Adrenocortical tumors in children

Childhood adrenocortical tumors (ACT) are rare. In the USA, only about 25 new cases occur each year. In Southern Brazil, however, approximately 10 times that many cases are diagnosed each year. Most cases occur in the contiguous states of São Paulo and Paraná. The cause of this higher rate has not been identified. Familial genetic predisposition to cancer (p53 mutations) and selected genetic syndromes (Beckwith-Wiedemann syndrome) have been associated with childhood ACT in general but not with the Brazilian counterpart. Most of the affected children are young girls with classic endocrine syndromes (virilizing and/or Cushing). Levels of urinary 17-ketosteroids and plasma dehydroepiandrosterone sulfate (DHEA-S), which are abnormal in approximately 90% of the cases, provide the pivotal clue to a diagnosis of ACT. Typical imaging findings of pediatric ACT consist of a large, well-defined suprarenal tumor containing calcifications with a thin capsule and central necrosis or hemorrhage. The pathologic classification of pediatric ACT is troublesome. Even an experienced pathologist can find it difficult to differentiate carcinoma from adenoma. Surgery is the single most important procedure in the successful treatment of ACT. The role of chemotherapy in the management of childhood ACT has not been established although occasional tumors are responsive to mitotane or cisplatin-containing regimens. Because of the heterogeneity and rarity of the disease, prognostic factors have been difficult to establish in pediatric ACT. Patients with incomplete tumor resection or with metastatic disease at diagnosis have a dismal prognosis. In patients with localized and completely resected tumors, the size of the tumor has predictive value. Patients with large tumors have a much higher relapse rate than those with small tumors.

The expression of the ACTH receptor

Adrenal glucocorticoid secretion is regulated by adrenocorticotropic hormone (ACTH) acting through a specific cell membrane receptor (ACTH-R). The ACTH-R is a member of the G protein superfamily-coupled receptors and belongs to the subfamily of melanocortin receptors. The ACTH-R is mainly expressed in the adrenocortical cells showing a restricted tissue specificity, although ACTH is recognized by the other four melanocortin receptors. The cloning of the ACTH-R was followed by the study of this gene in human diseases such as familial glucocorticoid deficiency (FGD) and adrenocortical tumors. FGD is a rare autosomal recessive disease characterized by glucocorticoid deficiency, elevated plasma ACTH levels and preserved renin/aldosterone secretion. This disorder has been ascribed to an impaired adrenal responsiveness to ACTH due to a defective ACTH-R, a defect in intracellular signal transduction or an abnormality in adrenal cortical development. Mutations of the ACTH-R have been described in patients with FGD in segregation with the disease. The functional characterization of these mutations has been prevented by difficulties in expressing human ACTH-R in cells that lack endogenous melanocortin receptor activity. To overcome these difficulties we used Y6 cells, a mutant variant of the Y1 cell line, which possesses a non-expressed ACTH-R gene allowing the functional study without any background activity. Our results demonstrated that the several mutations of the ACTH-R found in FGD result in an impaired cAMP response or loss of sensitivity to ACTH stimulation. An ACTH-binding study showed an impairment of ligand binding with loss of the high affinity site in most of the mutations studied.

Role of ACTH receptor in adrenocortical tumor formation

Adrenocorticotrophin (ACTH) is the major regulatory hormone of steroid synthesis and secretion by adrenocortical cells. The actions of ACTH are mediated by its specific membrane receptor (ACTH-R). The human ACTH-R gene was recently cloned, allowing systematic determination of its sequence, expression and function in adrenal tumorigenesis. The presence of oncogenic mutations of the ACTH-R gene in adrenocortical tumors has been reported. Direct sequencing of the entire coding region of the ACTH-R gene of sporadic adrenocortical adenomas and carcinomas did not reveal constitutive activating mutations, indicating that this mechanism is not frequent in human adrenocortical tumorigenesis. Recent studies demonstrated allelic loss of the ACTH-R gene in a subset of sporadic adrenocortical tumors using a PstI polymorphism located in the promoter region of the ACTH-R gene. Loss of heterozygosity of the ACTH-R was analyzed in 20 informative patients with a variety of benign and malignant adrenocortical tumors. Three of them showed loss of heterozygosity of the ACTH-R gene. In addition, Northern blot experiments demonstrated reduced expression of ACTH-R mRNA in these three tumors with loss of heterozygosity, suggesting the functional significance of this finding at the transcriptional level. Deletion of the ACTH-R gene seems to be involved in a subset of human adrenocortical tumors, contributing to cellular dedifferentiation.

Lack of mutations of exon 2 of the MEN1 gene in endocrine and nonendocrine sporadic tumors

In addition to the mutations that underlie most cases of the multiple endocrine neoplasia type 1 (MEN1) syndrome, somatic mutations of the MEN1 gene have also been described in sporadic tumors like gastrinomas, insulinomas and bronchial carcinoid neoplasm. We examined exon 2 of this gene, where most of the mutations have been described, in 148 endocrine and nonendocrine sporadic tumors. DNA was obtained by phenol/chloroform extraction and ethanol precipitation from 92 formalin-fixed, paraffin-embedded samples, and from 40 fresh tumor tissue samples. We used 5 pairs of primers to encompass the complete coding sequence of exon 2 of the MEN1 gene that was screened by the polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) technique in 78 sporadic thyroid cancers: 28 follicular adenomas, 35 papillary carcinomas, 14 follicular carcinomas, and 1 anaplastic thyroid carcinoma. We also examined 46 adrenal lesions (3 hyperplasias, 3 adenomas and 35 adrenocortical carcinomas, 2 pheochromocytomas, 2 ganglioneuroblastomas, and 1 lymphoma) and 24 breast cancers (6 noninvasive, 16 infiltrating ductal, and 2 invasive lobular tumors). The PCR product of 5 tumors suspected to present band shifts by SSCP was cloned. Direct sense and antisense sequencing did not identify mutations. These results suggest that the MEN1 gene is not important in breast, thyroid or adrenal sporadic tumorigenesis. Because the frequency of mutations varies significantly among tumor subgroups and allelic deletions are frequently observed at 11q13 in thyroid and adrenal cancers, another tumor suppressor gene residing in this region is likely to be involved in the tumorigenesis of these neoplasms.

Parathyroid hormone secretion in chronic human endogenous hypercortisolism

Osteoporosis is a common manifestation of Cushing's syndrome, but the mechanisms responsible for this abnormality have not been defined. With the objective of analyzing parathyroid hormone (PTH) secretion in chronic hypercortisolism (CH), we evaluated 11 healthy subjects and 8 patients with CH, 6 with Cushing's disease and 2 with adrenal adenoma. These volunteers were submitted to tests of PTH stimulation through hypocalcemia (EDTA), PTH suppression through hypercalcemia (iv and oral calcium), and evaluation of bone mineral density (BMD) by DEXA. During the test of PTH stimulation, the calcium and magnesium concentrations of the normal and CH groups were similar. Patients with CH showed an increased PTH response to the hypocalcemic stimulus compared to controls. PTH values were significantly higher in the CH group at 70 (17.5 ± 3.5 vs 10.2 ± 1.3 pmol/l, P = 0.04), and 120 min (26.1 ± 5.9 vs 11.3 ± 1.9 pmol/l, P = 0.008) of EDTA infusion. The area under the curve for PTH during EDTA infusion was also significantly higher in patients with CH than in normal subjects (1867 ± 453 and 805 ± 148 pmol l-1 2 h-1, P = 0.02). During the test of PTH suppression, calcium, magnesium and PTH levels of the patients with hypercortisolism and controls were similar. BMD was decreased in patients with hypercortisolism in the spine (0.977 ± 0.052 vs 1.205 ± 0.038 g/cm² in controls, P<0.01). In conclusion, our results show that subjects with CH present decreased bone mass mainly in trabecular bone. The use of dynamic tests permitted the detection of increased PTH secretion in response to a hypocalcemic stimulus in CH patients that may probably be involved in the occurrence of osteoporosis in this state.

p53 as a prognostic factor in adrenocortical tumors of adults and children

Mutations of the tumor suppressor gene p53 have been considered to be important determinants in several kinds of human cancer. Accumulation of p53 protein has been reported to correlate with more aggressive clinical behavior in some neoplasms. The role of p53 expression in adrenal cortical tumors (ACT) has not been elucidated but some studies have suggested its correlation with malignant behavior. Our objective was to determine if there is a correlation between the expression of immunoreactive p53 and the biological behavior of ACT. Fifty-seven ACT (21 from children and 36 from adults) were evaluated for p53 expression by immunohistochemistry in formalin-fixed paraffin-embedded tissue and analyzed in terms of outcome. The p53 parameter was utilized semiquantitatively. Tumors were classified as p53 negative when no positivity was observed, or when only few cells showed weak positivity (0/1+) and scored as p53 positive when there was a diffuse and strong nuclear positivity (2+/3+). In children, p53 positivity was associated with clinically malignant ACT and p53 negativity was associated with clinically benign ACT (P = 0.026). In adults' ACT, p53 positivity had an effect on disease-free survival (P<0.001) and also correlated with Weiss score, with a cutoff = 4 (P = 0.04). p53 expression was related to the clinical behavior of ACT in both children and adults and these findings seem to support a role for p53 in ACT progression.

Role of endogenous glucocorticoids in hyperalgesia and edema in old arthritic rats

We compared the intensity and frequency of arthritis in old (8-12 months, N = 12) and juvenile (2 months, N = 10) rats and determined the role played by adrenal glands in this disorder. Arthritis was induced by subcutaneous injection of Mycobacterium butyricum at the base of the tail of female Holtzman rats at day zero. Paw edema and hyperalgesia were monitored from day zero to day 21 after induction as signs of arthritis development. Some (N = 11) old animals were adrenalectomized bilaterally and treated with dexamethasone or celecoxib immediately following surgery. All bilaterally adrenalectomized old animals became susceptible to arthritis and the onset of disease was shortened from the 10th to the 5th day. Hyperalgesia and paw edema responses were less frequent in older animals (50 and 25% compared to control juvenile rats, respectively), although old responder animals showed responses of similar intensity to those of their juvenile counterparts: by the 14th day the data for hyperalgesia were juvenile = 0.8 ± 0.07/old = 0.8 ± 0.09, and for paw edema juvenile = 56.6 ± 6.04/old = 32.24 ± 12.7, reported as delta% increase in paw edema. Chronic treatment of adrenalectomized old animals with dexamethasone (0.01 or 0.1 mg/kg) but not celecoxib (3 mg/kg), once daily for 21 days by gavage, abolished the effects of adrenalectomy, in particular those related to the hyperalgesia response (old = 0.95 ± 0.03/dexamethasone = 0 ± 0; 14th day), thus suggesting a specific participation of circulating corticosteroids in the modulation of pain in old arthritic rats.

Mutations in the SRY, DAX1, SF1 and WNT4 genes in Brazilian sex-reversed patients

In most mammals, male development is triggered by the transient expression of the SRY gene, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Mutation studies have identified several genes essential for early gonadal development. We report here a molecular study of the SRY, DAX1, SF1 and WNT4 genes, mainly involved in sexual determination, in Brazilian 46,XX and 46,XY sex-reversed patients. The group of 46,XX sex-reversed patients consisted of thirteen 46,XX true hermaphrodites and four 46,XX males, and was examined for the presence of the SRY gene and for the loss of function (inactivating mutations and deletions) of DAX1 and WNT4 genes. In the second group consisting of thirty-three 46,XY sex-reversed patients we investigated the presence of inactivating mutations in the SRY and SF1 genes as well as the overexpression (duplication) of the DAX1 and WNT4 genes. The SRY gene was present in two 46,XX male patients and in none of the true hermaphrodites. Only one mutation, located outside homeobox domain of the 5' region of the HMG box of SRY (S18N), was identified in a patient with 46,XY sex reversal. A novel 8-bp microdeletion of the SF1 gene was identified in a 46,XY sex-reversed patient without adrenal insufficiency. The dosage of DAX1 and WNT4 was normal in the sex-reversed patients studied. We conclude that these genes are rarely involved in the etiology of male gonadal development in sex-reversed patients, a fact suggesting the presence of other genes in the sex determination cascade.

Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20ºC until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37ºC for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 ± 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350% above baseline). On one occasion, we did not detect any variation. Mean (± SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 ± 26.7, after: 213.7 ± 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.

Effects of stress on catecholamine stores in central and peripheral tissues of long-term socially isolated rats

Both the peripheral sympatho-adrenomedullary and central catecholaminergic systems are activated by various psycho-social and physical stressors. Catecholamine stores in the hypothalamus, hippocampus, adrenal glands, and heart auricles of long-term socially isolated (21 days) and control 3-month-old male Wistar rats, as well as their response to immobilization of all 4 limbs and head fixed for 2 h and cold stress (4ºC, 2 h), were studied. A simultaneous single isotope radioenzymatic assay based on the conversion of catecholamines to the corresponding O-methylated derivatives by catechol-O-methyl-transferase in the presence of S-adenosyl-l-(³H-methyl)-methionine was used. The O-methylated derivatives were oxidized to ³H-vanilline and the radioactivity measured. Social isolation produced depletion of hypothalamic norepinephrine (about 18%) and hippocampal dopamine (about 20%) stores and no changes in peripheral tissues. Immobilization decreased catecholamine stores (approximately 39%) in central and peripheral tissues of control animals. However, in socially isolated rats, these reductions were observed only in the hippocampus and peripheral tissues. Cold did not affect hypothalamic catecholamine stores but reduced hippocampal dopamine (about 20%) as well as norepinephrine stores in peripheral tissues both in control and socially isolated rats, while epinephrine levels were unchanged. Thus, immobilization was more efficient in reducing catecholamine stores in control and chronically isolated rats compared to cold stress. The differences in rearing conditions appear to influence the response of adult animals to additional stress. In addition, the influence of previous exposure to a stressor on catecholaminergic activity in the brainstem depends on both the particular catecholaminergic area studied and the properties of additional acute stress. Therefore, the sensitivity of the catecholaminergic system to habituation appears to be tissue-specific.

Cortisol and 17-a-hydroxy-progesterone levels in infants with refractory hypotension born at 30 weeks of gestation or less

Refractory hypotension is frequent in very low-birth weight infants, whose hypothalamic-pituitary-adrenal axis has been suggested to be immature. The objective of the present study was to evaluate basal cortisol and 17-a-OH-progesterone in the first 36 h of life in preterm infants with and without refractory hypotension (mean arterial blood pressure below the lower limit for gestational age throughout the study despite aggressive volume expansion and use of vasopressors). Thirty-five infants with £30 weeks of gestation and a birth weight £1250 g, with no postnatal use of corticosteroid or death in the first 48 h were studied. Mean arterial pressure was measured every 4 h during the first 48 h. Cortisol and 17-a-OH-progesterone were determined at 12 and 36 h and patients were divided into refractory hypotensive (N = 15) and control (N = 20) groups. The groups were not different regarding type of delivery, use of prenatal corticosteroid, requirement of mechanical ventilation, use of vasopressor drugs, morphine, fentanyl, prophylactic indomethacin, and mean sample timing. Although refractory hypotensive newborns were more immature, were smaller, suffered more deaths after 48 h of life and had a higher SNAPPE-2 score, their cortisol and 17-a-OH-progesterone levels were not different from controls at 12 h and at 36 h. The increase of cortisol in newborns with refractory hypotension 36 h after birth was significantly higher than in controls. Despite the fact that refractory hypotensive very low-birth weight neonates were submitted to a very stressful condition, their cortisol and 17-a-OH-progesterone levels were similar to controls.

The inhibitory role of sympathetic nervous system in the Ca2+-dependent proteolysis of skeletal muscle

Mammalian cells contain several proteolytic systems to carry out the degradative processes and complex regulatory mechanisms to prevent excessive protein breakdown. Among these systems, the Ca2+-activated proteolytic system involves the cysteine proteases denoted calpains, and their inhibitor, calpastatin. Despite the rapid progress in molecular research on calpains and calpastatin, the physiological role and regulatory mechanisms of these proteins remain obscure. Interest in the adrenergic effect on Ca2+-dependent proteolysis has been stimulated by the finding that the administration of β2-agonists induces muscle hypertrophy and prevents the loss of muscle mass in a variety of pathologic conditions in which calpains are activated. This review summarizes evidence indicating that the sympathetic nervous system produces anabolic, protein-sparing effects on skeletal muscle protein metabolism. Studies are reviewed, which indicate that epinephrine secreted by the adrenal medulla and norepinephrine released from adrenergic terminals have inhibitory effects on Ca2+-dependent protein degradation, mainly in oxidative muscles, by increasing calpastatin levels. Evidence is also presented that this antiproteolytic effect, which occurs under both basal conditions and in stress situations, seems to be mediated by β2- and β3-adrenoceptors and cAMP-dependent pathways. The understanding of the precise mechanisms by which catecholamines promote muscle anabolic effects may have therapeutic value for the treatment of muscle-wasting conditions and may enhance muscle growth in farm species for economic and nutritional purposes.

Central actions of glucocorticoids in the control of body fluid homeostasis: Review

The involvement of the hypothalamic-pituitary-adrenal axis in the control of body fluid homeostasis has been extensively investigated in the past few years. In the present study, we reviewed the recent results obtained using different approaches to investigate the effects of glucocorticoids on the mechanisms of oxytocin and vasopressin synthesis and secretion in response to acute and chronic plasma volume and osmolality changes. The data presented here suggest that glucocorticoids are not only involved in the mechanisms underlying the fast release but also in the transcriptional events that lead to decreased synthesis and secretion of these neuropeptides, particularly oxytocin, under diverse experimental conditions of altered fluid volume and tonicity. The endocannabinoid system, through its effects on glutamatergic neurotransmission within the hypothalamus and the nuclear factor κB-mediated transcriptional activity, seems to be also involved in the specific mechanisms by which glucocorticoids exert their central effects on neurohypophyseal hormone synthesis and secretion.

Glucocorticoids, master modulators of the thymic catecholaminergic system?

There is evidence that the major mediators of stress, i.e., catecholamines and glucocorticoids, play an important role in modulating thymopoiesis and consequently immune responses. Furthermore, there are data suggesting that glucocorticoids influence catecholamine action. Therefore, to assess the putative relevance of glucocorticoid-catecholamine interplay in the modulation of thymopoiesis we analyzed thymocyte differentiation/maturation in non-adrenalectomized and andrenalectomized rats subjected to treatment with propranolol (0.4 mg·100 g body weight-1·day-1) for 4 days. The effects of β-adrenoceptor blockade on thymopoiesis in non-adrenalectomized rats differed not only quantitatively but also qualitatively from those in adrenalectomized rats. In adrenalectomized rats, besides a more efficient thymopoiesis [judged by a more pronounced increase in the relative proportion of the most mature single-positive TCRαβhigh thymocytes as revealed by two-way ANOVA; for CD4+CD8- F (1,20) = 10.92, P < 0.01; for CD4-CD8+ F (1,20) = 7.47, P < 0.05], a skewed thymocyte maturation towards the CD4-CD8+ phenotype, and consequently a diminished CD4+CD8-/CD4-CD8+ mature TCRαβhigh thymocyte ratio (3.41 ± 0.21 in non-adrenalectomized rats vs 2.90 ± 0.31 in adrenalectomized rats, P < 0.05) were found. Therefore, we assumed that catecholaminergic modulation of thymopoiesis exhibits a substantial degree of glucocorticoid-dependent plasticity. Given that glucocorticoids, apart from catecholamine synthesis, influence adrenoceptor expression, we also hypothesized that the lack of adrenal glucocorticoids affected not only β-adrenoceptor- but also α-adrenoceptor-mediated modulation of thymopoiesis.

Maternal undernutrition and the offspring kidney: from fetal to adult life

Maternal dietary protein restriction during pregnancy is associated with low fetal birth weight and leads to renal morphological and physiological changes. Different mechanisms can contribute to this phenotype: exposure to fetal glucocorticoid, alterations in the components of the renin-angiotensin system, apoptosis, and DNA methylation. A low-protein diet during gestation decreases the activity of placental 11ß-hydroxysteroid dehydrogenase, exposing the fetus to glucocorticoids and resetting the hypothalamic-pituitary-adrenal axis in the offspring. The abnormal function/expression of type 1 (AT1R) or type 2 (AT2R) AngII receptors during any period of life may be the consequence or cause of renal adaptation. AT1R is up-regulated, compared with control, on the first day after birth of offspring born to low-protein diet mothers, but this protein appears to be down-regulated by 12 days of age and thereafter. In these offspring, AT2R expression differs from control at 1 day of age, but is also down-regulated thereafter, with low nephron numbers at all ages: from the fetal period, at the end of nephron formation, and during adulthood. However, during adulthood, the glomerular filtration rate is not altered, due to glomerulus and podocyte hypertrophy. Kidney tubule transporters are regulated by physiological mechanisms; Na+/K+-ATPase is inhibited by AngII and, in this model, the down-regulated AngII receptors fail to inhibit Na+/K+-ATPase, leading to increased Na+ reabsorption, contributing to the hypertensive status. We also considered the modulation of pro-apoptotic and anti-apoptotic factors during nephrogenesis, since organogenesis depends upon a tight balance between proliferation, differentiation and cell death.