Soybean isoflavones attenuate the expression of genes related to endometrial cancer risk.

OBJECTIVE: We evaluated whether genistein or estrogen treatment has the same effect when administered immediately or late to rats induced with menopause using ovariectomy. METHODS: Sixty adult female rats were divided into six treatment groups: GI = vehicle immediately after ovariectomy; GII = vehicle 30 days after ovariectomy; GIII = genistein immediately after ovariectomy; GIV = genistein 30 days after ovariectomy; GV = estrogen immediately after ovariectomy; and GVI = estrogen 30 days after ovariectomy. All animals were treated for 30 consecutive days. At the end of the treatment, part of the uteri was removed for subsequent histological studies and another part was used to evaluate estrogen receptors 1 and 2, cell proliferation (cyclin A1 and A2, cyclin D1, cyclin-dependent kinase inhibitors 1, 1B and 2, antigen identified by the monoclonal antibody Ki67) and angiogenesis (vascular endothelial growth factor, VEGF-A) gene expression. RESULTS: Late treatment after castration in rats resulted in more developed endometrium, enhanced cell proliferation and estrogen-signalling pathways, particularly the cyclin-related genes Ki67 and VEGF-A, compared to early treatment. Interestingly, these same effects were less intense with genistein compared to those induced by estrogen, especially when genistein was administered late. CONCLUSION: Our data show that isoflavone renders a lower risk of cancer when compared to estrogen in treatments.

Soybean concentrated extract counteracts oxidative stress in the uterus of rats.

OBJECTIVE: To evaluate the effects of soy isoflavone extract in the pro-oxidant/antioxidant balance in the uterus of ovariectomized rats. METHODS: Twenty 3-month-old adult female Wistar rats were divided into four equal groups: GI: sham-operated (estrous phase); GII: control ovariectomized rats; GIII: ovariectomized rats treated with genistein (50 µg/kg/day) by gavage; GIV: ovariectomized rats subcutaneously treated with estrogen (10 µg/kg/day). After 30 consecutive days of treatment, the rats were euthanized and the uterus removed. The distal thirds of the uterine horns were processed for histomorphometric analyses of endometrial and myometrial thicknesses and glandular area. Other regions of the uteri were kept in liquid nitrogen and subsequently processed for analysis of reactive species quantification (DCF), total antioxidant capacity (TAC) and lipid oxidation status (TBARS). Data were statistically analyzed by one-way ANOVA, complemented by the Tukey-Kramer test (p < 0.05). RESULTS: GII and GIII exhibited lower endometrial thickness, glandular area and myometrial thickness than GI and GIV, while a higher myometrial thickness was observed in GIV compared with the other groups. Moreover, the isoflavone-treated group showed lower DCF and TBARS compared to GII, and also an improvement of TAC compared to GI and GIV. Despite the significant decrease in TBARS, no significant difference in DCF nor a decrease in TAC were observed in GIV when compared to GII. CONCLUSION: Our data show that isoflavones improve antioxidant status and counteract oxidative stress, without promoting a trophic effect in the uterus of rats.

Glutamate-induced alterations in Ca2+ signaling are modulated by mitochondrial Ca2+ handling capacity in brain slices of R6/1 transgenic mice.

Huntington's disease is a neurodegenerative disorder caused by an expansion of CAGs repeats and characterized by alterations in mitochondrial functions. Although changes in Ca(2+) handling have been suggested, the mechanisms involved are not completely understood. The aim of this study was to investigate the possible alterations in Ca(2+) handling capacity and the relationship with mitochondrial dysfunction evaluated by NAD(P)H fluorescence, reactive oxygen species levels, mitochondrial membrane potential (DeltaPsi(m)) measurements and respiration in whole brain slices from R6/1 mice of different ages, evaluated in situ by real-time real-space microscopy. We show that the cortex and striatum of the 9-month-old R6/1 transgenic mice present a significant sustained increase in cytosolic Ca(2+) induced by glutamate (Glu). This difference in Glu response was partially reduced in R6/1 when in the absence of extracellular Ca(2+), indicating that N-methyl-D-aspartate receptors participation in this response is more important in transgenic mice. In addition, Glu also lead to a decrease in NAD(P)H fluorescence, a loss in DeltaPsi(m) and a further increase in respiration, which may have evoked a decrease in mitochondrial Ca(2+) Ca(2+)(m) uptake capacity. Taken together, these results show that alterations in Ca(2+) homeostasis in transgenic mice are associated with a decrease in Ca(2+)(m) uptake mechanism with a diminished Ca(2+) handling ability that ultimately causes dysfunctions and worsening of the neurodegenerative and the disease processes.

Alterations in calcium signaling and a decrease in Bcl-2 expression: possible correlation with apoptosis in aged striatum.

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca(2+) homeostasis. Thus, in the present work, we have investigated Ca(2+) signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca(2+) rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca(2+). Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca(2+) content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca(2+) handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.

Alterations in Calcium Signalingand a Decrease in Bcl-2 Expression: Possible Correlation With Apoptosisin Aged Striatum

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca2+ homeostasis. Thus, in the present work, we have investigated Ca2+ signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca2+ rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca2+. Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca2+ content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl-2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca2+ handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl-2 may contribute to apoptosis induction and cell death in rat striatum.

Increase in bax expression and apoptosis are associated in Huntington's disease progression.

Huntington's disease (HD) is a hereditary dominant neurodegenerative disorder and the progression of the disease may be associated with apoptosis and altered expression of apoptotic proteins. The aim of this study was to investigate gene expression of bax and bcl-2 in tissues from R6/1 transgenic (TGN) mice of different ages (3, 6 and 9 months). The mRNA expression was investigated and related to apoptotic cells measured by TUNEL. Results showed a significant and progressive increase in bax levels in the cortex of TGN (from 10 to 33%) when compared to control (CT) (8 to 20%) mice with 3, 6 and 9-month-old. The increase in bax was correlated with the elevation in the number of apoptotic nuclei, especially in the cortex of 6 (10%) and 9 (18%)-month-old mice. Increase in bax expression might be related to an apoptotic induction which contributes to the HD progression.

DNA strand breaks produced by oxidative stress in mammalian cells exhibit 3'-phosphoglycolate termini.

In recent years two mechanisms have been proposed for the production of DNA strand breaks in cells undergoing oxidative stress: (i) DNA attack by OH radical, produced by Fenton reaction catalyzed by DNA-bound iron; and (ii) DNA attack by calcium-activated nucleases, due to the increase of cytosolic and nuclear calcium induced by oxidative stress. We set out to investigate the participation of the former mechanism by detecting and quantifying 3'-phosphoglycolate, a 3' DNA terminus known to be formed by OH radical attack to the deoxyribose moiety, followed by sugar ring rupture and DNA strand rupture. These structures were found in DNA of monkey kidney cells exposed to hydrogen peroxide, iron nitrilotriacetate or ascorbate, all species known to favor a cellular pro-oxidant status. The method employed to measure 3' phosphoglycolate was the 32P-postlabeling assay. Repair time course experiments showed that it takes 10 h for 3'-phosphoglycolate to be removed from DNA. It was found that the DNA of both control cells and cells exposed to hydrogen peroxide had a very poor capacity of supporting in vitro DNA synthesis, catalyzed by DNA polymerase I. If the DNA was previously incubated with exonuclease III, an enzyme able to expose 3'-OH primers by removal of 3'-phosphoglycolate and 3'-phosphate termini the in vitro synthesis was substantially increased. This result shows that either of these termini are present at the break and that 3'-hydroxyl termini are virtually absent. At least 25% of the strand breaks exhibited 3'-phosphoglycolate termini as determined by the 32P-postlabeling assay, but due to the characteristic of the method this percentage is likely to be higher. These results favor the hypothesis that an oxidative agent generated by Fenton reaction is responsible for DNA strand breakage in cells undergoing oxidative stress.