Rodrigo Pinheiro Araldi

Using the comet and micronucleus assays for genotoxicity studies: A review

Physical, chemical and biological agents can act in the DNA, resulting in mutation involved in cancer. Thus, genotoxic tests are required by regulatory agencies in order to evaluate potential risk of cancer. Among these tests, the comet assay (CA) and micronucleus assay (MNA) are the most commonly used. However, there are different protocols and recommendations already published. This is the first review, after the inclusion of CA in S2R1 guidance and OECD 489, which summarizes the main technical recommendations of both CA and MNA.

Apoptotic effect of eugenol envolves G2/M phase abrogation accompanied by mitochondrial damage and clastogenic effect on cancer cell in vitro.

BACKGROUND Eugenol (EUG) is a major phenolic compound present in clove whose anti-cancer properties have been demonstrated previously. These anti-cancer properties may involves the modulation of different mechanisms, including α-estrogen receptor (αER) in luminal breast cancer cells, COX-2 inhibition in melanoma cells or p53 and caspase-3 activation in colon cancer cells. HYPOTHESIS EUG promotes a burst in ROS production causing cell-cycle perturbations, mitochondria toxicity and clastogenesis triggering apoptosis in melanoma breast- and cervix-cancer cells in vitro. METHODS Morphological changes were evaluated through the light- and electronic- microscopy. Cell-cycle, ROS, PCNA and Apoptosis was detected by flow cytometry and clastogenicity was evaluated by Comet-assay. RESULTS The results obtained herein pointed out that EUG promotes, increasing ROS production leading to abrogation of G2/M of phase of cell-cycle, and consecutively, clastogenesis in vitro. In addition, EUG induces Proliferation Cell Nuclear Antigen (PCNA) downregulation and decreasing in mitochondria potential (ΔΨm). Of note, a Bax up-regulation was also observed on cells treated with EUG. All of these findings cooperate in order to induce apoptosis in cancer cells. CONCLUSION These promising results presented herein shed new light on the mechanisms of action of EUG suggesting a possible applicability of this phenylpropanoid as adjuvant in anti-cancer therapy.

Genetics and metabolic deregulation following cancer initiation: A world to explore.

Cancer is a group of highly complex and heterogeneous diseases with several causes. According to the stochastic model, cancer initiates from mutation in somatic cells, leading to genomic instability and cell transformation. This canonical pathway of carcinogenesis is related to the discovery of important mechanisms that regulate cancer initiation. However, there are few studies describing genetic and metabolic alterations that deregulate transformed cells, resulting in epithelial-mesenchymal transition (EMT) and its most dramatic consequence, the metastasis. This review summarizes the main genetics and metabolic changes induced by reactive oxygen species (ROS) that lead to EMT.

The Roles of ROS in Cancer Heterogeneity and Therapy

Cancer comprises a group of heterogeneous diseases encompassing high rates of morbidity and mortality. Heterogeneity, which is a hallmark of cancer, is one of the main factors related to resistance to chemotherapeutic agents leading to poor prognosis. Heterogeneity is profoundly affected by increasing levels of ROS. Under low concentrations, ROS may function as signaling molecules favoring tumorigenesis and heterogeneity, while under high ROS concentrations, these species may work as cancer modulators due to their deleterious, genotoxic or even proapoptotic effect on cancer cells. This double-edged sword effect represented by ROS relies on their ability to cause genetic and epigenetic modifications in DNA structure. Antitumor therapeutic approaches may use molecules that prevent the ROS formation precluding carcinogenesis or use chemical agents that promote a sudden increase of ROS causing considerable oxidative stress inside tumor mass. Therefore, herein, we review what ROS are and how they are produced in normal and in cancer cells while providing an argumentative discussion about their role in cancer pathophysiology. We also describe the various sources of ROS in cancer and their role in tumor heterogeneity. Further, we also discuss some therapeutic strategies from the current landscape of cancer heterogeneity, ROS modulation, or ROS production.

Bovine papillomavirus productive infection in cell cultures: First evidences

Bovine papillomavirus (BPV) is the etiological agent of bovine papillomatosis (BP), infectious disease, characterized by the presence of multiples papillomas that can regress spontaneously or progress to malignances. Although recognized as mutagen, BPV action following cancer initiation remains few explored, since studies about cancer progression and metastasis are based on cell cultures. The lack of attention to in vitro models is a reflection of the papillomavirus replication paradigm, which is dependent of epithelium cell differentiation. Since 2008, we have explored the potential of cell lines derived from BPV-infected neoplasms as model to study the oncogenic process. In this study, we described BPV productive infection in cell lines derived from cutaneous papilloma, fibropapilloma and esophageal carcinoma (EC) in which BPV DNA sequences were previously detected by PCR. Considering that the immunodetection of L1 capsid protein is the main evidence of productive infection, we analyzed the expression of this protein by immunofluorescence and flow cytometry. Results showed the immunodetection of L1 protein in cell lines derived from cutaneous papilloma, fibropapilloma and EC, but not in cells derived from BPV-free normal skin. We also observed the presence of spherical and electron-dense particles, with 41.02-61.94 nm diameter in cytoplasmic vesicles of cells in the sixth passage of cutaneous papilloma, fibropapilloma and EC, being compatible with the expected BPV morphology. Cells derived from BPV-free normal skin, in turn, showed membranous particles up to 75.00 nm not compatible with BPV morphology. These results suggest the BPV productive infection in cells lines derived from BPV-infected neoplasm, reinforcing that these cells are useful models to study the viral biology and pathogenesis. Correspondence to: Rita de Cassia Stocco, Genetics Laboratory, Butantan Institute, São Paulo, 05503-900, Brazil, Tel: +55 11 2627-9701; e-mail: rita. stocco@butantan.gov.br

Integrated analysis of recombinant BPV-1 L1 protein for the production of a bovine papillomavirus VLP vaccine

Bovine papillomatosis is an infectious disease that is caused by bovine papillomavirus (BPV), which results in important economic losses. However, no BPV vaccines or effective treatment methods are commercially available to date. Moreover, the absence of papillomavirus replication in vitro makes the use of recombinant protein a promising candidate for vaccine formulations. Hence, we developed an integrated study on the L1 capsid protein of BPV-1, obtained from a bacterial expression system, regarding its purification, biosafety, thermostability and immunogenicity. The results indicated an absence of genotoxicity of the purified recombinant L1 protein, β-sheet prevalence of secondary structure folding, protein stability under high temperatures as well as the presence of capsomeres and VLPs. In addition, preliminary experimental vaccination of calves showed the production of specific antibodies against BPV-1 L1.

First detection of bovine papillomavirus type 2 in cutaneous wart lesions from ovines

This study diagnosed cutaneous wart lesions excised from three rams from a sheep farm in São Paulo State, Brazil. Histopathologically, these cases were diagnosed as papilloma. The amplification by PCR, sequencing and bioinformatics analysis showed that all the lesions presented DNA sequences of bovine papillomavirus type 2. This is the first report confirming the detection of BPV2 in papilloma warts from ovines.

Analysis of antioxidant, cytotoxic and mutagenic potential of Agave sisalana Perrine extracts using Vero cells, human lymphocytes and mice polychromatic erythrocytes

Brazilian Northeast is the worlds largest producer of Agave sisalana Perrine for the supply of the sisal fiber. About 95% of plant biomass, which comprise the mucilage and sisal juice, is considered a waste residual is discarded in the soil. However, the sisal juice is rich in steroidal saponins, which exhibits different pharmacological properties. Despite this, natural products are not necessarily safe. Based on this, this study analyzed the antioxidant, cytotoxic and mutagenic potential of three extracts derived from acid hydrolysis (AHAS), dried precipitate (DPAS) and hexanic of A. sisalana (HAS). These analyses were performed by in vitro and in vivo methods, using Vero cells, human lymphocytes and mice. Results showed that AHAS 50 and 100 can be considered a useful antineoplastic candidate due to their antioxidant and cytotoxic activity, with no genotoxic/clastogenic potential in Vero cells and mice. Although the comet assay in human lymphocytes has showed that the AHAS 25, AHAS 50 and AHAS 100 can lead to DNA breaks, these extracts did not promote DNA damages in mice bone marrow. Considering the different mutagenic responses obtained with the different methods employed, this study suggest that the metabolizing pathways can produce by-products harmful to health. For this reason, it is mandatory to analyze the mutagenic potential by both in vitro and in vivo techniques, using cells derived from different species and origins.