Jasmina Živanović

Citrus flavanones naringenin and hesperetin improve antioxidant status and membrane lipid compositions in the liver of old-aged Wistar rats

This study aimed to investigate effects of citrus flavanones naringenin (NAR) and hesperetin (HES) on liver antioxidant status and membrane phospholipid composition in 24-month-old rats. NAR and HES (15mg/kg) were administrated orally to male Wistar rats, once per day, for 4weeks. Control group received either vehicle (sunflower oil) or remained intact. The results showed decreased (p<0.05) activity of antioxidant enzymes (AOE), specifically catalase (CAT), superoxide dismutase (SOD) 1 and glutathione reductase (GR) in the liver of intact control old-aged rats in comparison to young intact controls. Flavanone administration to old-aged males increased (p<0.05) examined AOE activities in comparison to vehicle-administered animals. Namely, NAR was more potent in comparison to HES regarding the increase (p<0.05) in activities of examined antioxidant enzymes (SOD 1 and 2, glutathione peroxidase-GPx and GR) and the liver glutathione (GSH), while HES elevated (p<0.05) only activity of CAT and GR. Both flavanones significantly decreased (p<0.05) TBARS and improved (p<0.05) membrane phospholipid composition in favor of n-3 PUFA and n-6/n-3 PUFA ratio. Both flavanones did not affect liver histology and reduced (p<0.05) alanine aminotransferase and aspartate aminotransferase levels in serum. The results of this study indicate beneficial potential of citrus flavanones in the old-aged rat liver.

Soy isoflavones interfere with thyroid hormone homeostasis in orchidectomized middle-aged rats

We previously reported that genistein (G) and daidzein (D) administered subcutaneously (10mg/kg) induce changes in the angio-follicular units of the thyroid gland, reduce concentration of total thyroid hormones (TH) and increase thyrotropin (TSH) in serum of orchidectomized middle-aged (16-month-old) rats. To further investigate these effects, we now examined expression levels of the thyroglobulin (Tg), thyroperoxidase (Tpo), vascular endothelial growth factor A (Vegfa) and deiodinase type 1 (Dio 1) genes in the thyroid; in the pituitary, genes involved in TH feedback control (Tsh β, Dio 1, Dio 2, Trh receptor); and in the liver and kidney, expression of T3-activated genes Dio 1 and Spot 14, as well as transthyretin (Ttr), by quantitative real-time PCR. We also analyzed TPO-immunopositivity and immunofluorescence of T4 bound to Tg, determined thyroid T4 levels and measured deiodinase enzyme activities in examined organs. Decreased expression of Tg and Tpo genes (p<0.05) correlated with immunohistochemical staining results, and together with decreased serum total T4 levels, indicates decreased Tg and TH synthesis following treatments with both isoflavones. However, expression of Spot 14 (p<0.05) gene in liver and kidney was up-regulated, and liver Dio 1 expression and activity (p<0.05) increased. At the level of pituitary, no significant change in gene expression levels, or Dio 1 and 2 enzyme activities was observed. In conclusion, both G and D impaired Tg and TH synthesis, but at the same time increased tissue availability of TH in peripheral tissues of Orx middle-aged rats.

Testosterone application decreases the capacity for ACTH and corticosterone secretion in a rat model of the andropause.

The culminating phase of ageing in males-andropause is characterized by enhanced activity of the hypothalamic-pituitary-adrenal axis and frequent glucocorticoid excess. In parallel, free testosterone deficiency provides the baseline hormonal milieu for the ageing male. The aim of this study was to illustrate (using diverse microscopic and biochemical methodologies) the effects of testosterone application on the capacity for adrenocorticotropic hormone (ACTH) and corticosterone secretion in a rat model of the andropause. Middle-aged Wistar rats were divided into sham-operated (SO; n=8), orchidectomized (Orx; n=8) and testosterone treated orchidectomized (Orx+T; n=8) groups. Testosterone propionate (5 mg/kg b.w./day) was administered for three weeks, while SO and Orx groups received the vehicle alone. ACTH cells and the adrenal cortex were stained using immuno-histochemical, immuno-fluorescent and histochemical procedures. Circulating concentrations of testosterone, estradiol, ACTH and corticosterone, as well as the adrenal tissue corticosterone levels were measured by immunoassays. Testosterone application led to increased (p<0.05) serum concentrations of sex steroids. Consequently, in Orx+T rats the ACTH cell nuclei volume increased (p<0.05) by 34%, while the volume density of ACTH cells and their relative intensity of fluorescence decreased (p<0.05) by 46% and 21%, respectively, in comparison with the corresponding parameters in the Orx group. Testosterone also induced vasodilatation in the adrenocortical zona fasciculata, and decreased (p<0.05) the ACTH concentrations and adrenal tissue corticosterone levels by 38% and 31%, respectively, compared to the Orx group. In conclusion, testosterone administration caused a decrease in the capacity for ACTH and corticosterone secretion in a rat model of the andropause.

Membrane Steroid Receptor-Mediated Action of Soy Isoflavones: Tip of the Iceberg

Soy isoflavone’s (genistein and daidzein in particular) biological significance has been thoroughly studied for decades, so we started from the premise that refreshed investigation approach in this field should consider identification of their new molecular targets. In addition to recently described epigenetic aspects of polyphenole action, the cell membrane constituents-mediated effects of soy isoflavones are worthy of special attention. Accordingly, the expanding concept of membrane steroid receptors and rapid signaling from the cell surface may include the prominent role of these steroid-like compounds. It was observed that daidzein strongly interacts with membrane estrogen receptors in adrenal medullary cells. At low doses, daidzein was found to stimulate catecholamine synthesis through extracellular signal-regulated kinase 1/2 or protein kinase A pathways, but at high doses, it inhibited catecholamine synthesis and secretion induced by acetylcholine. Keeping in mind that catecholamine excess can contribute to the cardiovascular pathologies and that catecholamine lack may lead to depression, daidzein application promises to have a wide range of therapeutic effects. On the other hand, it was shown in vitro that genistein inhibits LNCaP prostate cancer cells invasiveness by decreasing the membrane fluidity along with immobilization of the androgen receptor containing membrane lipid rafts, with down regulation of the androgen receptors and Akt signaling. These data are promising in development of the molecular pharmacotherapy pertinent to balanced soy isoflavone treatment of cardiovascular, psychiatric, and steroid-related malignant diseases.

Immuno-histomorphometric and -fluorescent characteristics of GH cells after treatment with genistein or daidzein in an animal model of andropause

Abstract Somatopause, the complex aspect of andropause, is recognizable by reduced growth hormone - GH/insulin-like growth factor 1 axis function in the ageing male. Soy isoflavones (usually genistein and daidzein), which are known for their beneficial effects in the treatment of ageing symptoms, are active in the pituitary, as well. The immunohistomorphometric and -fluorescent characteristics of pituitary growth hormone secreting cells, in an animal model of andropause, were examined after a treatment with genistein or daidzein. Andropausal Wistar rats were divided into sham operated, orchidectomized and genistein or daidzein treated orchidectomized groups. Genistein or daidzein (30 mg/kg/day) were administered subcutaneously for three weeks, while sham operated and orchidectomized groups received the vehicle alone. Growth hormone secreting cells were identified by the peroxidase-antiperoxidase immuno-histochemical, and immuno-fluorescent procedure. The main characteristic of growth hormone secreting cells in soy isoflavones treated groups is a weaker immuno-histochemical staining and immuno-fluorescent signal compared to sham operated and orchidectomized groups. The growth hormone secreting cell volume in orchidectomized +genistein or +daidzein groups is by 13.8% and 11.9% (p<0.05) smaller respectively, in comparison with the orchidectomized group. In orchidectomized +genistein or +daidzein groups, the growth hormone secreting cells relative volume density is by 62.5% and 61.0% lower (p<0.05) respectively than for the sham operated group, and decreased by 65.4% and 64.0% (p<0.05) respectively, compared to the orchidectomized group. It can be concluded that chronic genistein or daidzein treatment, in an animal model of andropause, attenuates immunohistomorphometric and -fluorescent characteristics of growth hormone secreting cells.

Tamoxifen stimulates calcitonin-producing thyroid C-cells and prevents trabecular bone loss in a rat model of androgen deficiency

Thyroid C‐cells produce calcitonin (CT), a hypocalcemic hormone, that acts as an inhibitor of bone resorption. In this study, we investigated the effects of tamoxifen (TAM) as a selective estrogen receptor modulator on thyroid C‐cells, trabecular bone and biochemical markers of bone metabolism in an animal model of androgen deficiency, represented by middle‐aged orchidectomized (Orx) rats. Fifteen‐month‐old male Wistar rats were divided into: Orx and sham‐operated (SO) groups. Rats from one Orx group were injected subcutaneously with TAM citrate (Orx + TAM; 0.3 mg kg−1 b.w.), while the rats from SO and a second Orx group received vehicle alone, once a day for 3 weeks. The peroxidase–antiperoxidase method was applied for localization of CT in C‐cells. Thyroid C‐cells were morphometrically and ultrastructurally analyzed. An ImageJ image‐processing program was used to measure bone histomorphometric parameters. Blood serum samples were analyzed for CT, osteocalcin (OC), calcium (Ca2+) and phosphorus (P). Urinary Ca2+ concentrations were measured. TAM treatment significantly increased thyroid C‐cell volume (Vc) and serum CT when compared with vehicle‐treated Orx rats. Analysis of trabecular microarchitecture of the tibia showed that administration of TAM significantly increased cancellous bone area, trabecular thickness and trabecular number, whereas trabecular separation was significantly decreased compared with vehicle‐treated Orx rats. Serum OC and urinary Ca2+ concentrations were significantly lower in comparison with the control Orx group. These results indicate that in our rat model of androgen deficiency, TAM stimulated calcitonin‐producing thyroid C‐cells and increased trabecular bone mass.

Daidzein upregulates anti-aging protein Klotho and NaPi 2a cotransporter in a rat model of the andropause

In a rat model of the andropause we aimed to examine the influence of daidzein, soy isoflavone, on the structure and function of parathyroid glands (PTG) and the expression levels of some of the crucial regulators of Ca2+ and Pi homeostasis in the kidney, and to compare these effects with the effects of estradiol, serving as a positive control. Middle-aged (16-month-old) male Wistar rats were divided into the following groups: sham-operated (SO), orchidectomized (Orx), orchidectomized and estradiol-treated (Orx+E; 0.625mg/kg b.w./day, s.c.) as well as orchidectomized and daidzein-treated (Orx+D; 30mg/kg b.w./day, s.c.) group. Every treated group had a corresponding control group. PTH serum concentration was decreased in Orx+E and Orx+D groups by 10% and 21% (p<0.05) respectively, in comparison with the Orx. PTG volume was decreased in Orx+E group by 16% (p<0.05), when compared to the Orx. In Orx+E group expression of NaPi 2a was lower (p<0.05), while NaPi 2a abundance in Orx+D animals was increased (p<0.05), when compared to Orx. Expression of PTH1R was increased (p<0.05) in Orx+E group, while in Orx+D animals the same parameter was decreased (p<0.05), in comparison with Orx. Klotho expression was elevated (p<0.05) in Orx+D rats, in regard to Orx. Orx+D induced reduction in Ca2+/creatinine and Pi/creatinine ratio in urine by 32% and 16% (p<0.05) respectively, in comparison with Orx. In conclusion, presented results indicate the more coherent beneficial effects of daidzein compared to estradiol, on disturbed Ca2+ and Pi homeostasis, and presumably on bone health, in the aging male rats.

Genistein and daidzein treatments differently affect uterine homeostasis in the ovary-intact middle-aged rats.

&NA; This study aimed to investigate the effects of soy isoflavones, genistein (GEN) and daidzein, (DAI) on the uterine function in ovary‐intact middle‐aged rats. GEN and DAI (35 mg/kg) were subcutaneously administrated to acyclic (12‐month‐old) Wistar females, daily, for 4 weeks. Control group received either vehicle (olive oil and ethanol, 9:1) or remained intact. We found that GEN and DAI differently affect uterine morphophysiology. GEN significantly increased the uterine wet weight which was associated with hyperplastic changes, revealed by stereological and histomorphometrical analyses. Also, PCNA immunoexpression was increased, whereas expression of apoptotic marker (caspase‐3) was decreased. Protein and gene expressions of ER&agr; were down‐regulated, while PR and ER&bgr; were up‐regulated after GEN application. Also, GEN caused an increase of LAC and VEGF mRNA expression, together with an up‐regulation of Akt activity. In contrast, DAI did not change the uterine wet weight and stereological features of the main uterine compartments as well as LAC and VEGF gene expression. Absence of hyperplastic changes were illustrated by an increase in caspase‐3 immunoexpression, associated with reduced PCNA expression. DAI up‐regulated only the expression of ER&bgr;, while the expression levels of ER&agr; and PR remain unaffected. Also, DAI inhibited the activation of Akt due to down‐regulation of phosphorylated and total form of Akt protein expression. Compared to GEN, DAI did not promote events associated with the endometrial cell proliferation in the conducted study, figuring as the compound with a potential safety profile, which justifies further investigation. HighlightsDAI did not change the uterine wet weight and stereological features of the uterus.DAI up‐regulated the expression of ER&bgr;, while the expression levels of ER&agr; and PR remain unaffected.DAI inhibits the activation of Akt due to down‐regulation of phosphorylated form.Compared to GEN, DAI did not promote events associated with endometrial cell proliferation.

The phytoestrogen genistein prevents trabecular bone loss and affects thyroid follicular cells in a male rat model of osteoporosis.

As a major phytoestrogen of soy, genistein effectively prevents bone loss in both humans and rat models of osteoporosis. However, although the bone‐sparing effects of genistein are achieved directly through estrogen receptors, its mode of action on bone by modulation of other endocrine functions is not entirely clear. Thus, thyroid hormones and calcitonin (CT) have an essential influence on bone metabolism. Besides its action on bones, in this study we examined the effect of genistein on the activity of two different endocrine cell populations, thyroid follicular and C‐cells. Fifteen‐month‐old Wistar rats were either bilaterally orchidectomized (Orx) or sham‐operated (SO). Two weeks after surgery, half of the Orx rats were treated chronically with 30 mg kg−1 b.w. genistein (Orx + G) subcutaneously (s.c.) every day for 3 weeks, while the remaining Orx rats and the SO rats were given the same volume of sterile olive oil to serve as controls. For histomorphometrical analysis of the trabecular bone microarchitecture an ImageJ public domain image processing programme was used. Thyroid sections were analysed histologically and stereologically after visualization of follicular and C‐cells by immunohistochemical staining for thyroglobulin and CT. Thyroid follicular epithelium, interstitium, colloid and CT‐immunopositive C‐cells were examined morphometrically. Serum concentrations of osteocalcin (OC), triiodothyronine (T3), thyroxine (T4) and CT were determined as well as urinary calcium (Ca2+) concentrations. Genistein treatment significantly increased cancellous bone area (B.Ar), trabecular thickness (TbTh) and trabecular number (TbN) (P < 0.05), but trabecular separation (Tb.Sp) was decreased (P < 0.05) compared with control Orx rats. In the thyroid, genistein treatment significantly elevated the relative volume density (Vv) of the follicular cells (P < 0.05) compared with Orx, whereas Vv of the colloid was lower (P < 0.05) than in the Orx. Evaluation of the biochemical parameters showed significant reductions in serum OC, T3, T4 and urinary Ca2+ concentrations (P < 0.05), compared with Orx rats. These data indicate that genistein treatment improves the trabecular microarchitecture of proximal tibia, induces histomorphometrical changes in thyroid glands, and decreases circulating thyroid hormone levels in orchidectomized rat model of male osteoporosis.

Response of trabecular bone, thyroid C and follicular cells to synthetic salmon calcitonin in middle-aged orchidectomized male rats

In contrast to studies in women, male osteoporosis is poorly understood and strictly related to advancing age. Among the first antiresorptive substances used in the prevention and treatment of osteoporosis is calcitonin (CT), a hypocalcemic hormone that potently inhibits osteoclastic bone resorption. Natural CT is produced and secreted by thyroid C‐cells. The other endocrine population of thyroid cells produces thyroid hormones (TH), which also affect bone turnover. The aim of this study was to evaluate the influence of salmon CT on trabecular bone microarchitecture with special reference to effects on the structure and function of both CT‐ and TH‐producing thyroid cells in orchidectomized (Orx) middle‐aged rats. Twenty‐four male Wistar rats aged 15 months were randomly divided into Orx and sham‐operated (SO) groups. One group of Orx animals received (s.c.) synthetic salmon CT (Orx + CT; 100 IU kg−1 b.w.) subcutaneously every second day for 6 weeks. The second Orx group and SO rats were given the same volume of vehicle alone by the same schedule. Trabecular bone histomorphometrical parameters were: cancellous bone area (B.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were obtained with an ImageJ public‐domain image‐processing program. The peroxidase–antiperoxidase method was applied for localization of CT in C‐cells. Anti‐human CT antisera served as the primary antibodies. For immunohistochemical characterization of vascular endothelial growth factor (VEGF) in thyroid tissue, rabbit antisera against human VEGF, served as primary antibodies. CT‐immunopositive thyroid C‐cells, thyroid follicular epithelium, interstitium and colloid were evaluated morphometrically. Blood serum samples were analyzed for CT, osteocalcin (OC), and thyroxine (T4), and calcium (Ca2+) concentration was determined in urine samples. Salmon CT application significantly increased B.Ar, TbTh and TbN, but markedly decreased Tb.Sp. Administration of exogenous CT significantly decreased mean volume (Vc) and relative volume density (Vv) of thyroid C‐cells in relation to both SO and Orx groups. The Vv of the colloid was higher, whereas the VV of the follicular epithelium was lower after CT treatment compared with Orx alone. CT treatment markedly elevated serum CT, whereas serum OC, T4 and urinary Ca2+ concentrations were lower than in the Orx group. These results indicate that salmon CT stimulates trabecular bone microarchitecture, strongly inhibits thyroid C‐cells and changes the structure of the thyroid gland, indicating hypoactivity.