Valeria Rodríguez

Molecular mechanisms triggered by low-calcium diets.

Ca is not only essential for bone mineralisation, but also for regulation of extracellular and intracellular processes. When the Ca2+ intake is low, the efficiency of intestinal Ca2+ absorption and renal Ca2+ reabsorption is increased. This adaptive mechanism involves calcitriol enhancement via parathyroid hormone stimulation. Bone is also highly affected. Low Ca2+ intake is considered a risk factor for osteoporosis. Patients with renal lithiasis may be at higher risk of recurrence of stone formation when they have low Ca2+ intake. The role of dietary Ca2+ on the regulation of lipid metabolism and lipogenic genes in adipocytes might explain an inverse relationship between dairy intake and BMI. Dietary Ca2+ restriction produces impairment of the adipocyte apoptosis and dysregulation of glucocorticosteroid metabolism in the adipose tissue. An inverse relationship between hypertension and a low-Ca2+ diet has been described. Ca2+ facilitates weight loss and stimulates insulin sensitivity, which contributes to the decrease in the blood pressure. There is also evidence that dietary Ca2+ is associated with colorectal cancer. Dietary Ca2+ could alter the ratio of faecal bile acids, reducing the cytotoxicity of faecal water, or it could activate Ca2+-sensing receptors, triggering intracellular signalling pathways. Also it could bind luminal antigens, transporting them into mucosal mononuclear cells as a mechanism of immunosurveillance and promotion of tolerance. Data relative to nutritional Ca2+ and incidences of other human cancers are controversial. Health professionals should be aware of these nutritional complications and reinforce the dairy intakes to ensure the recommended Ca2+ requirements and prevent diseases.

A mitochondrial mechanism is involved in apoptosis of Robertsonian mouse male germ cells

The aim of this study was to determine whether the intrinsic mechanism of apoptosis is involved in the death of germ cells in Robertsonian (Rb) heterozygous adult male mice. Testes from 5-month-old Rb heterozygous CD1 x Milano II mice were obtained and compared with those from homozygous CD1 (2n=40) and Milano II (2n=24) mice. For histological evaluation of apoptosis, TUNEL labelling and immunohistochemistry were used to localise Bax and cytochrome c. Expression of calbindin D(28k) (CB), an anti-apoptotic molecule, was also analysed by immunohistochemistry and immunoblotting. Testicular ultrastructure was visualised by electron microscopy. Morphology and cell associations were abnormal in the Rb heterozygous seminiferous epithelium. An intense apoptotic process was observed in tubules at stage XII, mainly in metaphase spermatocytes. Metaphase spermatocytes also showed Bax and cytochrome c redistributions. Mitochondria relocated close to the paranuclear region of spermatocytes. CB was mainly expressed in metaphase spermatocytes, but also in pachytene spermatocytes, spermatids and Sertoli cells at stage XII. The co-localisation of CB and TUNEL labelling was very limited. Sixty per cent of metaphase spermatocytes were apoptotic and calbindin negative, while 40% were calbindin positive without signs of apoptosis. Ten per cent of the Bax- and cytochrome c-positive cells were also calbindin positive. These data suggest that apoptosis of the germ cells in heterozygous mice occurs, at least in part, through a mitochondrial-dependent mechanism. Calbindin overexpression might prevent or reduce the apoptosis of germ cells caused by Rb heterozygosity, which could partially explain the subfertility of these mice.

Ursodeoxycholic and deoxycholic acids: A good and a bad bile acid for intestinal calcium absorption

The aim of this study was to investigate the effect of ursodeoxycholic acid (UDCA) on intestinal Ca(2+) absorption and to find out whether the inhibition of this process caused by NaDOC could be prevented by UDCA. Chicks were employed and divided into four groups: (a) controls, (b) treated with 10mM NaDOC, (c) treated with 60 μg UDCA/100g of b.w., and (d) treated with 10mM NaDOC and 60 μg UDCA/100g of b.w. UDCA enhanced intestinal Ca(2+) absorption, which was time and dose-dependent. UDCA avoided the inhibition of intestinal Ca(2+) absorption caused by NaDOC. Both bile acids altered protein and gene expression of molecules involved in the transcellular pathway of intestinal Ca(2+) absorption, but in the opposite way. UDCA aborted the oxidative stress produced by NaDOC in the intestine. UDCA and UDCA plus NaDOC increased vitamin D receptor protein expression. In conclusion, UDCA is a beneficial bile acid for intestinal Ca(2+) absorption. Contrarily, NaDOC inhibits the intestinal cation absorption through triggering oxidative stress. The use of UDCA in patients with cholestasis would be benefited because of the protective effect on the intestinal Ca(2+) absorption, avoiding the inhibition caused by hydrophobic bile acids and neutralizing the oxidative stress.

Time dependent changes in the intestinal Ca2 + absorption in rats with type I diabetes mellitus are associated with alterations in the intestinal redox state

The aim was to determine the intestinal Ca²⁺ absorption in type I diabetic rats after different times of STZ induction, as well as the gene and protein expression of molecules involved in both the transcellular and paracellular Ca²⁺ pathways. The redox state and the antioxidant enzymes of the enterocytes were also evaluated in duodenum from either diabetic or insulin-treated diabetic rats as compared to control rats. Male Wistar rats (150-200 g) were divided into two groups: 1) controls and 2) STZ-induced diabetic rats (60 mg/kg b.w.). A group of diabetic rats received insulin for five days. The insulin was adjusted daily to maintain a normal blood glucose level. Five 5 d after STZ injection, there was a reduction in the intestinal Ca²⁺ absorption, which was maintained for 30 d and disappeared at 60 d. Similar changes occurred in the GSH and (˙)O(2)(-) levels. The protein expression of molecules involved in the transcellular pathway increased at 5 and 30 d returning to control values at 60 d. Their mRNA levels declined considerably at 60 d. The gene and protein expression of claudin 2 was upregulated at 30 d. Catalase activity increased at 5 and 30 d normalizing at 60 d. To conclude, type I D.m. inhibits the intestinal Ca²⁺ absorption, which is transient leading to a time dependent adaptation and returning the absorptive process to normal values. The inhibition is accompanied by oxidative stress. When insulin is administered, the duodenal redox state returns to control values and the intestinal Ca²⁺ absorption normalizes.

Spermatocyte apoptosis, which involves both intrinsic and extrinsic pathways, explains the sterility of Graomys griseoflavus × Graomys centralis male hybrids

Spermatogenic impairment and the apoptotic pathways involved in establishing sterility of male hybrids obtained from crossing Graomys griseoflavus females with Graomys centralis males were studied. Testes from G. centralis, G. griseoflavus and hybrids were compared at different ages. Terminal transferase-mediated dUTP nick-end labelling assay (TUNEL), Fas, Bax and cytochrome c labelling were used for apoptosis evaluation, and calbindin D(28k) staining as an anti-apoptotic molecule. In 1-month-old animals, spermatocytes were positive for all apoptotic markers, but moderate TUNEL (+) spermatocyte frequency was only found in G. centralis. At subsequent ages, the apoptotic markers were downregulated in testes from parental cytotypes, but not in hybrid testes. TUNEL (+) spermatocytes were present at 78% and 44% per tubule cross-section in 2- and 3-month-old hybrid animals, respectively. Pachytene spermatocyte death in adult hybrids occurs via apoptosis, as revealed by high caspase-3 expression. Calbindin was highly expressed in spermatocytes of adult hybrids, in which massive cell death occurs via apoptosis. Calbindin co-localisation with TUNEL or Fas, Bax and cytochrome c was very limited, suggesting an inverse regulation of calbindin and apoptotic markers. Hybrid sterility is due to breakdown of spermatogenesis at the pachytene spermatocyte stage. Both extrinsic and intrinsic pathways are involved in apoptosis of spermatocytes, which are the most sensitive cell type to apoptotic stimuli.

Ursodeoxycholic and deoxycholic acids: Differential effects on intestinal Ca2+ uptake, apoptosis and autophagy of rat intestine

The aim of this work was to study the effect of sodium deoxycholate (NaDOC) and ursodeoxycholic acid (UDCA) on Ca(2+) uptake by enterocytes and the underlying mechanisms. Rats were divided into four groups: a) controls, b) treated with NaDOC, c) treated with UDCA d) treated with NaDOC and UDCA. Ca(2+) uptake was studied in enterocytes with different degrees of maturation. Apoptosis, autophagy and NO content and iNOS protein expression were evaluated. NaDOC decreased and UDCA increased Ca(2+) uptake only in mature enterocytes. The enhancement of protein expression of Fas, FasL, caspase-8 and caspase-3 activity by NaDOC indicates triggering of the apoptotic extrinsic pathway, which was blocked by UDCA. NO content and iNOS protein expression were enhanced by NaDOC, and avoided by UDCA. The increment of acidic vesicular organelles and LC3 II produced by NaDOC was also prevented by UDCA. In conclusion, the inhibitory effects of NaDOC on intestinal Ca(2+) absorption occur by decreasing the Ca(2+) uptake by mature enterocytes. NaDOC triggers apoptosis and autophagy, in part as a result of nitrosative stress. In contrast, UDCA increases the Ca(2+) uptake by mature enterocytes, and in combination with NaDOC acts as an antiapoptotic and antiautophagic agent normalizing the transcellular Ca(2+) pathway.

Melatonin not only restores but also prevents the inhibition of the intestinal Ca2 + absorption caused by glutathione depleting drugs

We have previously demonstrated that melatonin (MEL) blocks the inhibition of the intestinal Ca(2+) absorption caused by menadione (MEN). The purpose of this study were to determine whether MEL not only restores but also prevents the intestinal Ca(2+) absorption inhibited either by MEN or BSO, two drugs that deplete glutathione (GSH) in different ways, and to analyze the mechanisms by which MEN and MEL alter the movement of Ca(2+) across the duodenum. To know this, chicks were divided into four groups: 1) controls, 2) MEN treated, 3) MEL treated, and 4) treated sequentially with MEN and MEL or with MEN and MEL at the same time. In a set of experiments, chicks treated with BSO or sequentially with BSO and MEL or with BSO and MEL at the same time were used. MEL not only restored but also prevented the inhibition of the chick intestinal Ca(2+) absorption produced by either MEN or BSO. MEN altered the protein expression of molecules involved in the transcellular as well as in the paracellular pathway of the intestinal Ca(2+) absorption. MEL restored partially both pathways through normalization of the O2(-) levels. The nitrergic system was not altered by any treatment. In conclusion, MEL prevents or restores the inhibition of the intestinal Ca(2+) absorption caused by different GSH depleting drugs. It might become one drug for the treatment of intestinal Ca(2+) absorption under oxidant conditions having the advantage of low or null side effects.

Buthionine Sulfoximine and 1,25-Dihydroxyvitamin D Induce Apoptosis in Breast Cancer Cells via Induction of Reactive Oxygen Species

Calcitriol or 1,25(OH)2D3 is a negative growth regulator of breast cancer cells. The aim of this study was to determine whether L-buthionine-S,R-sulfoximine, a glutathione-depleting drug, modifies the antiproliferative effects of 1,25(OH)2D3 on MCF-7 cells. For comparison, we included studies in MCF-7 cells selected for vitamin D resistance and in human mammary epithelial cells transformed with SV40 and ras. Our data indicate that L-buthionine-S,R-sulfoximine enhances the growth inhibition of 1,25(OH)2D3 in all transformed breast cell lines. This effect is mediated by ROS leading to apoptosis. In conclusion, BSO alters redox state and sensitizes breast cancer cells to 1,25(OH)2D3-mediated apoptosis.

Association of cellular and molecular alterations in Leydig cells with apoptotic changes in germ cells from testes of Graomys griseoflavus×Graomys centralis male hybrids.

Spermatogenesis is disrupted in Graomys griseoflavus×Graomys centralis male hybrids. This study was aimed to determine whether morphological alterations in Leydig cells from hybrids accompany the arrest of spermatogenesis and cell death of germ cells and whether apoptotic pathways are also involved in the response of these interstitial cells. We used three groups of 1-, 2- and 3-month-old male animals: (1) G. centralis, (2) G. griseoflavus and (3) hybrids obtained by crossing G. griseoflavus females with G. centralis males. Testicular ultrastructure was analyzed by transmission electron microscopy. TUNEL was studied using an in situ cell death detection kit and the expression of apoptotic molecules by immunohistochemistry. The data confirmed arrest of spermatogenesis and intense apoptotic processes of germ cells in hybrids. These animals also showed ultrastructural alterations in the Leydig cells. Fas, FasL and calbindin D28k overexpression without an increase in DNA fragmentation was detected in the Leydig cells from hybrids. In conclusion, the sterility of Graomys hybrids occurs with ultrastructural changes in germ and Leydig cells. The enhancement of Fas and FasL is not associated with cell death in the Leydig cells. Probably the apoptosis in these interstitial cells is inhibited by the high expression of the antiapoptotic molecule calbindin D28k.

Naringin attenuates liver damage in streptozotocin-induced diabetic rats

The aim of this study was to evaluate whether NAR has a hepatoprotective role in a model of STZ-induced diabetes and to elucidate the underlying mechanisms triggered by the flavonoid. Male Wistar rats were divided into three groups: 1) controls, 2) STZ rats 3) STZ rats treated daily with NAR (40 mg/kg b.w.) for 30 days. NAR prevented increases in serum aminotransferases and alkaline phosphatase activities in STZ rats. The flavonoid blocked serum lipid alterations, but not the biometric parameters in STZ rats. Microscopic examination in liver from STZ rats revealed morphological changes indicative of increased adipogenesis and cell death and inflammation, which were all mitigated by the flavonoid. NAR inhibited the NFκB/IL-6/Cox-2 overexpressions triggered by oxidative stress in STZ rats. The iNOS/NO/nitrosylated protein pathway was also blocked by NAR. The increment in the protein expression of Fas/FasL/caspase-3 and in the Bax/Bcl-2 ratio showed that both pathways of apoptosis were increased by the diabetes, effects that were abrogated by NAR treatment. In conclusion, NAR protects against the liver damage caused by STZ-induced diabetes and it could be a novel therapeutic strategy to prevent the non alcoholic fatty liver disease associated with the type 1Diabetes mellitus.