Impact of citrus flavanones on thyroid homeostasis and lipid status of old-aged rats

Citrusni flavanoni, naringenin (NAR) i hesperetin (HES), su polifenolne supstance koje zbog sterne sličnosti sa endogenim tiroksinom (T4) mogu da interferiraju sa aktivnošću tiroidne peroksidaze, ključnog enzima u biosintezi tiroidnih hormona. Sa starenjem se povećava osetljivost tiroidne osovine na efekte egzogenih supstanci i raste rizik od nastanka hroničnih bolesti usled opadanja detoksikacione sposobnosti i akumulacije lipida u jetri. Cilj ove doktorske disertacije bio je da se ispita uticaj NAR i HES na tiroidnu homeostazu i lipidni status 24 meseca starih mužjaka pacova Wistar soja. Supstance su aplikovane per os u dozi od 15 mg/kg t.m., tokom četiri nedelje. Rezultati ove disertacije su pokazali da NAR i HES uzrokuju strukturne i ultrastrukturne promene štitaste žlezde starih mužjaka pacova, povećavaju imunohistohemijsku ekspresiju tiroglobulina za koji je vezan T4 i vaskularnog endotelijalnog faktora rasta. Ekspresija trijodtironinom (T3) regulisanih gena u jetri je snižena nakon tretmana citrusnim flavanonima. U serumu, NAR je povisio koncentracije TSH i HDL, dok je koncentracija antioksidativnih enzima jetre snižena nakon oba tretmana. Uočava se desaturacija masnih kiselina u fosfolipidima membrane hepatocita, povećanje udela n-3, a sniženja zastupljenosti n-6 masnih kiselina nakon primene oba citrusna flavanona. Smanjuje se lipidna peroksidacija membrane hepatocita i poboljšava antioksidativna zaštita u jetri nakon NAR i HES tretmana. NAR je ispolji jači efekat, jer je uticao na aktivnost, gensku i proteinsku ekspresiju antioksidativnih enzima, i parametre redoks ravnoteže, dok je HES pojačao antioksidativnu zaštitu, ali u manjoj meri. Upotreba citrusnih flavanona uzrokuje neželjene efekte - subkličničku hipotireozu koju prate povišen nivo TSH i smanjena ekspresija T3 regulisanih gena u jetri. Međutim, sa stanovišta desaturacije membrane hepatocita i poboljšanja antioksidativne zaštite u jetri, upotreba NAR i HES se može smatrati korisnom, imajući u vidu uticaj ovih promena na produženje dužine životnog veka.Citrus flavanones, naringenin (NAR) and hesperetin (HES), are polyphenolic substances, structurally similar to endogenous thyroxine (T4), that can interfere with the activity of thyroid peroxidase, the key enzyme in thyroid hormone biosynthesis. With aging, thyroid axis sensitivity to the effects of exogenously applied substances increases and the risk of chronic diseases raises due to reduced detoxification ability and the accumulation of lipids in the liver. The aim of this doctoral dissertation was to examine the impact of NAR and HES on thyroid homeostasis and the lipid status of 24 monthsold Wistar male rats. The substances were administered per os at a dose of 15 mg/kg b.w., for four weeks. The results of this dissertation have shown that NAR and HES cause structural and ultrastructural changes in the thyroid gland of old male rats, increasing the immunohistochemical expression of T4 bounded to thyroglobulin as well as of vascular endothelial growth factor. Expression of triiodothyronine (T3) regulated genes in the liver were decreased after treatment with citrus flavanones. In the serum, the NAR increased concentrations of TSH and HDL, while the concentration of liver antioxidative enzymes was reduced after both treatments. The desaturation of fatty acids in the hepatocyte membrane phospholipids, the increase in the proportion of n-3, and the reduction in the presence of n-6 fatty acids after application of both citrus flavanones is observed. Lipid peroxidation of the hepatocyte membrane is reduced and the antioxidant protection is improved in the liver after NAR and HES treatments. The NAR has a stronger effect, as it has affected the activity, gene and protein expression of antioxidant enzymes, as well as the parameters of redox balance, while HES enhanced antioxidant protection, but to a less degree. The use of citrus flavanones causes unwanted effects - subclinical hypothyrosis followed by elevated TSH and reduced expression of T3 regulated genes in the liver. However, from the perspective of the hepatocyte membrane desaturation and the enhancement of antioxidant protection in the liver, the use of NAR and HES can be considered useful, given that the effects of these changes are associated with the extend life span

Effects of calcium administration on parathyroid gland, NaPi 2a cotransporter and PTH1R in an animal model of the andropause

Background: Increased risk of osteoporotic bone fractures represents the adverse event in andropausal men. Due to diminished calcium absorption in elderly, its supplementation is used for prevention and treatment of advanced-age osteoporosis. Methods: Sixteen-month-old Wistar rats were divided into sham-operated (SO), orchidectomized (Orx) and Ca2+treated orchidectomized (Orx+Ca) groups. Ca2+ (28.55 mg/kg b.w.) was administered intramuscularly for 3 weeks, while the SO and Orx received vehicle alone. Parathyroid glands (PTG) were analyzed histomorphometrically, while the expression of NaPi 2a mRNA from kidneys was determined by real time PCR. NaPi 2a and PTH1R abundance was detected immunofluorescently. Serum and urine parameters were determined biochemically. Results: The PTG volume was 15% (p<0.05) greater in Orx rats than in the SO group. In Orx+Ca2+ animals, PTG volume was decreased by 17% (p<0.05), when compared to the Orx rats. Orchidectomy led to an increment of serum PTH of 13% (p<0.05) compared to the SO group, while Orx+Ca decreased it by 10% (p<0.05) when compared to Orx animals. The intensity of the NaPi 2a signal was reduced in Orx rats, in comparison with the SO group. Orx+Ca2+ treatment increased the abundance of NaPi 2a, compared to the Orx group. In Orx rats, the staining for PTH1R was stronger when compared to the SO group, while the Orx+Ca2+ treatment induced reduction of the PTH1R immunofluorescence, compared to Orx animals. Orchidectomy increased Pi urinary concentrations by 8% (p<0.05), in comparison with the SO control, while in the Orx+Ca2+ group urinary Pi concentration was 5% lower (p<0.05) than for the Orx rats. Conclusions: Our results indicate that Ca2+ administration reduces the PTH serum level and the presence of PTH1R, while increased abundance of NaPi 2a cotransporter positively regulates Pi urine reabsorption in an animal model of the andropause.Uvod: Povećan rizik od preloma kostiju, izazvan osteoporozom, uobičajen je u andropauzi. Usled smanjene apsorpcije kalcijuma kod starijih osoba, on se često koristi u prevenciji i tretmanu osteoporoze u poznom dobu. Metode: Wistar pacovi stari 16 meseci podeljeni su na lažno operisanu (SO), orhidektomisanu (Orx) i Ca2+ tretiranu orhidektomisanu (Orx+Ca) grupu. Ca2+ je aplikovan intramuskularno u dozi od 28,55 mg/kg telesne mase, dok su kontrolne grupe SO i Orx primile odgovarajući volumen rastvarača. Paratireoidne žlezde (PTŽ) analizirane su histomorfometrijski, dok je nivo ekspresije gena za NaPi 2a određen pomoću PCR metode u realnom vremenu. Prisustvo NaPi 2a i PTH1R identifikovano je imunofluorescentnom metodom. Parametri iz seruma i urina određeni su biohemijskim metodama. Rezultati: Volumen PTŽ je povećan za 15% (p<0,05) kod Orx pacova u odnosu na SO grupu. Kod Orx+Ca tretiranih životinja, volumen PTŽ je smanjen za 17% (p<0,05) u poređenju sa Orx pacovima. Orhidektomija je povecala nivo PTH u serumu za 13% (p<0,05) u odnosu na SO grupu, dok je nivo ovog hormona kod Orx+Ca pacova smanjen za 10% (p<0,05) u poređenju sa Orx životinjama. Intenzitet NaPi 2a signala je redukovan kod Orx životinja u odnosu na SO grupu, dok Orx+Ca tretman povecava prisustvo NaPi 2a, u poređenju sa Orx grupom. Kod Orx pacova, obojenost PTH1R je jača u odnosu na SO grupu, dok je Orx+Ca tretman redukovao imunofluorescencu u poređenju sa Orx životinjama. Orhidektomija je povećala koncentraciju Pi u urinu za 8% (p<0,05) u poređenju sa SO kontrolom, dok je Orx+Ca tretman smanjio koncentraciju Pi u urinu za 5% (p<0,05) u odnosu na koncentraciju kod Orx pacova. Zaključak: Prikazani rezultati ukazuju na to da tretman Ca2+ smanjuje nivo PTH u serumu kao i prisustvo PTH1R, dok je zastupljenost NaPi 2a kotransportera povećana, što pozitivno reguliše reapsorpciju Pi iz urina u animalnom modelu andropauze.Projekat ministarstva br. 17300

Old age-associated impairment of the rat liver antioxidant defense system: the basis for affirmation of the experimental model

Letter to the Edito

Somatopause, weaknesses of the therapeutic approaches and the cautious optimism based on experimental ageing studies with soy isoflavones

The pathological phenomenon of somatopause, noticeable in hypogonadal ageing subjects, is based on the growth hormone (GH) production and secretion decrease along with the fall in GH binding protein and insulin-like growth factor 1 (IGF-1) levels, causing different musculoskeletal, metabolic and mental issues. From the perspective of safety and efficacy, GH treatment is considered to be highly controversial, while some other therapeutic approaches (application of IGF-1, GH secretagogues, gonadal steroids, cholinesterase-inhibitors or various combinations) exhibit more or less pronounced weaknesses in this respect. Soy isoflavones, phytochemicals that have already demonstrated the health benefits in treated elderly, at least experimentally reveal their potential for the somatopausal symptoms remediation. Namely, genistein enhanced GHRH-stimulated cAMP accumulation and GH release in rat anterior pituitary cells; refreshed and stimulated the somatotropic system (hypothalamic nuclei and pituitary GH cells) function in a rat model of the mild andropause, and stimulated the GH output in ovariectomized ewes as well as the amplitude of GH pulses in the rams. Daidzein, on the other hand, increased body mass, trabecular bone mass and decreased bone turnover in the animal model of severe andropause, while both isoflavones demonstrated blood cholesterol-lowering effect in the same model. These data, which necessarily need to be preclinically and clinically filtered, hint some cautious optimism and call for further innovative designing of balanced soy isoflavone-based therapeutics

Evaluation of rapd markers as a marker-assisted selection tool for variety type and erucic acid content in rapeseed

Random amplification of polymorphic DNA (RAPD) analysis was performed on twelve rapeseed genotypes from Institute of Field and Vegetable Crops, Novi Sad, Serbia, genepool in order to identify markers that could be used in marker assisted selection (MAS) for different growing type and selection of the varieties with low or zero level of erucic acid. Out of fifteen RAPD markers, three were monomorphic, whereas twelve had polymorphic profiles. Three primers amplified specific fragments in spring varieties. UBC 25 and UBC 191 amplified the fragments of 450 and 750 bp, respectively, in all tested spring varieties, except in JR-NS-36. Primer UBC 72 generated a fragment of 700 bp that was present in all spring varieties. These fragments were not present in any of winter varieties. None of the tested RAPD primers amplified fragment(s) uniquely present either in varieties with or without (0%) erucic acid or with different erucic acid content. Cluster analysis showed a concordance between the position of varieties in the cluster and their pedigree information, but also enabled separation of spring and winter varieties. Contingency analysis confirmed that fragment UBC 72_700 is specific for spring varieties, while for erucic acid content, only moderate association was found with UBC 137_750

Immunohistomorphometric changes of the pituitary gonadotropic cells after testosterone application in a rat model of the andropause

Andropause, the culminating phase of male ageing, is characterized by deregulation of the hypothalamic-pituitarygonadal axis and low circulating free testosterone. The aim of this study was to investigate the immunohistomorphometric characteristics of the pituitary gonadotropic i.e. follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) producing cells after testosterone application in a rat model of the andropause. Middle-aged Wistar rats were divided into orchidectomized (ORX; n=8) and testosterone treated orchidectomized (ORX+T; n=8) groups. Testosterone propionate (5 mg/kg b.m. /day) was administered for three weeks, while the ORX group received the vehicle alone. Immunohistochemically stained FSH and LH cells underwent morphometric and optical density-related analysis, while circulating concentrations of the sex steroids were measured by immunoassays. Serum concentrations of testosterone and estradiol were significantly (p<0.05) increased by 24 and 2.7 fold respectively, compared to the ORX group. The volume of FSH and LH cells was significantly (p<0.05) decreased by 51.3% and 56.6% respectively, in comparison with ORX rats. Relative volume density of FSH and LH cells was also significantly (p<0.05) decreased by 54.0% and 72.8% respectively, compared to the ORX group. Results related to the optical density of gonadotropic cells (reflecting their hormonal content) were in line with the morphometric findings i.e. this parameter of FSH and LH cells was significantly (p<0.05) decreased by 25.7% and 16.2% respectively, in comparison with ORX rats. Conclusion: In conclusion, applied testosterone increased the serum concentrations of sex steroids, as well as it decreased morphometric parameters and optical density of gonadotropic cells in ORX rats

Citrus flavanones mildly interfere with pituitary-thyroid axis in old-aged male rats.

Citrus flavanones naringenin (NAR) and hesperetin (HES) are potent antioxidants that may contribute to maintenance of health at old age by improving cardiovascular and metabolic status. However, they may also affect thyroid hormone economy. Keeping in mind impaired thyroid function at older age, in this study we tested wheather NAR or HES administration potentiate this decline. NAR or HES were administrated orally (15mg/kg) to male 24-month-old Wistar rats during 4 weeks. Control groups received vehicle, sunflower oil. Qualitative and quantitative immunohistochemical and immunofluorescent expression of specific proteins and stereological analyses of thyroid tissue were performed. Thyroid stimulating hormone (TSH) and total thyroxine (T4) concentrations were measured in serum. Thyroid parenchyma of both flavanone-treated groups was characterized by lower (p<0.05) absolute and relative volume of luminal colloid, accompanied by elevated (p<0.05) relative volume of stroma in comparison with the controls. No hypertrophy or absolute thyroid volume change was detected. Intensity of immunopositive signal for thyroglobulin (Tg) and T4 bound to Tg (T4-Tg) increased (p<0.05) in the colloid of thyroid follicles after both flavanone treatments. Serum TSH increased (p<0.05) after NAR, while T4 remained unchanged after both treatments. In conclusion, NAR elevated serum TSH in old-aged males, thus being more potent than HES in altering pituitary-thyroid axis. However, changes in thyroid structure, namely moderate colloid depletion and higher Tg and T4-Tg protein expressions after both treatments, indicate preserved capacity of the gland to compensate flavanone interfering, and maintain T4 production in old-aged males

Effects of calcium administration on parathyroid gland, NaPi 2a cotransporter and PTH1R in an animal model of the andropause

Background: Increased risk of osteoporotic bone fractures represents the adverse event in andropausal men. Due to diminished calcium absorption in elderly, its supplementation is used for prevention and treatment of advanced-age osteoporosis. Methods: Sixteen-month-old Wistar rats were divided into sham-operated (SO), orchidectomized (Orx) and Ca2+treated orchidectomized (Orx+Ca) groups. Ca2+ (28.55 mg/kg b.w.) was administered intramuscularly for 3 weeks, while the SO and Orx received vehicle alone. Parathyroid glands (PTG) were analyzed histomorphometrically, while the expression of NaPi 2a mRNA from kidneys was determined by real time PCR. NaPi 2a and PTH1R abundance was detected immunofluorescently. Serum and urine parameters were determined biochemically. Results: The PTG volume was 15% (p<0.05) greater in Orx rats than in the SO group. In Orx+Ca2+ animals, PTG volume was decreased by 17% (p<0.05), when compared to the Orx rats. Orchidectomy led to an increment of serum PTH of 13% (p<0.05) compared to the SO group, while Orx+Ca decreased it by 10% (p<0.05) when compared to Orx animals. The intensity of the NaPi 2a signal was reduced in Orx rats, in comparison with the SO group. Orx+Ca2+ treatment increased the abundance of NaPi 2a, compared to the Orx group. In Orx rats, the staining for PTH1R was stronger when compared to the SO group, while the Orx+Ca2+ treatment induced reduction of the PTH1R immunofluorescence, compared to Orx animals. Orchidectomy increased Pi urinary concentrations by 8% (p<0.05), in comparison with the SO control, while in the Orx+Ca2+ group urinary Pi concentration was 5% lower (p<0.05) than for the Orx rats. Conclusions: Our results indicate that Ca2+ administration reduces the PTH serum level and the presence of PTH1R, while increased abundance of NaPi 2a cotransporter positively regulates Pi urine reabsorption in an animal model of the andropause.Uvod: Povećan rizik od preloma kostiju, izazvan osteoporozom, uobičajen je u andropauzi. Usled smanjene apsorpcije kalcijuma kod starijih osoba, on se često koristi u prevenciji i tretmanu osteoporoze u poznom dobu. Metode: Wistar pacovi stari 16 meseci podeljeni su na lažno operisanu (SO), orhidektomisanu (Orx) i Ca2+ tretiranu orhidektomisanu (Orx+Ca) grupu. Ca2+ je aplikovan intramuskularno u dozi od 28,55 mg/kg telesne mase, dok su kontrolne grupe SO i Orx primile odgovarajući volumen rastvarača. Paratireoidne žlezde (PTŽ) analizirane su histomorfometrijski, dok je nivo ekspresije gena za NaPi 2a određen pomoću PCR metode u realnom vremenu. Prisustvo NaPi 2a i PTH1R identifikovano je imunofluorescentnom metodom. Parametri iz seruma i urina određeni su biohemijskim metodama. Rezultati: Volumen PTŽ je povećan za 15% (p<0,05) kod Orx pacova u odnosu na SO grupu. Kod Orx+Ca tretiranih životinja, volumen PTŽ je smanjen za 17% (p<0,05) u poređenju sa Orx pacovima. Orhidektomija je povecala nivo PTH u serumu za 13% (p<0,05) u odnosu na SO grupu, dok je nivo ovog hormona kod Orx+Ca pacova smanjen za 10% (p<0,05) u poređenju sa Orx životinjama. Intenzitet NaPi 2a signala je redukovan kod Orx životinja u odnosu na SO grupu, dok Orx+Ca tretman povecava prisustvo NaPi 2a, u poređenju sa Orx grupom. Kod Orx pacova, obojenost PTH1R je jača u odnosu na SO grupu, dok je Orx+Ca tretman redukovao imunofluorescencu u poređenju sa Orx životinjama. Orhidektomija je povećala koncentraciju Pi u urinu za 8% (p<0,05) u poređenju sa SO kontrolom, dok je Orx+Ca tretman smanjio koncentraciju Pi u urinu za 5% (p<0,05) u odnosu na koncentraciju kod Orx pacova. Zaključak: Prikazani rezultati ukazuju na to da tretman Ca2+ smanjuje nivo PTH u serumu kao i prisustvo PTH1R, dok je zastupljenost NaPi 2a kotransportera povećana, što pozitivno reguliše reapsorpciju Pi iz urina u animalnom modelu andropauze.Projekat ministarstva br. 17300

Determination of the Stages of Deep Pectoral Myopathy Induced in Broilers Fed with Supplemental Coenzyme Q10

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

Prostate cancer metastasis and soy isoflavones: a dogfight over a bone

Prostate cancer is a complex, progressive, bone-tropic disease, which is usually associated with skeletal issues, poor mobility and a fatal outcome when it reaches the metastatic phase. Soy isoflavones, steroid-like compounds from soy-based food/dietary supplements, have been found to decrease the risk of prostate cancer in frequent consumers. Herein, we present a systematization of the data on soy isoflavone effects at different stages of metastatic prostate cancer progression, with a particular interest in the context of bone-related molecular events. Specifically, soy isoflavones have been determined to downregulate the prostate cancer cell androgen receptors, reverse the epithelial to mesenchymal transition of these cells, decrease the expressions of prostate-specific antigen, matrix metalloproteinase and serine proteinase, and reduce the superficial membrane fluidity in prostate cancer cells. In addition, soy isoflavones suppress the angiogenesis that follows prostate cancer growth, obstruct prostate cancer cells adhesion to the vascular endothelium and their extravasation in the area of future bone lesions, improve the general bone morphofunctional status, have a beneficial effect on prostate cancer metastasiscaused osteolytic/osteoblastic lesions and possibly affect the pre-metastatic niche formation. The observed, multilevel antimetastatic properties of soy isoflavones imply that they should be considered as promising components of combined therapeutic approaches to advanced prostate cancer