Increase in the Antioxidant and Anti-Inflammatory Activity of Euterpe oleracea Martius Oil Complexed in ß-Cyclodextrin and Hydroxypropyl-ß-Cyclodextrin.

Reactive oxygen species (ROS) are aerobic products generated during cellular respiration, but in the case of oxidative stress, they become key factors in the development of inflammatory processes and chronic diseases such as diabetes and rheumatoid arthritis. In this work, Euterpe oleracea oil (EOO), as well as the complexes produced by slurry (S) and kneading (K), were analyzed for antioxidant capacity in vitro, while only the ß-cyclodextrin complex obtained by kneading (EOO-ßCD-K), which showed better complexation, was selected for anti-inflammatory assays in vivo. In the scavenging activity of OH·, the hydroxypropyl-ß-cyclodextrin complex obtained by kneading (EOO-HPßCD-K) exhibited an activity 437% higher than the pure oil. In the paw edema assay, EOO-ßCD-K reduced edema by 200% and myeloperoxidase (MPO) activity by 112%. In an air pouch model, this treatment showed a reduction in leukocyte, MPO, and Interleukin-1ß (IL-1ß) levels; meanwhile those of glutathione and IL-10 were increased, demonstrating its ability to potentiate the anti-inflammatory effect of EOO.

Low-level laser irradiation induces in vitro proliferation of stem cells from human exfoliated deciduous teeth.

The aim of this study was to evaluate the effect of low-level laser irradiation (LLLI) on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHED). Cells were irradiated or not (control) with an InGaAlP laser diode (660 nm, 30 mW, continuous action mode) using two different energy densities (0.5 J/cm2-16 s; 1.0 J/cm2-33 s). Irradiation was performed at 0 and 48 h, with the laser probe fixed at a distance of 0.5 cm from the cells. Cell proliferation was analyzed at 0, 24, 48, and 72 h by the Trypan blue exclusion method and MTT assay. Cell cycle and Ki67 expression were analyzed by flow cytometry. Apoptosis-related events were evaluated by expression of annexin V/PI and nuclear morphological changes by staining with DAPI. Differences between groups at each time were analyzed by the Kruskal-Wallis and Mann-Whitney tests, adopting a level of significance of 5% (p < 0.05). The results showed that an energy density of 1.0 J/cm2 promoted an increase in cell proliferation at 48 and 72 h compared to the control and 0.5 J/cm2 groups. Cell cycle analysis revealed a predominance of cells in the S and G2/M phases in the irradiated groups. This finding was confirmed by the increased expression of Ki67. Low positive staining for annexin V and PI was observed in all groups, and no nuclear changes were detected, indicating that cell viability was not affected by the energy densities tested. It can be concluded that the LLLI parameters used (660 nm, 30 mW, 1.0 J/cm2) promote the proliferation of SHEDs and the maintenance of cell viability.

Characterization and Antiproliferative Activity of a Novel 2-Aminothiophene Derivative-ß-Cyclodextrin Binary System.

The novel 2-aminothiophene derivative 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (6CN) has shown potential anti-proliferative activity in human cancer cell lines. However, the poor aqueous solubility of 6CN impairs its clinical use. This work aimed to develop binary 6CN-ß-cyclodextrin (ßCD) systems with the purpose of increasing 6CN solubility in water and therefore, to improve its pharmacological activity. The 6CN-ßCD binary systems were prepared by physical mixing, kneading and rotary evaporation methods and further characterized by FTIR, XRD, DSC, TG and SEM. In addition, molecular modeling and phase solubility studies were performed. Finally, MTT assays were performed to investigate the cytostatic and anti-proliferative effects of 6CN-ßCD binary systems. The characterization results show evident changes in the physicochemical properties of 6CN after the formation of the binary systems with ßCD. In addition, 6CN was associated with ßCD in aqueous solution and the solid state, which was confirmed by molecular modeling and the aforementioned characterization techniques. Phase solubility studies indicated that ßCD forms stable 1:1 complexes with 6CN. The MTT assay demonstrated the cytostatic and anti-proliferative activities of 6CN-ßCD binary systems and therefore, these might be considered as promising candidates for new anticancer drugs.

Morphology of the genital organs of the female red-rumped agouti (Dasyprocta leporina, Linnaeus, 1758) during estrous cycle phases and in advanced pregnancy.

The study investigated the gross and microscopic anatomy of the genital organs of 20 agoutis at different stages of the estrous cycle and four in the final trimester of pregnancy. Specimens were euthanized and their reproductive organs were fixed in a 4% paraformaldehyde or 2.5% glutaraldehyde solution and submitted to routine histological techniques for light and scanning electron microscopy. In the ovary, during the proestrus phase, we observed developing follicles and corpus luteum (CL) in regression; during estrus, there were Graafian follicles; during metestrus, there was a hemorrhagic corpus, whereas in diestrus, there was a mature CL. The uterus was partially double because the cervix was cranially septate but caudally, the septum disappeared, forming a single ostium that opened into the vagina. Changes occurred along the estrous cycle in the uterine and vaginal epithelia, that is, an increase in the uterine epithelium height accompanied by an increase of thickness of the vaginal epithelium during the follicular phase and a decrease of thickness of both epithelia during the luteal phase. The endometrial lining was composed of a simple cuboidal epithelium to simple columnar epithelium with basal nuclei. The vaginal mucosa consisted of epithelium that varied from nonkeratinized stratified squamous (luteal phase) to keratinized stratified squamous (follicular phase). The clitoris was external to the vagina. It presented two protruding lateral keratinized spicules and a centralized urethra, with no common parts between the urinary and genital tracts. Anatomical and histological changes were observed mainly in the cervix, vagina and spicules of the clitoris during the EC.

Diosgenin induces genotoxic and mutagenic effects on HepG2 cells.

Yam roots and other plants from Dioscorea genus have cultural, nutritional and economic importance to tropical and subtropical regions and have a great amount of diosgenin in its composition. In the present study the cytotoxic, genotoxic and mutagenic potential of diosgenin on HepG2 cells was investigated. Cytotoxicity was assessed using MTT and clonogenic assay. Genotoxic and mutagenic effects were performed using single cell gel electrophoresis and cytokinesis-block micronucleus assay, respectively. A reduction on cell viability was observed due to diosgenin treatment at concentrations higher than 30 µM. A genotoxic effect was shown by comet assay and CBMN. Besides, an increase in micronucleus frequency along with a significant cytostatic effect were observed. Diosgenin elicited DNA damage on HepG2 cells which could not be efficiently repaired contributing to the mutagenic effect observed. Those results suggest that diosgenin deleterious effect could take place through genetic instability, fact that affects the normal cell cycle, leading to cell's death.

Use of Zymography in Trypanosomiasis Studies.

Zymography assay is a semiquantitative technique, very sensitive, and commonly used to determine metalloproteinase levels in different types of biological samples, including tissues, cells, and extracts of protein. Samples containing metalloproteinases are loaded onto a polyacrylamide gel containing sodium dodecyl sulphate (SDS) and a specific substrate (gelatin, casein, collagen, etc.). Then proteins are allowed to migrate under an electric current and the distance of migration is inversely correlated with the molecular weight. After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity (metalloproteinase activity). In the context of infections caused by trypanosomatids (Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei), the characterization of metalloproteinase by zymography can contribute to the comprehension of the pathogenesis mechanisms and host-parasite interaction.

Post-implantation development of red-rumped agouti (Dasyprocta leporina Linnaeus, 1758).

Fourteen pregnant agouti (Dasyprocta leporina Linnaeus, 1758) females at gestational ages of 30-100days and two at gestational ages of 55-100days, were studied, including 24 embryos/fetuses and two neonates (single offspring from two females). Females were euthanized and embryos/fetuses were examined, measured, and photographed. Embryos at 30days post-copulation were "C-shaped," with paddle-shaped limb buds and 10.75±0.11mm crown-rump length (CRL). In embryos at 35days post-copulation, paddle-shaped forelimbs, fin-shaped pelvic limbs, and a liver bud were evident, with 15.6±0.16mm CRL; at 45days, mouth and nostrils were evident, lower incisors were emerging, and tactile hairs could be seen near the nostrils, with 40.8±0.4mm CRL; at 55days, cranial sutures had fused, digits were totally separated and claws formation was beginning, with 58.8±0.1mm CRL; at 65days, the genital bud had differentiated, with 83±0.08mm CRL; at 85days, the body was covered with short hair, with 127.7±0.6mm CRL; and at 100days, the mature fetus had fully formed teeth and claws and an open external acoustic meatus, with 164.3±10.4mm CRL. Neonates had bristly hair and open eyes, with 179.1±0.5mm CRL. In conclusion, characteristics of the embryo/fetus during pregnancy can be used to estimate gestational age, and can be used as parameters during ultrasound examinations to help identify developmental pathologies and to test effects of nutrition, drugs, and pollution on pregnancy.

Evaluation of the antiproliferative activity of 2-amino thiophene derivatives against human cancer cells lines.

In spite of great progress in understanding cancer biology, current therapeutic procedures remain unsatisfactory. Chemotherapy is often followed by secondary effects with cellular toxicity negatively affecting the results. The discovery and development of new safe and efficient antitumor agents is necessary. Derivatives of 2-amino thiophene have been a topic of constant investigation due to their versatile synthetic applicability and broad spectrum of biological applications; among which are antifungal and antiproliferative activity shown in prior studies of our group. In the current study, compounds 6CN09, 6CN10, 6CN12, 6CN14, 7CN09 and 7CN11 were analyzed as to antiproliferative effect in human cells of cervical adenocarcinoma (HeLa), human pancreatic adenocarcinoma (PANC-1) and mice fibroblasts (3T3), which were exposed to the compounds in concentrations of 5, 10, 25 and 50µM during 24 and 48h. They were submitted to MTT assay. In order to elucidate the action mechanism of antitumor thiophene derivatives flow cytometry was performed to evaluate cell death and cell cycle analysis. The results showed that thiophene derivatives demonstrated great antiproliferative potential in the HeLa and PANC-1 cell lines when compared with the control, and the percentage of cell proliferation inhibition approximated or was higher than the standard drug used; doxorubicin (Dox). In highlight were the derivatives 6CN14 and 7CN09 that showed greater efficiency in the antiproliferative evaluation. Further, all compounds had a protective effect on the non-tumor 3T3 cell line. The flow cytometry analysis showed few cells in apoptosis in both the HeLa and PANC-1 lines, although the compounds interfered with the progression of the cell cycle, and avoided cell growth and multiplication in the HeLa tumor line. These thiophene derivatives demonstrated cytostatic and antiproliferative effects and may be considered as promising molecular candidates for anticancer drugs.

Effect of Hecogenin on DNA instability.

Hecogenin is a sapogenin found in Agave species in high quantities and is responsible for the many therapeutic effects of these medicinal plants. In addition, this compound is also widely used in the pharmaceutical industry as a precursor for the synthesis of steroidal hormones and anti-inflammatory drugs. Despite Hecogenin being widely used, little is known about its toxicological properties. Therefore, the present study aimed to investigate the cytotoxic, genotoxic and mutagenic effects of Hecogenin on HepG2 cells. Cytotoxicity was analyzed using the MTT test. Then, genotoxic and mutagenic potentials were assessed by comet assay and cytokinesis-block micronucleus assay, respectively. Cytotoxic effect was observed only when cells were exposed to concentrations of Hecogenin equal or higher than 100 µM. Although a lower concentration of Hecogenin caused DNA damage, a reduction on nuclear mutagenic markers in HepG2 cells was observed. The results indicated that Hecogenin treatment generated DNA damage, but in fact it would be repaired, avoiding dissemination of the damage throughout the cell division. Further studies need to be performed to confirm the observed protective effect of Hecogenin against genomic instability.

Telmisartan induces apoptosis and regulates Bcl-2 in human renal cancer cells.

It has been well-characterized that the renin-angiotensin system (RAS) physiologically regulates systemic arterial pressure. However, RAS signaling has also been shown to increase cell proliferation during malignancy, and angiotensin receptor blockers (ARBs) are able to decrease pro-survival signaling by inhibiting anti-apoptotic molecules and suppressing caspase activity. In this study, the apoptotic effects of telmisartan, a type of ARB, was evaluated using a non-cancerous human renal cell line (HEK) and a human renal cell carcinoma (RCC) cell line (786). Both types of cells were treated with telmisartan for 4 h, 24 h, and 48 h, and then were assayed for levels of apoptosis, caspase-3, and Bcl-2 using MTT assays, flow cytometry, and immunostaining studies. Analysis of variance was used to identify significant differences between these data (P < 0.05). Following the treatment of 786 cells with 100 µM and 200 µM telmisartan, a marked inhibition of cell proliferation was observed. 50 µM cisplatin also caused high inhibition of these cells. Moreover, these inhibitions were both concentration- and time-dependent (P < 0.05). Various apoptotic effects were also observed compared with control cells at the 24 h and 48 h timepoints assayed (P < 0.001). Furthermore, positive caspase-3 staining and down-regulation of Bcl-2 were detected, consistent with induction of cell death. In contrast, treatment of HEK cells with telmisartan did not produce an apoptotic effect compared with control cells at the 24 h timepoint (P > 0.05). Treatment with cisplatin promoted in HEK cells high index of apoptosis (P < 0.001). Taken together, these results suggest that telmisartan induces apoptosis via down-regulation of Bcl-2 and involvement of caspase-3 in human RCC cells.