V. Centeno

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.

Genotypes and clinical aspects associated with bone mineral density in Argentine postmenopausal women

The aim of this study was to determine genotypes and clinical aspects associated with bone mineral density (BMD) in postmenopausal women from Córdoba, Argentina. Polymorphisms were assessed by RFLP-PCR technique using BsmI and FokI for vitamin D receptor gene (VDR) and XbaI and PvuII for estrogen receptor-α gene (ERα) as restrictases. Sixty-eight healthy, 54 osteopenic, and 64 osteoporotic postmenopausal women were recruited. Femoral neck and lumbar spine BMD were inversely correlated with age in the entire analyzed population. Height was lower in osteopenic and osteoporotic women as compared to healthy women (P < 0.05). Weight and body mass index (BMI) were the lowest in osteoporotic women (P < 0.01 versus healthy group). Serum procollagen type I Nterminal propeptide (PINP) was higher in osteoporotic women as compared to the other groups. Distribution of VDR and ERα genotypes was similar in the three groups. Genotype bb (VDR) was associated with low values of lumbar BMD in the healthy group (P < 0.05 versus genotype Bb), and with low values of femoral BMD (P < 0.05 versus genotype BB) in osteoporotic women. BB*Pp interaction was associated with the highest femoral neck BMD (P < 0.05), whereas the bb*xx interaction was associated with the lowest femoral neck BMD in the total population analyzed (P < 0.05). In conclusion, parameters such as age, height, weight, BMI, serum PINP, VDR genotypes, and interactions between VDR and ERá genotypes could be useful to predict a decrease in BMD in Argentine postmenopausal women.

Effects of a single dose of menadione on the intestinal calcium absorption and associated variables

The effect of a single large dose of menadione on intestinal calcium absorption and associated variables was investigated in chicks fed a normal diet. The data show that 2.5 micro mol of menadione/kg of b.w. causes inhibition of calcium transfer from lumen-to-blood within 30 min. This effect seems to be related to oxidative stress provoked by menadione as judged by glutathione depletion and an increment in the total carbonyl group content produced at the same time. Two enzymes presumably involved in calcium transcellular movement, such as alkaline phosphatase, located in the brush border membrane, and Ca(2+)- pump ATPase, which sits in the basolateral membrane, were also inhibited. The enzyme inhibition could be due to alterations caused by the appearance of free hydroxyl groups, which are triggered by glutathione depletion. Addition of glutathione monoester to the duodenal loop caused reversion of the menadione effect on both intestinal calcium absorption and alkaline phosphatase activity. In conclusion, menadione shifts the balance of oxidative and reductive processes in the enterocyte towards oxidation causing deleterious effects on intestinal Ca(2+) absorption and associated variables, which could be prevented by administration of oral glutathione monoester.

Sodium deoxycholate inhibits chick duodenal calcium absorption through oxidative stress and apoptosis

High concentrations of sodium deoxycholate (NaDOC) produce toxic effects. This study explores the effect of a single high concentration of NaDOC on the intestinal Ca(2+) absorption and the underlying mechanisms. Chicks were divided into two groups: 1) controls and 2) treated with different concentrations of NaDOC in the duodenal loop for variable times. Intestinal Ca(2+) absorption was measured as well as the gene and protein expressions of molecules involved in the Ca(2+) transcellular pathway. NaDOC inhibited the intestinal Ca(2+) absorption, which was concentration dependent. Ca(2+)-ATPase mRNA decreased by the bile salt and the same occurred with the protein expression of Ca(2+)-ATPase, calbindin D(28k) and Na(+)/Ca(2+) exchanger. NaDOC produced oxidative stress as judged by ROS generation, mitochondrial swelling and glutathione depletion. Furthermore, the antioxidant quercetin blocked the inhibitory effect of NaDOC on the intestinal Ca(2+) absorption. Apoptosis was also triggered by the bile salt, as indicated by the TUNEL staining and the cytochrome c release from the mitochondria. As a compensatory mechanism, enzyme activities of the antioxidant system were all increased. In conclusion, a single high concentration of NaDOC inhibits intestinal Ca(2+) absorption through downregulation of proteins involved in the transcellular pathway, as a consequence of oxidative stress and mitochondria mediated apoptosis.

Intestinal Na+/Ca2+ exchanger protein and gene expression are regulated by 1,25(OH)2D3 in vitamin D-deficient chicks

The role of 1,25(OH)(2)D(3) on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)(2)D(3) were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)(2)D(3) simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)(2)D(3). NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)(2)D(3). Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)(2)D(3) treatment. Rapid effects of 1,25(OH)(2)D(3) on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)(2)D(3) on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)(2)D(3) enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.

Vitamin D receptor genotypes are associated with bone mass in patients with Turner syndrome.

Abstract Background: Turner syndrome (TS) patients present low bone mineral density (BMD) and increased fracture risk, probably due to a genetic defect aggravated by hormonal deficiency. Aim: To study the relationship between vitamin D receptor (VDR) gene polymorphisms and BMD and bone parameters in TS patients. Methods: DNA from 65 TS patients and 110 controls was amplified by PCR and digested with Fok I, Bsm I and Apa I restrictases. Lumbar and femoral BMD were determined by DEXA and serum intact parathyroid hormone, osteocalcin and β-CrossLaps by electrochemiluminescence. Results: Genotype distribution within the Apa I site was different in both groups: genotype Aa was more abundant in TS (63.8% vs. 41.3%; p<0.01), whereas AA predominated in controls (33.9% vs. 15.5%; p<0.01). Patients carrying genotype bb (Bsm I) or ff (Fok I) had lower BMD than those with other genotypes (p<0.01 and p<0.05, respectively). Conclusion: Bsm I and Fok I polymorphic sites of VDR could be genetic determinants of BMD in TS patients.

Association of vitamin D receptor gene Cdx2 polymorphism with bone markers in Turner syndrome patients.

Abstract Background: Turner syndrome (TS) patients usually have low bone mineral density (BMD) and increased risk of osteoporotic fractures. We have previously demonstrated an association of bb (BsmI polymorphic site) and ff (FokI polymorphic site) vitamin D receptor (VDR) genotypes with reduced BMD in TS patients. Aim: To analyze the relationship between VDR-Cdx2 polymorphism and BMD as well as bone metabolic variables in TS patients. Methods: Fifty-five TS patients and 59 control women were studied. VDR-Cdx2 genotypes were determined using TaqMan probes in a real time thermocycler. Lumbar and femoral BMD were determined by dual-energy X-ray absorptiometry (DEXA) and serum intact parathyroid hormone, osteocalcin and β-CrossLaps were determined by electrochemiluminescence. Results: Patients with genotype GG had higher levels of both osteocalcin and β-CrossLaps as compared to patients with genotype GA (p<0.01 and p<0.05, respectively). Conclusion: Patients carrying genotype GG have higher levels of bone formation and resorption markers. This indicates a more active bone turnover that could impact on their future bone mineral density.

Fluoride alters connexin expression in rat incisor pulp.

OBJECTIVE Connexins (Cxs) are important to control growth and cell differentiation of dental tissues. The aim of the present study was to assess the impact of chronic exposure to sodium fluoride (NaF) on Cxs expression and alkaline phosphatase (ALP) activity in dental pulp, and on morphometric parameters of adult rat mandible and incisors. DESIGN Three groups of male Wistar rats (22 days-old) were given water containing: (a) 0.3 mg/L (Control), (b) 10 mg/L and (c) 50 mg/L of NaF for eight weeks. Incisor pulp homogenates were prepared for determination of Cx32, Cx43 and Cx45 gene expression, using semi-quantitative RT-PCR, and of ALP activity. Morphometric parameters of mandible and incisors were determined on radiographs. RESULTS Cx43 gene expression increased with exposure to NaF in a dose-dependent manner. Cx32 mRNA levels were higher than controls in the 10mg/L NaF group only; Cx45 mRNA levels were lower in groups given 10 and 50mg/L of NaF than in controls. ALP activity was higher in both high-NaF dose groups compared to the control group (p<0.05). Lower incisor diameter was lower in the 50 mg/L NaF than in the control group (p<0.01). None of the mandibular growth parameters were affected by NaF treatment. CONCLUSION Our results showed that fluorotic alterations in rat incisor were associated with increased Cx43 expression and ALP activity, as well as with changes in the expression pattern of different Cxs in pulp tissue. The observed changes may have a stimulating effect on dentin mineralization.

Molecular mechanisms involved in the enhancement of mitochondrial malate dehydrogenase activity by calcitriol in chick intestine

Mitochondrial malate dehydrogenase (mMDH) from the intestine is the NAD-linked oxidoreductase of the tricarboxylic acid cycle with the highest activity and response to vitamin D treatment in vitamin D-deficient chicks (-D). The aim of this study was to elucidate potential molecular mechanisms by which cholecalciferol or calcitriol enhances the activity of this enzyme. One group of animals used was composed of -D and -D treated with cholecalciferol or with calcitriol. A second group consisted of -D and -D supplemented with high Ca(2+) diet. A third group included chicks receiving either a normal or a low Ca(2+) diet. In some experiments, animals were injected with cycloheximide. Data showed that either vitamin D (cholecalciferol or calcitriol) or a low Ca(2+) diet increases mMDH activity. High Ca(2+) diet did not modify the intestinal mMDH activity from -D. The mMDH activity from -D remained unaltered when duodenal cells were exposed to 10(-8) mol/L calcitriol for 15 min. The enhancement of mMDH activity by calcitriol was completely abolished by simultaneous cycloheximide injection to -D. mMDH mRNA levels, detected by RT-PCR, indicate that calcitriol did not affect gene expression. In contrast, Western blots show that calcitriol enhanced the protein expression. In conclusion, calcitriol stimulates intestinal mMDH activity by increasing protein synthesis. No response of mMDH activity by rapid effects of calcitriol or activation through increment of serum Ca(2+) was demonstrated. Consequently, ATP production would be increased, facilitating the Ca(2+) exit from the enterocytes via the Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger, which participate in the intestinal Ca(2+) absorption.

Chronic Exposure to Fluoride During Gestation and Lactation Increases Mandibular Bone Volume of Suckling Rats

We aimed to investigate the effect of maternal exposure to NaF on mandibular bone microarchitecture and phosphocalcic plasma parameters of the offspring. For this purpose, 10-, 15-, and 21-day-old pups (n = 6–8 per group) from two groups of mothers, control and NaF 50mg/L treated dams, were used. Plasma calcium (Ca) and phosphorus (P) levels and alkaline phosphatase activity (ALP) were measured. Fluoride concentration (F−) in bone and in stomach content was measured using potentiometry after isothermal distillation. Morphometric, histological, and histomorphometric analyses of the jaw bones were performed. Plasma Ca and P levels and ALP activity increased in 10-day and decreased in 21-day-old pups from NaF-treated mothers. Fluoride concentration in stomach content samples of 15- and 21-day-old nursing pups from mothers exposed to NaF in their drinking water was higher compared to that observed in control dam offspring. Mandibular F− content was higher in 21-day-old pups born to F−-exposed dams compared to those observed in age-matched control pups. Mandibular area increased in 21-day-old pups born to treated mothers as compared to controls. Mandibular bone volume BV/TV (%) was higher in offspring from NaF-exposed dams than in controls at all the studied times. The increase in bone volume after exposure to F− was concomitant with the increase in trabecular thickness and the decrease in trabecular separation. Altogether, our results showed that exposure to NaF during gestation and lactation increased mandibular area and bone volume of pups, with concomitant changes in phosphocalcic parameters associated with the bone modeling process.