Effect of N-stearoylethanolamine on the DNA fragmentation intensity in tumour and extratumoral tissues of the human adrenal cortex

The effect of different concentrations of N-stearoylethanolamine (NSE 18:0) on fragmentation of DNA in the tumoural and extratumour tissues of the adrenal glands in vitro was studied. In this work the following types of tissue were investigated: extratumoural tissue from patients with hormonally active tumours, benign tumour tissue (hormonally active and hormonally inactive), tissue of malignant tumours and hyperplasic tissue of the adrenal glands (Itsenko-Cushing disease). It has been established that the NSE increases the intensity of DNA fragmentation only in the tissue of hormonally inactive tumours. Benign hormonally active tumours, malignant tumours and hyperplastic tissue of the adrenal glands were resistant to the NSE. The possible mechanisms of resistance to the drug are discussed.     

Vascular endothelial growth factor and basic fibroblast growth factor evaluation in blood serum of patients with hormonally active and inactive adrenal gland tumours

The vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) levels of 36 patients with adrenal gland tumours were analysed. The mean age of patients was 43 years (29-67 years), and there were 25 women (69.4%) and 11 men (31.6%). In 34 patients adrenalectomy was performed and in two cases lesions were considered inoperable. In all cases VEGF and bFGF were measured preoperatively and in all operated patients the level of VEGF was measured at 1 month postoperatively. A statistically significant increase in VEGF levels before surgery in comparison with the controls was recorded in all patients with adrenal tumours. No correlation between the size of a tumour and VEGF levels was observed. The serum level of VEGF decreased in patients after surgical removal of the tumour, no matter which type of tumour, with the exception of a patient showing a recurrence of cortex cancer. A statistically significant decrease was found only in patients operated on for cortex cancers and hormonally active and inactive cortex and medulla inactive benign tumours. The postoperative recurrence of the malignant tumour may be preceded by an increase in plasma VEGF levels. Such correlations were not found with bFGF.     

Potentialities of computed tomography and ultrasound in diagnosis of hormonally active adrenal diseases: results of comparison CT and US with operative adn histological data

The data given in the paper suggest that X-ray computed tomography (CT) is highly effective in detecting all types of hormonally active adrenal abnormalities. CT used in hormonally active adrenal diseases yielded data on major quantitative and qualitative (primarily densitometric) criteria that could be used in assessing the images of the adrenal area in these patients. Ultrasound study (USS) made at the first stage of topical diagnostic searches was of informative value in detecting adrenal tumor lesions, the technique being highly sensitive in the diagnosis of adrenal pheochromocytomas and adenocarcinomas, but less informative in the detection of hormonally active adrenocortical adenomas (aldesterone-producing ones in particular) than CT. The diagnosis of various adrenocortical hyperplasies and the differentiation of hyperplastic and tumor forms of hypercorticoidism are a prerogative of CT that substantially supplements USS findings in such cases.     

Role of magnetic resonance imaging in the complex radiation diagnosis of hormonally active adrenal tumors

Based on the results of examination of 30 patients with hormonally active adrenal tumors, the authors consider the MRI sympatomatology of their different types and the potentialities of the technique in the complex radiation diagnosis of this pathology. The authors present their assessments of the relative intensity of a signal and the structure of each type of hormone-producing tumors of the glands by using different MRI pulse sequences that may be useful in establishing a presumptive morphological diagnosis. They identify MRI sequences that are of the greatest informative value for the diagnosis of each type of hormonally active adrenal tumors. There is evidence for that MRI is highly effective in detecting all types of hormonally active adrenal tumors and, in the context of their topographic and anatomic diagnosis, has an unquestionable advantage over ultrasonography and X-ray computed tomography in some cases.     

Effect of o,p'-DDD and Li+ on apoptotic DNA fragmentation in conventionally normal and tumour tissues of human adrenal cortex

The actions of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane (o,p'-DDD), potassium and lithium ions upon apoptotic processes in conventionally normal and tumour tissues of human adrenal cortex were studied. There was no effect of K+ on the apoptosis in tumour tissue. o,p'-DDD--the specific drug for conservative therapy of adrenocortical cancer--enhanced the apoptotic DNA fragmentation in all tested tissues. The conclusion was made that apoptosis may be involved in curative effect of o,p'-DDD in adrenal cortex. Lithium ions, which are used in clinic as antidepressant, inhibited the apoptosis in conventionally normal tissue and in most tumours. On the other hand, lithium enhanced the DNA fragmentation in the postoperative tissue of patients with Cushing disease. The possible mechanisms mediating lithium effects on the adrenal cortex are discussed.     

Adenylate cyclase activity and cyclic nucleotide content in human adrenal tumors under Icenko-Cushing syndrome

The adenylate cyclase activity and cyclic nucleotide content in excised human adrenal tumours (Icenko-Cushing syndrome) were determined. The experimental data were compared to those obtained for hyperplastic adrenals. All adrenal tumours under study revealed a decreased cAMP level, an increased cGMP level and a resulting decrease of the cAMP/cGMP ratio. In malignant adrenal tumours the adenylate cyclase activity was sharply increased in comparison with that in hyperplastic adrenals. In the majority of malignant tumours the adenylate cyclase response to ACTH was either altogether absent or sharply decreased. In benign adrenal tumours the basal activity of the enzyme was unchanged and the enzyme response to ACTH was essentially normal. The decrease of adenylate cyclase response to ACTH in malignant tumours is apparently not due to the impaired catalytic activity of the enzyme, since its response to stimulation by sodium fluoride remains unaffected. In some tumours (one malignant and two benign ones) a non-specific stimulation of adenylate cyclase by hormones, which are not natural activators of the enzyme was observed. It was assumed that these changes are due to the damage of hormonal receptors in adrenal tumours.     

Effect of adrenocorticotropic hormone on the caspase-3 level and DNA fragmentation in hyperplasia tissue of human adrenals

The effect of corticotropin and inhibitor of protein kinase C, chelerythrine chloride, on the change of caspase-3 level and on the rate of DNA laddering in hyperplasia adrenal cortex tissue of patient with Cushing's syndrome was studied. It was established that ACTH caused significant antiapoptotic effect in the human adrenal cortex. The adding of ACTH to incubation medium caused a sharp decrease of the caspase-3 level in adrenocorticocytes. Chelerythrine chloride, on the contrary, increases the caspase-3 level by 22%. ACTH influence decreases DNA laddering. Either the adding of chelerythrine chloride to incubation medium, or the adding of chelerythrine chloride simultaneously with ACTH, led to the enhancing of DNA fragmentation. The obtained data suggest that antiapoptotic effect of ACTH in adrenal cortex tissue estimated according by the caspase-3 level and by the rate of DNA fragmentation depends on activation of protein kinase C. However, the intensification of DNA laddering can be also explained by cytotoxic effect, high level of interaction with DNA and strong intercalation ability of this alkaloid.     

MEN I gene mutations in sporadic adrenal adenomas

Loss of heterozygosity (LOH) on chromosome 11q13 occurs in about 20% of sporadic adrenal neoplasms. Adrenal lesions, mostly benign, occur in up to 40% of patients from MEN I kindreds. The MEN I gene, positioned on 11q13, has been considered a primary candidate gene in these lesions. We studied a group of 15 patients with sporadic adrenal adenoma, and 1 patient with multinodular hyperplasia. Of the 16 patients, 4 had incidentally discovered masses, 5 had Conn's syndrome, 6 suffered from Cushing's syndrome, and 9 had high sex hormone production. Studies with the markers D11S480, PYGM, D11S449, and D11S987 in 13 patients (12 of whom were from our group of 16) revealed 4 losses of heterozygosity on D11 S480 on 11q13, but the deletion did not affect the MEN I gene in any case. We present complete direct DNA sequencing data of the menin gene in 14 sporadic adrenal adenomas and one with adrenal hyperplasia. We identified one heterozygous missense mutation, T552S, in a hormonally inactive adrenal adenoma. One base exchange was identified close to the intron-exon boundary in intron 9 of a nodular adrenal hyperplasia. mRNA expression studies found that MEN I was transcribed in all 13 samples analyzed. In summary, our study identified the second patient with sporadic benign adrenal tumor presenting a menin gene mutation. Our complete direct sequencing approach adds evidence that menin gene mutations may account only for a minority of benign adrenal tumors if at all. Another tumor-suppressor gene inactivated in sporadic adrenal neoplasms may be located on chromosome 11q13.     

Inhibin immunoreactivity in gonadal and non-gonadal tumors

Inhibin immunoreactivity was estimated in a number of gonadal and non-gonadal tumors. Dog Sertoli cell tumors and human granulosa cell and Leydig cell tumors contained high concentrations of inhibin-like material. Levels, comparable with those in normal testes and ovaries were detected in human testicular non-seminomas and in ovarian cystadenomas, thecomas and adenofibromas. No activity was found in human testicular Sertoli/Leydig cell tumors and seminomas and in ovarian adenocarcinomas, teratomas and a dysgerminoma. Furthermore, human adrenal cortical tissue (tumor and hyperplastic adrenal) contained inhibin immunoreactivity. No activity was found in human tumors of the stomach, gut, liver, kidney, pancreas and mammary gland or in meningiomas. It is concluded that inhibin is not a good marker for specific gonadal tumors. Inhibin might have intratumor actions a growth or differentiation factor.     

Changes in rat adrenal cyclic nucleotides during normal and neoplastic growth

In an attempt to correlate changes in cyclic nucleotide levels with in vivo growth of the rat adrenal gland we have measured adrenal cyclic AMP and cyclic GMP in normal, hyperplastic, and neoplastic rat adrenals. The first group of animals were subject to either unilateral adrenalectomy (ADX) or acute hypophysectomy 1 h prior to unilateral adrenalectomy (HADX). Cyclic nucleotides were measured in the contralateral adrenal post-operatively. In HADX rats cyclic GMP rose steadily throughout the 7 day study period, while ADX rats exhibited significant decreases in adrenal cyclic GMP. Cyclic AMP remained approximately 1.5 pm/mg tissue in HADX rats, while in ADX rats there was significant elevation of adrenal cyclic AMP at all time points. Cyclic GMP/cyclic AMP ratios remained constant in HADX animals; however, the growing adrenals of ADX animals exhibited depressed cyclic GMP/cyclic AMP ratios at all time periods.Adrenal hyperplasia was induced in a seond group of animals by a transplantable, corticotropin-secreting, pituitary tumor. Adrenals from age-matched animals served as controls. Adrenal cyclic AMP was significantly elevated in tumor-bearers at a time correspinding to the peak accumulation of adrenal weight, protein and DNA in these animals. In contrast, adrenal cyclic GMP in both tumor-beares and control animals fell steadily throughout the study period. Cyclic GMP/cyclic AMP ratios of control animals decreased from 2 to 3 weeks post-transplant remaining at the 3 week value during the period corresponding to rapid adrenal growth in tumor-bearers. The cyclic GMP/cyclic AMP ratio in the hyperplastic adrenals of tumor-bearers decreased steadily throughout their rapid growth period, suggesting a positive correlation between adrenal growth and depression of the cyclic GMP/cyclic AMP ratio.Cyclic nucleotide levels in neoplastic adrenals of rats bearing the transplantable adrenocortical carcinoma 494 were compared with cyclic nucleotides in normal rat adrenal glands. Cyclic AMP was not different in the two groups. However, the cyclic GMP content of neoplastic adrenals was significantly lower than that of normal adrenal tissue, causing a suppression of the cyclic GMP/cyclic AMP ratio in the neoplastic tissue. Thus, measurement of adrenal cyclic nucleotides in both hyperplastic and neoplastic rat adrenal glands suggests that adrenal growth in vivo may be characterized by a depression of the cyclic GMP/cyclic AMP ratio.     

The role of progesterone metabolism and androgen synthesis in renal blood pressure regulation.

11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) plays a crucial role in converting hormonally active cortisol into inactive cortisone, conferring specificity onto the human mineralocorticoid receptor (MR). Progesterone binds with even higher affinity to the MR, but acts as an MR antagonist. How aldosterone is able to keep its function as predominant MR ligand in clinical situations with high progesterone concentrations, such as pregnancy, is not clear. We have shown in vitro that the human kidney possesses an effective enzyme system that metabolizes progesterone to inactive metabolites in a process similar to the inactivation of cortisol by 11beta-HSD2. In studies on patients with adrenal insufficiency, we have shown that the in vivo anti-mineralocorticoid activity of progesterone is diminished by inactivating metabolism of progesterone, local formation of the deoxycorticosterone mineralocorticoid from progesterone, and inhibition of 11beta-HSD2 by progesterone and its metabolites resulting in decreased inactivation of cortisol and hence increased MR binding by cortisol. The enzymes involved in progesterone metabolism are also responsible for the capability of the human kidney to convert pregnenolone to DHEA and androstenedione leading to the formation of active androgens, testosterone and 5alpha-DH-testosterone. Locally produced androgens might be responsible for the observed difference in blood pressure between men and women and higher susceptibility to hypertension in men.     

Complete sequencing and mRNA expression analysis of the MEN-I gene in adrenal myelolipoma.

The molecular pathogenesis of adrenal myelolipoma is unclear. Endocrine activity of these tumors and association with other endocrine tumors have stimulated the hypothesis that it may belong to the group of sporadic tumors caused by defects of the gene responsible for multiple endocrine neoplasia type I (MEN-I). DNA of blood and tumoral sections from two patients with adrenal myelolipoma were analyzed by examination of variable number of tandem repeats (VNTR) loci PYGM, D11S987, D11S480, and D11S449 on chromosome 11q13 and by complete direct DNA sequencing of all coding exons and splice junctions of the MEN-I gene. Menin expression was examined by RT-PCR. RT-PCR did not detect menin expression in one adrenal myelolipoma. No loss of heterozygozity on chromosome 11q13 was identified. Intragenic heterozygozity was retained in codon 418 of the menin gene in both patients. No mutation was identified in the coding exons of the menin gene. Complete DNA sequencing yielded no hint that defects of the MEN-I gene are responsible for the formation of adrenal myelolipomas. Adrenal myelolipomas do not share the loss of heterozygozity on chromosome 11q13 observed in some benign adenomatous and many malignant adrenocortical tumors.     

Metabolism of androgens in human hyperplastic prostate: evidence for a differential localization of the enzymes involved in the metabolism

The subcellular distribution of 5 alpha-reductase, 17 beta-hydroxy steroid dehydrogenase, 3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities was studied in human hyperplastic prostate. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities are located in the nuclear envelope. 3 alpha-hydroxysteroid dehydrogenase activity was almost equally distributed between cytosol and membranes, 3 beta-hydroxysteroid dehydrogenase activity was linked to all membranes. Direct testosterone metabolism (transformation into its active metabolite 5 alpha-DHT and into androstenedione, an inactive androgen) takes place only in the nucleus whereas indirect metabolism takes place mainly in the cytoplasm. These findings add new evidence for the mechanism of action of testosterone in prostatic tissue. Testosterone diffuses into the cell, migrates toward the nucleus and is transformed at the nuclear envelope level into two metabolites, DHT and androstenedione. After transformation into its active form, the hormone enters the nucleus whereas the inactive form is released into the cytoplasm. This metabolism could be seen as a control of the amount of active hormone entering the nucleus and being able to bind the androgen receptor.     

Adrenal Cortical Ribonuclease H (hybrid) (38494).

Ribonuclease Hybrid (RNaseH) from adrenal cortical tissue has been characterized. RNase H specifically degrades the RNA strand of purified RNA:DNA hybrids but is inactive on single- or double-stranded RNA or on DNA. The mode of clevage by RNase H is endonucleolytic, producing oligoribonucleoties with 3'-hydroxyl and 5'- phosphate termini. ACTH administration to guinea pigs results in no detectable change in adrenal cortical RNase H activity at times when changes in DNA synthesis are marked.     

Assessment of pro- and antiangiogenic factors blood serum concentrations in patients with hormonal inactive adrenal tumors

INTRODUCTION: The growth and persistence of solid tumors and their metastases is connected with angiogenesis. This process is determined by activity of pro- and antyangiogenic factors. VEGF is the one of the most important factors having a stimulant effect on angiogenesis. Soluble forms of VEGF receptors are inhibitors of angiogenesis. The soluble forms of VEGF receptors containing extra cellular part of receptor, which binds ligand, seem to be real inhibitors of VEGF. THE AIM OF THE STUDY: Evaluation the value of serum VEGF and soluble forms of VEGF receptors concentration as a marker of malignancy in patients with hormonal inactive adrenal tumors. MATERIAL AND METHODS: Twenty seven patients (18 female, 9 male; mean age 48+/-4.3 years) with adrenocortical carcinoma (N=8), adrenal metastases (N=4) and adrenocortical adenoma (N=15) were included in this study. Age- and gender-matched control samples were acquired from healthy volunteers (N=10). Serum VEGF and sVEGFR levels were determinated by means of ELISA assay. Statistical analysis was performed using the Student-t test, the Pearson's test and the series test. RESULTS: In healthy controls mean VEGF level was 197.2 pg/ml, sVEGFR-1 43.5 pg/ml and sVEGFR-2 8976.3 pg/ml. Patients with adrenocortical carcinoma had the levels of VEGF (1263.8 pg/ml) significantly higher and of sVEGFR-2 (5893.7 pg/ml) significantly lower in comparison to control group (p<0.05). On the other hand the mean VEGF (334.2 pg/ml) concentration in patients with benign adrenocortical adenoma wasn't significant different than in control group (p>0.05) but mean sVEGFR-1 (21.7 pg/ml) and sVEGFR-2 (7106.4 pg/ml) concentrations were significantly lower than in the control (p<0.05). In metastases group mean VEGF (485.9 pg/ml) level was higher and sVEGFR-2 (5455.2 pg/ml) was lower than in control group (p<0.05). CONCLUSION: These data suggest that determination of VEGF and sVEGFR concentration in the serum of patients with hormonal inactive adrenal tumors may be applied as an additional marker of malignancy.     

The endogenous concentration of estradiol and estrone in pathological human postmenopausal endometrium

The endogenous estrone (E1) and estradiol (E2) levels (in pg/g tissue) were measured in 7 postmenopausal patients with a hyperplastic endometrium, in 3 with an atypical adenomatous hyperplastic endometrium and in 13 with a carcinomatous endometrium. These tissue concentrations were compared with the E1 and E2 concentrations in plasma (pg/ml) and with data from a control group of postmenopausal women with atrophic endometria. The tissue levels of both steroids showed large variations and there were no significant correlations with their plasma levels. In the hyperplastic and the atypical adenomatous hyperplastic group the mean E2 tissue level was higher compared with the mean E1 tissue level, despite the excess of E1 over E2 in peripheral plasma. In the carcinomatous group the mean E1 tissue level was higher, although not significantly, than the mean E2 tissue level. There were no significant differences between the E1 and E2 tissue levels in the three different pathological groups as compared to the atrophic control group. We conclude that it is unlikely that estrogens alone play a crucial role in the development of a pathological endometrium.     

Diagnostic difficulties in adrenal incidentaloma--analysis of 125 cases

INTRODUCTION: Therapeutic approach to incidentaloma, in spite of existing algorithms, is not always obvious due to diagnostic difficulties. The aim of the study was to assess the validity of the initial diagnoses of incidentaloma which determined the qualification for the operation. MATERIAL AND METHODS: 125 patients hospitalised in the Endocrinology Dept. of the Medical University of Bialystok in the years 2003-2005 and in the Endocrinology dept. of Voivodeship Hospital of Bialystok. The patients were clinically and hormonally examined (metanephrines in daily urine collection, daily cortisol rhythm, short dexamethasone test, aldosterone, and renin plasma activity, Na, K levels in the serum) as well as computer tomography of the adrenal glands were performed. RESULTS: 42 patients were qualified for adrenalectomy. Adenoma was confirmed in 25 patients (in 7 subclinical Cushing syndrome was diagnosed, in 2 Conn disease, in 16 inactive changes), phaeochromocytoma in 6 patients, cysts in 3, lipoma in 2, carcinoma in one, in 4 patients metastases (in 2 of kidney carcinoma, in 1 of malignant melanoma and in 1 of planocellular carcinoma) and in one oncocytoma. Metanephrines urine measurements showed 33% of false positive results. CONCLUSIONS: Qualification for adrenalectomy requires an assessment of tumor's enlargement, its tissue density, morphology and growth dynamics. To reduce the percentage of false positive results of metanephrine measurement there is to eliminate an influence of some drugs, victuals, beverages and nicotine and eventually to carry out additional tests. The decision as to proceeding with adrenal incidentaloma should be individualized based on clinical symptoms, hormonal tests and tumor morphology.     

Detection of inactive or less active forms of tyrosine hydroxylase in human adrenals by a sandwich enzyme immunoassay

A sensitive sandwich enzyme immunoassay for tyrosine hydroxylase (TH) from bovine and human adrenals has been developed. Anti-TH antibody was prepared from bovine adrenal TH. The assay system consisted of an antibody F(ab')2 immobilized on polystyrene beads as a solid phase and of beta-D-galactosidase-conjugated antibody. This method was highly sensitive and specific for the assay of TH. Human adrenal TH level was determined by similar sensitivity as bovine adrenal TH, suggesting the presence of common antigenic sites between human and bovine adrenal enzymes. The presence of inactive or less active forms of TH in human adrenals was revealed by purification of the enzyme and monitoring with this enzyme immunoassay as well as with enzyme activity assay.     

Testicular adrenal rest tumours in salt wasting congenital adrenal hyperplasia (in vivo and in vitro studies)

We describe the case of a 20-year-old patient with salt-wasting congenital adrenal hyperplasia (CAH) related to 21-hydroxylase deficiency. Bilateral craggy testicular tumours were found, requiring histological evaluation. Prior to the surgical procedure, the patient was treated with dexamethasone (he presented cortisol deficiency) and was stimulated with ACTH. High levels of 11beta-OH steroids measured in the gonadal vein, compared with peripheral blood samples suggested the presence of adrenal rests. Incubation of the tumours (which could not be differentiated histologically, from Leydig tissue), with radioactive steroid precursors was carried out. The results revealed the testicular tumours were of adrenal tissue origin, associated with 21-hydroxylase deficiency. The patient's non-compliance to glucocorticoid treatment was the main cause of his hypogonadotropic hypogonadism.     

Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells.

Removal of ultraviolet light induced cyclobutane pyrimidine dimers (CPD) from active and inactive genes was analyzed in cells derived from patients suffering from the hereditary disease Cockayne's syndrome (CS) using strand specific probes. The results indicate that the defect in CS cells affects two levels of repair of lesions in active genes. Firstly, CS cells are deficient in selective repair of the transcribed strand of active genes. In these cells the rate and efficiency of repair of CPD are equal for the transcribed and the nontranscribed strand of the active ADA and DHFR genes. In normal cells on the other hand, the transcribed strand of these genes is repaired faster than the nontranscribed strand. However, the nontranscribed strand is still repaired more efficiently than the inactive 754 gene and the gene coding for coagulation factor IX. Secondly, the repair level of active genes in CS cells exceeds that of inactive loci but is slower than the nontranscribed strand of active genes in normal cells. Our results support the model that CS cells lack a factor which is involved in targeting repair enzymes specifically towards DNA damage located in (potentially) active DNA.