Marko Miler

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.

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.

Testosterone and estradiol treatments differently affect pituitary-thyroid axis and liver deiodinase 1 activity in orchidectomized middle-aged rats.

We previously reported that orchidectomy (Orx) of middle-aged rats (15-16-month-old; MA) slightly affected pituitary-thyroid axis, but decreased liver deiodinase (Dio) type 1 and pituitary Dio2 enzyme activities. At present, we examined the effects of subsequent testosterone-propionate treatment (5mg/kg; Orx+T), and compared the effects of testosterone with the effects of estradiol-dipropionate (0.06mg/kg; Orx+E) treatment. Hormones were subcutaneously administered, daily, for three weeks, while Orx and sham-operated (SO) controls received only the vehicle. The applied dose of T did not alter serum TSH, T4 and T3 concentrations in Orx- MA, though it increased TSH when administrated to Orx young adults (2.5-month-old; Orx-YA). However, pituitaries of Orx-MA+T rats had higher relative intensity of immunofluorescence (RIF) for TSHβ; in their thyroids we found increased volume and height of follicular epithelium, decreased volume of the colloid and higher RIF for T4-bound to thyroglobulin (Tg-T4). Liver Dio1 activity was increased. E-treatment did not affect serum hormone levels, pituitary RIF for TSHβ, or liver Dio1 activity in Orx-MA rats. Thyroids had decreased relative volume and height of follicular epithelium, increased relative volume of the colloid, decreased volume of sodium-iodide symporter-immunopositive epithelium and lower RIF for Tg-T4. Detected changes were statistically significant. In conclusion, androgenization enhanced pituitary TSHβ RIF, thyroid activation and liver Dio1 enzyme activity in Orx-MA, without elevating serum TSH as in Orx-YA rats. Estrogenization induced pituitary enlargement with no effect on pituitary TSHβ RIF, serum TSH or liver Dio1 activity. E also induced alterations in thyroid histology that indicate mild suppression of its functioning, and contributed to thyroid blood vessel enlargement in Orx-MA rats.

Morphological and functional changes in pituitary-thyroid axis following prolonged exposure of female rats to constant light

Light regulates numerous physiological functions and synchronizes them with the environment, in part by adjusting secretion of different hormones. We hypothesized that constant light (CL) would disturb pituitary‐thyroid axis. Our aim was to determine morphological and functional changes in this endocrine system in such extreme conditions and, based on the obtained results, to propose the underlying mechanism(s). Starting from the thirtieth postnatal day, female Wistar rats were exposed to CL (600 lx) for the following 95 days. The controls were maintained under the regular laboratory lighting conditions. After decapitation, pituitaries and thyroids were prepared for further histomorphometric, immunohistochemical, and immunofluorescence examinations. Concentration of thyroid stimulating hormone (TSH), total T4 and T3 (TH) were determined. Thyroid tissue of light‐treated rats was characterized by microfollicular structure. We detected no change in total thyroid volume, localization and accumulation of thyroglobulin, thyroid peroxidase, and sodium‐iodide symporter in the follicular epithelium of CL rats. The volume of follicular epithelium and activation index were increased, while volume of the colloid and serum levels of TH decreased. In the pituitary, the relative intensity of TSH β‐immunofluorescence signal within the cytoplasm of thyrotrophs increased, but their average cell volume and the relative volume density decreased. Serum TSH was unaltered. We conclude that exposure of female rats to CL induced alterations in pituitary‐thyroid axis. Thyroid tissue was characterized by microfollicular structure. Serum TH levels were reduced without accompanying increase in serum TSH. We hypothesize that increased secretion and clearance of TH together with unchanged or even decreased hormonal synthesis, resulted in decreased serum TH levels in CL group. We assume this decrease consequently led to increased synthesis and/or accumulation of pituitary TSH. However, decreased average TSH cell volume and relative volume density, together with unchanged serum TSH, point to additional, negative regulation of thyrotrophs. J. Morphol. 275:1161–1172, 2014.

Effects of age and soybean isoflavones on hepatic cholesterol metabolism and thyroid hormone availability in acyclic female rats

ABSTRACT Soy‐food and its isoflavones, genistein (G) and daidzein (D), were reported to exert mild cholesterol‐lowering effect, but the underlying mechanism is still unclear. In this research, first we studied age‐related alterations in hepatic cholesterol metabolism of acyclic middle‐aged (MA) female rats. Then we tested if purified isoflavones may prevent or reverse these changes, and whether putative changes in hepatic thyroid hormone availability may be associated with this effect. Serum and hepatic total cholesterol (TChol), bile acid and cholesterol precursors, as well as serum TSH and T4 concentrations, hepatic deiodinase (Dio) 1 enzyme activity and MCT8 protein expression were determined by comparing data obtained for MA with young adult (YA) intact (IC) females. Effects of subcutaneously administered G or D (35 mg/kg) to MA rats were evaluated versus vehicle‐treated MA females. MA IC females were characterized by: higher (p < 0.05) serum TChol, lower (p < 0.05) hepatic TChol and its biosynthetic precursors, lower (p < 0.05) hepatic 7&agr;‐hydroxycholesterol but elevated (p < 0.05) 27‐ and 24‐hydroxycholesterol in comparison to YA IC. Both isoflavone treatments decreased (p < 0.05) hepatic 27‐hydroxycholesterol, G being more effective than D, without affecting any other parameter of Chol metabolism. Only G elevated hepatic Dio1 activity (p < 0.05). In conclusion, age‐related hypercholesteremia was associated with lower hepatic Chol synthesis and shift from main neutral (lower 7&agr;‐hydroxycholesterol) to alternative acidic pathway (higher 27‐hydroxycholesterol) of Chol degradation to bile acid. Both isoflavones lowered hepatic 27‐hydroxycholesterol, which may be considered beneficial. Only G treatment increased hepatic Dio1 activity, thus indicating local increase in thyroid hormones, obviously insufficient to induce prominent cholesterol‐lowering effect. HIGHLIGHTSAge‐related hypercholesteremia in middle‐aged acyclic female rats (13‐month‐old) is associated with:(i) lower hepatic cholesterol biosynthesis and(ii) shift from neutral to acydic pathway of cholesterol degradation to bile acid in the liver.(iii) unchanged TSH and total T4 in serum, as well as Dio1 enzyme activity and MCT8 protein expression in the liverSoy isoflavones reversed only hepatic 27‐hydroxycholesterol to the level of young adults.Only genistein elevated hepatic Dio1 activity, thus indicating local increase in thyroid hormones.

Diosgenin-caused changes of the adrenal gland histological parameters in a rat model of the menopause

Diosgenin, a steroidal sapogenin of natural origin, has demonstrated benefits when it comes to the treatment of malignancies, cardiovascular issues and menopausal symptoms. In this study, we investigated the histological changes of the adrenal gland after diosgenin application in a rat model of the menopause. Middle-aged, acyclic female Wistar rats were divided into control (C; n=6) and diosgenin treated (D; n=6) groups. Diosgenin (100mg/kg b.w./day) was orally administered for four weeks, while C group received the vehicle alone. A histological approach included design-based stereology, histochemistry and immunohistochemistry. The adrenal cortex volume decreased in D females by 15% (p<0.05) while the volume of adrenal medulla increased (p<0.05) by 64%, compared to the same parameters in C group. Volume density of the zona glomerulosa (expressed per absolute adrenal gland volume) in D rats increased (p<0.05) by 22% in comparison with C animals. Diosgenin treatment decreased (p<0.05) the volume density of the zona fasciculata (expressed per volume of adrenal cortex) by 15% when compared to C females. Absolute volume of the zona reticularis in D group decreased (p<0.05) by 38% in comparison with the same parameter in C rats. Also, after diosgenin application, the volume density of the zona reticularis (expressed per volume of adrenal cortex) and the zona reticularis cell volume were decreased by 51% and 20% (p<0.05) respectively, compared to C animals. Our results, reflecting a decrease in many stereological parameters of the adrenal cortex, indicate that diosgenin took over the role of corticosteroid precursors and became incorporated into steroidogenesis.

Effects of antipsychotic drug administration on antioxidative defense enzymes in male rat kidney

ABSTRACT The use of atypical antipsychotic drugs (APD) was reported to be associated with adverse effects on the kidneys. Thus, the aim of this study was to examine whether APD exerted their adverse effects by interfering with the renal antioxidant defense system. Male 3-mo-old Wistar rats were treated for 28 d with ziprasidone (ZIP), clozapine (CLO), or sertindole (SER) using a daily dose recommended for antipsychotic drug therapy. The expression and activities of antioxidant enzymes superoxide dismutase (SOD) type 1 and type 2, catalase (CAT), glutathione reductase (GR), and glutathione S-transferases (GSTs) activity were measured in the kidneys. Changes in the kidneys were also evaluated histologically. Ziprasidone, CLO, and SER reduced renal SOD type 1 and type 2 activities. Decreased CAT activity was observed only in SER-treated rats. An inhibition in GR activity and increased activity of GST was found only after treatment with CLO. Histological analysis showed dilatation of proximal tubules in kidneys with all three drugs. In conclusion, data indicate that redox disturbances may contribute to renal morphologic alterations in proximal tubules in rats treated with all APD.

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.

İlave Koenzim Q10 ile Beslenerek Derin Pektoral Myopati Oluşturulan Etlik Piliçlerde Miyopatinin Devrelerinin Belirlenmesi

The aim of this study was to examine the structural characteristics and incidence of different stages of deep pectoral myopathy (DPM) that was induced in broilers fed a coenzyme Q10 (CoQ10) supplemented diet. A total of 288 1-day-old chicks (Cobb 500) were equally divided among 8 pens (pens 1 to 8). The diet was the same for all chicks until day 35 post-hatching. Subsequently, broilers in pens 5 to 8 were fed the 20 mg of CoQ10/kg finisher diet until the end of the experiment (day 42 post-hatching). To induce DPM, 5 male birds from each of the pens 1 to 8 were subjected to encouraged wing flapping (EWF) at the end of their 37th day. At the end of the trial, the incidence of DPM stages in broilers was determined and an analysis of the histological parameters of deep pectoral muscles was performed. Results showed that, in the groups subjected to EWF, broilers with the CoQ10 supplement had a lower average DPM stage and volume density of necrotic muscle cells, as well as a higher volume density of non-necrotic muscle cells. These results can be related to the antioxidant properties of CoQ10, which, in chickens subjected to EWF, reduced the effects of DPM on cell necrosis and muscle tissue damage.