Enhanced ERbeta immunoexpression and apoptosis in the germ cells of cimetidine-treated rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In Vitro Evaluation of the Genotoxic Activity and Apoptosis Induction of the Extracts of Roots and Leaves from the Medicinal Plant Coccoloba mollis (Polygonaceae)

Coccoloba mollis (Family Polygonaceae) is a medicinal plant popularly used in cases of memory loss, stress, insomnia, anemia, impaired vision, and sexual impotence, but the scientific literature, to date, lacks studies on the biological effects of this species, particularly with regard to cytotoxicity and induction of DNA damage. The aim of the present study was to assess in vitro (in hepatic HTC cells) ethanolic extracts of the roots and leaves of C. mollis for cytotoxicity, genotoxicity, and induction of apoptosis. For these evaluations the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay, comet assay, micronucleus test with cytokinesis block, and an in situ test for detection of apoptotic cells with acridine orange staining were used. The results showed that the extract obtained from the roots of C. mollis is more cytotoxic than that obtained from the leaves and that the reduction in cell viability observed in the MTT assay was a result, at least in part, from the induction of apoptosis. Both extracts induced DNA damage at a concentration of 20 mu g/mL in the comet assay, but no genotoxicity was detected with any of the treatments carried out in the micronucleus test.

Effects of uracil calculi on cell growth and apoptosis in the BBN- initiated Wistar rat urinary bladder mucosa

The different potential of initiated and non-initiated urinary bladder mucosa (UBM) to develop neoplasia was quantitatively evaluated in the male Wistar rat. Initiation of carcinogenesis was accomplished with N-butyl-N-(4- hydroxybutyl)-nitrosamine (BBN). Stimuli for cell proliferation and apoptosis were obtained by exposure followed by withdrawal of 3% Uracil in the diet. The proliferation index (PI) was estimated in UBM immunostained for the proliferating nuclear cell antigen (PCNA). The apoptotic index (AI) and the density of papillary/nodular hyperplasia (PNH) were estimated in hematoxilin- eosin stained sections. PNH was the main proliferative response to the mechanical irritation by uracil, irrespective of previous initiation with BBN. Uracil exposure induced higher PI and PNH density in the initiated rats. After uracil withdrawal, there was a significant increase of the AI in both uracil-treated groups, which correlated well to the respective PNH density. However, at the end of the experiment, PNH incidence and density were significantly higher in the BBN-initiated mucosa, which also presented 18% incidence of papillomas and 27% of carcinomas. Therefore, under prolonged uracil calculi trauma, the UBM of BBN-initiated Wistar rats gives rise to epithelial proliferative lesions that progress to neoplasia through acquired resistance to apoptosis.

Apoptosis in the early developing periodontium of rat molars

Development of the periodontium involves a series of complex steps that result in the formation of root dentine, cementum, bone and fibres of the ligament. These precisely controlled and timed events require the participation of the enamel organ derived epithelial cells of Hertwig's (HRS) and ectomesenchymal cells of the dental follicle. These events involve rapid turnover of the tissues and cells, including disappearance of epithelial cells of HRS. Thus, it seemed likely to us that programmed cell death (apoptosis) may play a role in the development of the periodontium. Fragments of first molars, obtained from 14- and 29-day-old rats, were fixed in glutaraldehyde-formaldehyde and processed for light and electron microscopy. For the TUNEL method for detection of apoptosis, specimens were fixed in 4% formaldehyde and embedded in paraffin. Results confirmed that epithelial cells of HRS maintain a close relationship with the forming dentine root, and that they may become trapped in the dentino-cemental junction. Some of the epithelial cells exhibited ultrastructural features which are consistent with the interpretation that they were undergoing programmed cell death, i.e. apoptosis. Periodontal fibroblast-like cells showed typical images of apoptosis and engulfed apoptotic bodies. TUNEL positive structures were present in all corresponding regions. It seems therefore that apoptosis of epithelial cells of HRS and fibroblast-like cells of the periodontal ligament constitutes an integral part of the developmental process of the tissues of the periodontium. (C) 2000 Wiley-Liss, Inc.

Apoptosis in different gastric lesions and gastric cancer: Relationship with Helicobacter pylori, overexpression of p53 and aneuploidy

Apoptosis has an essential function in maintaining the integrity of the gastrointestinal mucosa. Its deregulation is associated with the occurrence of lesions such as in atrophic gastritis, peptic ulcers, intestinal metaplasia, and stomach tumorigenesis. Thus, the aim of the present study was to investigate the frequency of apoptotic cells (apoptotic index, AI) by using two different immunohistochemical techniques, TUNEL and anti-activated caspase-3 antibody (CPP32), in gastric dyspepsia [chronic gastritis (CG, N = 34), chronic atrophic gastritis (CAG, N = 11), gastric ulcer (GU, N = 17), and intestinal metaplasia (IM, N = 15)], normal gastric mucosae (NM, N = 8), and gastric adenocarcinoma (GC, N = 12). The relationship was investigated between the AI and Helicobacter pylori infection, diagnosed by PCR, overexpression of p53 protein determined by immunohistochemistry, and aneuploidy by fluorescence in situ hybridization, as performed by our laboratory in previous studies. No significant differences were observed in AI between the different groups, whether by the TUNEL technique (F = 1.60; P = 0.1670) or by CPP32 antibody (F = 1.70; P = 0.1420). Nonetheless, CAG and CG groups had AI statistically higher than those of normal mucosae. These two groups (CAG and CG) also showed a higher frequency of apoptosis-positive cases (TUNEL+ or CPP32+). Generally, there was no correlation between the AI detected by the TUNEL and CPP32 techniques in the groups studied, except in the GC group (r = 0.70). Moreover, there was no significant association between apoptosis and H. pylori infection, overexpression of p53 protein and aneuploidy, but the H. pylori-positive cases only of GU (P = 0.0233) and IM (P = 0.0253) groups displayed a statistically higher AI compared to H. pylori-negative NM, when the CPP32 antibody technique was used. Thus, CG and CAG have increased apoptosis, which may occur independent of an association with H. pylori infection, aneuploidy and overexpression of p53 protein. ©FUNPEC-RP.

Cellular and molecular mechanisms of apoptosis in skeletal muscle atrophy

Apoptosis is necessary for maintaining the integrity of proliferative tissues, such as epithelial cells of the gastrointestinal and integumentary systems. The role of apoptosis in post-mitotic tissues, such as skeletal muscle, is less well defined, but several lines of evidence suggest that it occurs in both myofiber and other interstitial muscle cell types. Apoptosis of myonuclei likely contributes to the loss of muscle mass, but the mechanisms underlying this process are largely unknown. Caspase-dependent as well as caspase-independent pathways have been implicated, and the mode by which atrophy is induced likely determines the apoptotic mechanisms that are utilized. It remains to be determined whether a decrease in apoptosis will alleviate atrophy and distinct research strategies may be required to clarify the different causes of skeletal muscle mass loss. In this review, it was also speculated that apoptosis is a normal regulatory process that the myofiber can use to reduce the number of nuclear domains, thus ensuring optimal cell functions according to the mechanical load imposed on the muscle. ©FUNPEC-RP.

Selenium supplementation enhances weight gain in cattle

To evaluate the effect of selenium on cattle growth in a continuous grazed pasture system. Selenium was added to protein-mineral salt and given to male, around-12-month old, non-castrated Nellore calves. Animals were randomly and equally distributed into 4 groups (15 calves/group) which received supplementation containing 0, 3.6, 5.4 or 6.4 mg selenium/animal/day (groups Gc, G 3.6, G 5.4 and G 6.4, respectively). The animals were weighed on day zero and 120, and the pasture forages were collected at day 0 for chemical analyses. Weight gain was higher in cattle from G 5,4 (45.58%) than in the other groups, and higher in G 3,6 (24.97%) and G 6,4 (22.67%) than in Gc. The supplementation with 5.4 mg selenium/animal/day enhanced weight gain in cattle fed on selenium poor diet (0.04 mg Se/kg dry matter).

γ-Radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells

We investigated the effects of γ-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca2+ handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca2+, reduced amount of intrareticular Ca2+, and reduced capacitive Ca2+ entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca2+, 1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca2+, amount of intrareticular Ca2+, capacitative Ca2+ entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca2+ handling, and apoptosis appear due to a toxic action of intracellular Ca2+. Ca2+-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca2+ handling and apoptosis induced by γ-radiation. © 2008 Elsevier B.V. All rights reserved.

Smoking enhances bone loss in anterior teeth in a Brazilian population: A retrospective cross-sectional study

The aim of the present study was to radiographically evaluate the effect of smoking on bone loss resulting from chronic periodontitis. Periapical radiographs were analyzed of 80 patients with chronic periodontitis (40 current or former smokers and 40 never-smokers) that attended a private periodontal practice. The smokers or former-smokers with a minimum consumption of 10 cigarettes/day for a period of over 10 years were selected. Interproximal radiographic bone loss was considered as the distance between the cementum-enamel junction and the alveolar bone crest. Bone loss for smokers was higher than that observed in never-smokers (p < 0.05) (3.33 ± 1.09 mm and 2.24 ± 0.76 mm; mean ± standard deviation for smokers and non-smokers, respectively). When each region of the mouth was comparatively evaluated, it was observed that the smokers' incisors presented the highest bone loss when compared with the other groups of teeth (p < 0.01). Within the limits of the present investigation it can be concluded that smoking enhances the bone loss resulting from periodontitis and that the incisors are the teeth most affected.

Inhibition of human neutrophil apoptosis by Paracoccidioides brasiliensis: Role of interleukin-8

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production. © 2008 The Authors.

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd.

Cimetidine-induced vascular cell apoptosis impairs testicular microvasculature in adult rats

Cimetidine, an H2 receptor antagonist used for treatment of gastric ulcers, exerts antiandrogenic and antiangiogenic effects. In the testes cimetidine impairs spermatogenesis, Sertoli cells and peritubular tissue, inducing apoptosis in the myoid cells. Regarding the importance of histamine and androgens for vascular maintenance, the effect of cimetidine on the structural integrity of the testicular vasculature was evaluated. Adult male rats received cimetidine (CMTG) and saline (CG) for 50 days. The testes were fixed in buffered 4% formaldehyde and embedded in historesin and paraffin. In the PAS-stained sections, the microvascular density (MVD) and the vascular luminal area (VLA) were obtained. TUNEL method was performed for detection of cell death. Testicular fragments embedded in Araldite were analyzed under transmission electron microscopy. A significant decrease in the MVD and VLA and a high number of collapsed blood vessel profiles were observed in CMTG. Endothelial cells and vascular muscle cells were TUNEL-positive and showed ultrastructural features of apoptosis. These results indicate that cimetidine induces apoptosis in vascular cells, leading to testicular vascular atrophy. A possible antagonist effect of cimetidine on the H2 receptors and/or androgen receptors in the vascular cells may be responsible for the impairment of the testicular microvasculature.

Caffeine reduces cadmium accumulation in the organism and enhances the levels of antioxidant protein expression in the epididymis

The aim of this study was to investigate the effects of caffeine (20. mg/L) intake on cadmium (15. mg/L) accumulation in the rat blood, testes, epididymis and prostate as well as cadmium-induced changes to the antioxidant defense system of the epididymis. Caffeine reduced the cadmium concentration in all tissues analyzed. Meanwhile, cadmium reduced catalase activity and increased superoxide dismutase (SOD) activity in the epididymis. Caffeine increased SOD activity, catalase and glutathione tissue expression and sustains the cadmium's effect on catalase and GSP-Px activity. No differences in the expression of metallothionein and lipid peroxidation were observed among the different treatments in the epididymis. In conclusion, low doses of cadmium alter the antioxidant enzymatic profile of the epididymis, but not induced oxidative lipid damage. Caffeine intake reduces overall cadmium accumulation in the organism and enhances the levels of antioxidant protein expression in the epididymis, thus exerting a protective effect against this metal. © 2012 Elsevier Inc.