character of changes in L catepsine-like activity under these diseases was not so natural. In diaseses of the adrenal glands the changes in the B- and L-catepsine-like activity were only shown in the blood plasma of patients with cerebral layer tumors but not the gland cortex: B-catepsine-like activity increased in the blood plasma of patients with benign or malignant tumors, and L-catepsine-like activity decreased under benign tumor from chromaffin tissue. The established picture of changes in enzymatic activity in the blood plasma of patients does not allow to think that the determination of blood B- and L- catepsine-like activity can be recommended for obtaining additional information at diagnosis thyroid diseases.

Caspase-3 activity in papillary thyroid carcinomas.

AIM: The aim of this work was to assess caspase-3 activity in the tissue of papillary thyroid carcinomas of patients and analyze the peculiarities of changes in this activity depending upon a number of pathomorphological and clinical features of tumoral process. METHODS: Caspase-3 activity was determined by spectrophotometry with regard to acetyl-asp-glu-val-asp-paranitroanilide. RESULTS: At initial stages of tumor development, in the absence of metastases to lymph nodes, blood and lymphatic vessel invasion by tumor cells, extrathyroid spreading of tumor, sclerotic and fibrous changes in tumor stroma, and in the presence of tumor capsule, caspase-3 activity in papillary carcinoma tissue was higher compared to unchanged thyroid tissue of normofollicular structure. In case of a more aggressive behaviour of tumor, enzyme activity in carcinoma tissue did not differ significantly or (in case of extrathyroid spreading of tumor) was decreased compared to that in extratumoral tissue. In combination, this was expressed by a progressive decrease in caspase-3 activity in tumor tissue with increasing T category. Сaspase 3 activity was found to be much higher in the tissue of papillary carcinomas of follicular-papillary structure and lower in the tissue of tumors of mixed structure with solid areas, compared to that in the tissue of papillary carcinomas of typical papillary structure. CONCLUSIONS: The data obtainеd in assessing caspase-3 activity suggest that the intensity of spontaneous apoptosis of human papillary thyroid carcinoma cells depends upon the stage and aggressiveness of tumoral process.

[Cathepsin-like activity in blood plasma of patients with papillary thyroid carcinoma].

The peculiarities of the thyroid changes depending on clinical characteristics of disease and histological structure of tumor were fixed by the studies of cathepsin-like activity in blood plasma of patients with papillary thyroid carcinoma. H-cathepsin-like activity had a tendence to more significant increase especially in patients with concomitant benign thyroid disease with increasing the tumor category. In a case of metastatic tumor without other concomitant thyroid pathology, the degree of an increase in enzymatic activity was significantly higher comparing to the blood of patients without tumor metastases. B-cathepsin-like activity in blood plasma of patients only with tumor carcinoma was at the level of that of healthy subjects. B-cathepsin-like activity in blood plasma had especially significant increased under tumor T1 category that was connected with nodular goiter or adenoma in a case concominant carcinoma by other thyroid pathology. In patients with carcinoma that was diagnosed against a background of concomitant pathology, the insignificant increase in L-cathepsin-like activity was not dependent on clinical features of disease, while in a case of carcinoma without concomitant pathology a degree of its increase was certainly dependent on a category of tumor T, its histological structure and the presence of oxyphilic cell metaplasia. We failed to get an evidence linking the changes in cathepsin-like activity in blood with the processes of intra- or extrathyroid invasion and invasion into the lymphatic or blood vessels. The results of the studies do not provide a rationale to consider that the cathepsin-like activity in blood of patients with papillary thyroid carcinoma can be the valuable additional biochemical criterion for differential diagnosis, estimation of the severity, and other characteristics of the disease.

[Mechanism of apoptosis in the thyroid cells in thyroid pathology].

The literature data and the results of own examinations concerning the changes in programmed cell death processes of goiter alterated thyroid tissue in patients with euthyroid or toxic goiter and also the extranodular tissue of the gland in the presence of pathological changes in it (the pronounced hyperplastic process, sclerotic and/or dystrophic changes, lymphoid infiltration, chronic or autoimmune thyroiditis) were analyzed. It has been shown that the significant changes in the processes ofmitochondria and postmitochondria regulation and apoptosis realization are occurred under thyroid pathology. The character and the degree of disorders in the apoptosis mitochondria mechanisms and the activity of destructive processes in the patient thyroid cells are dependent on many factors such as the type of pathology, the severity of its occurrence, the peculiarities of tissue follicular structure and the patient sex. Obtained resistance of the apoptosis mitochondria mechanisms can play an important role in the pathogenesis of thyroid nodular pathology or in the development of inflammatory and autoimmune processes.

[Effect of antioxidants on internucleosomal DNA fragmentation in thyroid tissue of patients with various thyroid pathology].

The in vitro effects of a-tocopherol and melatonin on the intensity of DNA internucleosomal fragmentation in tissue and neoplastic tissue of patients with thyroid pathology was studied. alpha-Tocopherol at a concentration of 10(-7) M and melatonin at a concentration of 10(-3) M were shown to inhibit internucleosomal DNA fragmentation in unchanged thyroid tissue, while in pathologically changed tissue and neoplastic tissue the effect of antioxidants was either absent, or those effects of preparations were prevailing, which were aimed at an increase in the intensity of internucleosomal DNA fragmentation. Some peculiarities of changes in the content of certain oligonucleosome fractions depending on the preparation, its dose, and thyroid disorder have been identified. The possible mechanisms of the effect of antioxidants on internucleosomal DNA fragmentation in normal thyroid tissue and in the presence of thyroid pathology are discussed.

[Specific binding of GABA in the adrenal glands and catecholamine blood and adrenal gland levels in rats under stress with inhibited functioning of the hypothalamus-hypophyseal-adrenocortical systems]. / Spetsyfichne zv'iazuvannia GABA u nadnyrkovykh zalozakh i vmist katekholaminiv u krovi ta nadnyrkovykh zalozakh pry stresuvanni shchuriv za umov hal'muvannia hipotalamo-hipofizarno-adrenokortykal'noï systemy.

Forced swimming of intact rats was accompanied by an activation of the hormonal link in sympatho-adrenal system and by a decrease in the level of specific binding of 14C-GABA by adrenal plasma membranes; the former was prevented with the administration of GABAergic preparations (baclofen, GABA-taurine) before stress. Under inhibition of the function of hypothalamo-pituitary-adrenocortical axis following multiple hydrocortisone administration and, in presence of developing metabolic disorders and adrenal secretion of catecholamines, the latter reacted to stress by a decrease in adrenalin, noradrenaline and dopamine secretion without changing the intensity of specific binding of GABA. Administration of GABAergic preparations before stress prevented the development of stress disorders of noradrenaline and dopamine secretion partially or completely, and sodium thiosulphate administration increased acutely specific binding of GABA with adrenal plasma membranes simultaneously preventing the accumulation of adrenal noradrenaline and the decrease in blood DOPA level.