Ezequiel Viveros-Valdez

Induction of virulence factors, apoptosis, and cytokines in precision-cut hamster liver slices infected with Entamoeba histolytica

Precision-cut liver slices (PCLS) are mainly used to evaluate hepatotoxicity and metabolism of chemicals, as well as to study mechanisms of liver damage and repair. However, recently they have been used as a system to study amoebic infections. The aim of this study was to validate this model as an alternative for experimental amoebic liver absess (ALA) in animals. To do this, the PCLS was analyzed for the expression of amoebapore and cysteine proteinases 1 and 5, three of the most studied virulence factors of Entamoeba histolytica, as well as the induction of apoptosis and cytokines production in response to the ex vivo infection. PCHLS were prepared with the Brendel-Vitron tissue slicer and then, infected with 200,000 trophozoites of E. histolytica. Samples were taken at 0, 6, 12, 18, and 24 h and compared to control non-infected slices. Morphological studies were performed in order to verify the infection; while apoptosis was studied by TUNEL and PAS techniques. The expression of cysteine proteinases (1 and 5), and amoebapore, was analyzed by real-time PCR. By using ELISA assays, the production of cytokines was also studied. PCHLS were found to be a reproducible infection system, and E. histolytica caused the expression of cysteine proteinases and amoebapore in infected slices. At the same time, trophozoites induce release of cytokines and apoptotic death of the hepatocytes close to them. PCHLS represent a new and suitable alternative model to study the pathogenesis of hepatic amoebiasis.

Organotypic Culture of Breast Tumor Explants as a Multicellular System for the Screening of Natural Compounds with Antineoplastic Potential

Breast cancer is the leading cause of death in women worldwide. The search for novel compounds with antitumor activity, with less adverse effects and higher efficacy, and the development of methods to evaluate their toxicity is an area of intense research. In this study we implemented the preparation and culture of breast tumor explants, which were obtained from precision-cut breast tumor slices. In order to validate the model we are proposing to screen antineoplastic effect of natural compounds, we selected caffeic acid, ursolic acid, and rosmarinic acid. Using the Krumdieck tissue slicer, precision-cut tissue slices were prepared from breast cancer samples; from these slices, 4 mm explants were obtained and incubated with the selected compounds. Viability was assessed by Alamar Blue assay, LDH release, and histopathological criteria. Results showed that the viability of the explants cultured in the presence of paclitaxel (positive control) decreased significantly (P < 0.05); however, tumor samples responded differently to each compound. When the explants were coincubated with paclitaxel and compounds, a synergic effect was observed. This study shows that ex vivo culture of breast cancer explants offers a suitable alternative model for evaluating natural or synthetic compounds with antitumor properties within the complex microenvironment of the tumor.

Free radical scavengers from the Mexican herbal tea "poleo" (Hedeoma drummondii).

The aerial parts of the Lamiaceae Hedeoma drummondii (Benth.) are used in Mexico to prepare a herbal tea and by North American Amerindians as a spice. The methanolic extract of the aerial parts exhibited a strong antioxidant effect measured by the scavenging of the free diphenyl picrylhydrazyl (DPPH) radical. Assay-guided fractionation of the crude methanolic extract allowed the identification of three major active constituents, chlorogenic, caffeic and rosmarinic acid, as well as sideritoflavone derivatives and simple phenolics. The TEAC, FRAP, total phenolic and flavonoid content were determined. The high content of caffeic acid and rosmarinic acid relates to the antioxidant activity of H. drummondii.

Breast Organotypic Cancer Models

Breast cancer is the most common cancer type diagnosed in women, it represents a critical public health problem worldwide, with 1,671,149 estimated new cases and nearly 571,000 related deaths. Research on breast cancer has mainly been conducted using two-dimensional (2D) cell cultures and animal models. The usefulness of these models is reflected in the vast knowledge accumulated over the past decades. However, considering that animal models are three-dimensional (3D) in nature, the validity of the studies using 2D cell cultures has recently been questioned. Although animal models are important in cancer research, ethical questions arise about their use and usefulness as there is no clear predictivity of human disease outcome and they are very expensive and take too much time to obtain results. The poor performance or failure of most cancer drugs suggests that preclinical research on cancer has been based on an over-dependence on inadequate animal models. For these reasons, in the last few years development of alternative models has been prioritized to study human breast cancer behavior, while maintaining a 3D microenvironment, and to reduce the number of experiments conducted in animals. One way to achieve this is using organotypic cultures, which are being more frequently explored in cancer research because they mimic tissue architecture in vivo. These characteristics make organotypic cultures a valuable tool in cancer research as an alternative to replace animal models and for predicting risk assessment in humans. This chapter describes the cultures of multicellular spheroids, organoids, 3D bioreactors, and tumor slices, which are the most widely used organotypic models in breast cancer research.

Formaldehyde induces DNA strand breaks on spermatozoa and lymphocytes of Wistar rats

Formaldehyde (FA) interacts with biological molecules such as DNA and it induces DNA-protein cross-links (DPCs), oxidative stress, reactive oxygen species (ROS), methylation, chromosomal damage, fragmentation, and adducts of DNA, which are considered the most important genotoxic effects caused by exposure to FA. The purpose of this study was to evaluate the percentage of DNA fragmentation on lymphocytes and spermatozoa from Wistar rats exposed to different doses of FA. The results about the percentage of fragmentation of DNA in lymphocytes and spermatozoa, were statistical different from controlled group versus treated groups respectively to (p < 0.05). Pathological changes were observed in the seminiferous tubules, especially in rats exposed to 30 mg/kg of FA. This study provided additional evidence supporting that FA induces DNA strand breaks in both cells and therefore genotoxic damage in Wistar rats.

HIGH SPEED CENTRIFUGAL COUNTERCURRENT CHROMATOGRAPHY (HSCCC) ISOLATION AND IDENTIFICATION BY LC-MSn ANALYSIS OF THE POLAR PHENOLICS FROM VASCONCELLEA QUERCIFOLIA

High speed centrifugal countercurrent chromatography (HSCCC) was successfully applied to preparative isolation of the main polar phenolics from the Peruvian Caricaceae Vasconcellea quercifolia A. St.-Hil. High-performance liquid chromatography with diode array (HPLC-DAD) and electrospray ionisation mass spectrometric detection (ESI-MS) was used to carry out a comprehensive characterisation of phenolic compounds from fruits and leaves of this plant. The main phenolics were the quercetin diglycoside rutin and the triglycoside manghaslin. Nine minor flavonoids were tentatively identified as kaempferol, quercetin, isorhamnetin and methoxymyricetin glycosides. HSCCC combined with other chromatographic methods allowed the isolation of the main phenolics from the plant. HPLC-DAD-MS analysis shows that the main phenolic compounds in V. quercifolia are similar to those described for V. pubescens but differs in the identity of the minor constituents.