Avaliação da atividade mutagênica de complexos heterolépticos de Rutênio(II) com atividade anti - Mycobacterium tuberculosis

A tuberculose (TB) é classificada como a segunda causa de morte por um único agente infeccioso, o Mycobacterium tuberculosis. Crescentes incidências de cepas resistêntes a múltiplas drogas (MDR) estão emergindo como uma ameaça global à saúde. Além disso, os medicamentos padrões da terapia são incapazes de controlar o surto de TBMDR e os casos de co-infecção com HIV. Nos últimos anos, um grande número de grupos de pesquisa têm dedicado a sua atenção para o desenvolvimento de agentes antimicobacterianos específicos e de baixo custo, direcionados às cepas MDR. Novos estudos apontam os complexos de rutênio(II) como uma promissora alternativa na terapia anti-TB. Esses complexos vêm sendo testados contra diversas bactérias e estudos atuais indicaram excelentes resultados contra cepas de TB-MDR. Todavia, mesmo com resultados promissores, é imprescindível avaliar seus efeitos toxicológicos, a fim de garantir um uso seguro à saúde humana. Nesse contexto, a avaliação do potencial mutagênico é fundamental para assegurar seu uso sem demonstrar riscos no desenvolvimento de cânceres, bem como outras doenças desencadeadas por alterações no DNA. Baseados nisso, este trabalho teve como objetivo, avaliar os efeitos mutagênicos de três complexos de rutênio(II), com promissora atividade anti-TB-MDR, denominados de SCAR 4, SCAR 5 e SCAR 6 por meio dos ensaios de mutação gênica reversa nas cepas TA1535 TA98, TA97a, TA100 e TA102 de Salmonella typhimurium (Teste de Ames) e pelo ensaio do micronúcleo citoma com bloqueio da citocinese (CBMN-cit) em cultura de células CHO-K1 e HepG2. Em ambos os ensaios, foram utilizados modelos onde foi possível avaliar o efeito da metabolização dos complexos. O ensaio de sobrevivência clonogênica foi utilizado para avaliar o efeito antiproliferativo nas linhagens CHO-K1 e HepG2, além de selecionar...Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Avaliação toxicogenética e atividade antitumoral in vitro de complexos heterolépticos de Rutênio(II): Atividades citotóxicas, genotóxicas e interação com biomoléculas

Os ensaios de toxicologia genética são utilizados como base no desenvolvimento de novos fármacos, uma vez que podem identificar rapidamente agentes que causam danos ao material genético. Agências regulatórias preconizam a utilização de ensaios que identificam danos genotóxicos, de forma que possam servir como triagem de processos que levam à carcinogênese. Na busca por novos fármacos, a química inorgânica medicinal representa um campo de grande promessa, com potencial de expansão devido a diversidade química e reatividade dos metais. Especialmente, os complexos de rutênio, têm recebido destaque no tratamento de doenças como o câncer e a tuberculose multi-droga resistente (TB-MDR). Neste contexto, este trabalho teve como objetivo, avaliar os efeitos toxicogenéticos e antitumorais de três complexos de rutênio(II), com promissora atividade anti-TB-MDR, denominados de SCAR 4, SCAR 5 e SCAR 6 e, diante da importância do conhecimento farmacocinético de novos fármacos, outro objetivo foi avaliar a permeabilidade in vitro desses complexos. A avaliação toxicogenética foi realizada por meio dos ensaios de mutação gênica reversa com Salmonella typhimurium (Teste de Ames) e pelo ensaio do micronúcleo citoma com bloqueio da citocinese (CBMN-cit) em ausência e em presença do sistema de metabolização. Ensaios de sobrevivência clonogênica foram utilizados para avaliar a viabilidade das células CHO-K1 e HepG2, utilizadas no ensaio do CBMN-cit. A avaliação da atividade antitumoral foi investigada por meio de ensaios de citotoxicidade frente às linhagens tumorais humanas Caco-2, DU-145, HeLa, HepG2 e MDA-MB-231, pela capacidade de inibição da topoisomerase I humana (Top IB) e por ensaios físico-químicos de interação com o calf thymus DNA (ct-DNA). A permeabilidade foi analisada por meio do ensaio in vitro de permeação em monocamadas de células Caco-2 na presença e ausência de albumina do soro bovino (BSA). A interação dos complexos com albumina foi analisada por meio da medida da supressão da fluorescência dos resíduos de triptofano da BSA. Os resultados mostraram que apenas o complexo SCAR 6 apresentou efeito mutagênico, observado no Teste de Ames e no CBMN-cit e, em ambos os modelos, o efeito foi apresentado apenas quando o complexo fora submetido a sistemas metabolizadores. Na avaliação da citotoxicidade frente às linhagens tumorais, todos os complexos se mostraram seletivos contra as linhagens de câncer de próstata e mama, com destaque para o complexo SCAR 6, que se apresentou seletivo em quatro diferentes linhagens celulares tumorais. Por outro lado, o complexo SCAR 5 foi altamente ativo na inibição da Top IB, seguido do complexo SCAR 4. Ensaios físico-químicos apontaram os efeitos de interação dos complexos com o ct-DNA, confirmando a capacidade do complexo SCAR 6 se ligar ao DNA de forma covalente, enquanto SCAR 4 e 5 apresentaram um perfil de interação eletrostática com o ácido nucleico. SCAR 4 e 5 também apresentaram alta permeabilidade aparente (Papp) in vitro. O complexo SCAR 6 apresentou baixa Papp, sobretudo com a adição de BSA no tampão de permeabilidade, provavelmente devido a alta afinidade pela albumina apresentada nos ensaios de interação com a BSA, onde, os complexos SCAR 4 e 5, apresentaram constante de ligação moderada. Nossos estudos mostraram que os complexos SCAR 4, 5 e 6 apresentam uma afinidade relevante por biomoléculas como DNA, Top IB e albumina. A interação, mesmo que ainda pouco elucidada por esses alvos biológicos, pode sugerir a aplicação terapêutica desses complexos de rutênio, seja interrompendo vias bioquímicas, causando danos no DNA ou, inclusive, interagindo de forma sinérgica, promovendo terapias mais seguras e eficazes.The genetic toxicology assays are used as the basis in the development of new drugs since it’s may rapidly identify agents that cause damage to the genetic material. Regulatory agencies have recommend the use of assays that identify genotoxic effcts, thus they may serve as a screening processes that lead to carcinogenesis. In the search of new molecules for therapeutic purposes, medicinal inorganic chemistry is a major promise of the field, with potential expansion due to chemical diversity and reactivity of metals. Especially, ruthenium complexes, have been highlighted in the treatment of growing diseases such as cancer and multi-drug resistant tuberculosis (MDR-TB). In this context, this study aimed to evaluate the toxicogenetic effects of three ruthenium(II) complex, that showed promising anti-MDR-TB activity, called SCAR 4, SCAR 5 and SCAR 6. In addition to this safety profile, and cosidering the traditionally antitumor activity of ruthenium complexes, this study also aimed to evaluate the antitumor activity of these complexes. In front of the importance of pharmacokinetic knowledge of new drugs, another aim of this study was to evaluate the in vitro permeability of these complexes. The toxicogenetics evaluation was performed by reverse gene mutation assays with Salmonella typhimurium (Ames test) and by Cytokinesis-block micronucleus cytome assay (CBMN-cyt). In both assays, we used models to assess the effect of complexes’s metabolization. The clonogenic survival assays were used to assess the viability of HepG2 and CHO-K1 cells, that it has been used in CBMN-cyt assay. The evaluation of the antitumor activity was investigated by means of cytotoxicity assays with the human tumor cell lines Caco-2, DU-145, HeLa, HepG2 and MDA-MB-231, by the ability to inhibit human topoisomerase I (Top IB) and physico-chemical assays of interaction with the thymus calf DNA (ct-DNA). The permeability was examined by means of in vitro permeation assay with Caco-2 cell monolayers in presence and absence of Bovine Serum Albumin (BSA). The interaction of the complex with albumin was analyzed by measuring the suppression of the fluorescence of tryptophan residues from BSA. In genotoxicity evaluation, only SCAR 6 complex showed mutagenic effect. This effect was observed in the Ames test and the CBMN-cyt and, in both models, the effects were observed only when the complex was subjected to metabolizers systems. In cytotoxicity evaluation, all the complexes showed selective index against prostate and breast cancer cell lines, especially SCAR 6, which was selective in four different tumor cell lines. Moreover, the SCAR 5 complex was highly active in inhibiting Top IB, followed by SCAR 4. Physico-chemical assays showed the effects of interaction of the complexes with ct-DNA, confirming the capacity of SCAR 6 to bind covalently to DNA, while SCAR 4 and 5 showed an electrostatic interaction profile with DNA. SCAR 4 and 5 also showed high apparent permeability (Papp) in vitro. The SCAR 6 complex showed low Papp, especially with the addition of BSA in the permeability buffer, probably due to the high affinity for albumin presented in interaction assays with BSA, where the complexes SCAR 4 and 5 have showed moderate binding. Our studies showed that SCAR 4, 5 and 6 have presented a relevant affinity for biomolecules such as DNA, Top IB and albumin. The interaction, even middling elucidated, by these biological targets, may suggest the therapeutic application of these ruthenium complexes, such as disrupting biochemical pathways, causing DNA damage, or even interacting synergistically, promoting more safer and effective therapies.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

In Vitro Evaluation of Acellular Collagen Matrices Derived from Porcine Pericardium: Influence of the Sterilization Method on Its Biological Properties

The aim of this study were characterize acellular collagen matrices derived from porcine pericardium (PP) and to evaluate their properties after sterilization by ethylene oxide and gamma ray. PP matrices were subjected to alkaline hydrolysis (AH), and samples were characterized for biological stability, membrane thickness measurements, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). Subsequently, the matrices were frozen, lyophilized and sterilized by ethylene oxide or gamma radiation. For in vitro assays, CHO-K1 cell culture was used and evaluated for cytotoxicity, clonogenic survival assay, genotoxicity and mutagenicity. Analysis of variance (ANOVA) was used, followed by Dunnett’s post-test, with a significance level of 5%. After AH, there was no significant change in matrix thickness. The relative biodegradability of the material after implantation was observed. Morphology and dimensions had small changes after AH. As for cell viability, none of the tested matrices showed a statistically significant difference (p > 0.05; Dunnett) regardless of the sterilization method. Furthermore, it was found that PP matrices did not interfere with the proliferation capacity of CHO-K1 cells (p > 0.05; Dunnett). As for genotoxicity, when sterilized with ethylene oxide (NP, P12 and P24), it showed genotoxic potential, but it was not genotoxic when sterilized by gamma radiation. No mutagenic effects were observed in either group. PP-derived collagen matrices hydrolyzed at different times were not cytotoxic. It is concluded that the best method of sterilization is through gamma radiation, since no significant changes were observed in the properties of the PP matrices

Characterization and Quantification of the Compounds of the Ethanolic Extract from Caesalpinia ferrea Stem Bark and Evaluation of Their Mutagenic Activity

Caesalpinia ferrea Martius has traditionally been used in Brazil for many medicinal purposes, such as the treatment of bronchitis, diabetes and wounds. Despite its use as a medicinal plant, there is still no data regarding the genotoxic effect of the stem bark. This present work aims to assess the qualitative and quantitative profiles of the ethanolic extract from the stem bark of C. ferrea and to evaluate its mutagenic activity, using a Salmonella/microsome assay for this species. As a result, a total of twenty compounds were identified by Flow Injection Analysis Electrospray Ionization Ion Trap Mass Spectrometry (FIA-ESI-IT-MS/MSn) in the ethanolic extract from the stem bark of C. ferrea. Hydrolyzable tannins predominated, principally gallic acid derivatives. The HPLC-DAD method was developed for rapid quantification of six gallic acid compounds and ellagic acid derivatives. C. ferrea is widely used in Brazil, and the absence of any mutagenic effect in the Salmonella/microsome assay is important for pharmacological purposes and the safe use of this plant

Mutagenicity and chemopreventive activities of Astronium species assessed by Ames test

In the neotropical savannah, Astronium species are used in popular medicine to treat allergies, inflammation, diarrhea and ulcers. Given that natural products are promising starting points for the discovery of novel potentially therapeutic agents, the aim of the present study was to investigate the mutagenic and antimutagenic activities of hydroalcoholic extracts of Astronium spp. The mutagenicity was determined by the Ames test on Salmonella typhimurium strains TA98, TA97a, TA100 and TA102. The antimutagenicity was tested against the direct-acting and indirect-acting mutagens. The results showed that none of the extracts induce any increase in the number of revertants, demonstrating the absence of mutagenic activity. On the other hand, the results on the antimutagenic potential showed a moderate inhibitory effect against NPD and a strong protective effect against B[a]P and AFB1. This study highlights the importance of screening species of Astronium for new medicinal compounds. The promising results obtained open up new avenues for further study and provide a better understanding the mechanisms by which these species act in protecting DNA from damage. However, further pharmacological and toxicological investigations of crude extracts of Astronium spp., as well as of its secondary metabolites, are necessary to determine the mechanism(s) of action to guarantee their safer and more effective application to human health

Mutagenicity and antimutagenicity of six Brazilian Byrsonima species assessed by the Ames test

Background: In various regions of Brazil, several species of the genus Byrsonima (Malpighiaceae) are widely used to treat gastrointestinal complications. This genus has about 150 species of shrubs and trees distributed over the entire Neotropical region. Various biological activities have been identified in these plants, especially antioxidant, antimicrobial and topical and systemic anti-inflammatory activities. The aim of this study was to investigate the mutagenicity and antimutagenicity of hydroalcoholic leaf extracts of six species of Byrsonima: B. verbascifolia, B. correifolia, B. coccolobifolia, B. ligustrifolia, B. fagifolia and B. intermedia by the Salmonella microsome assay (Ames test).Methods: Mutagenic and antimutagenic activity was assessed by the Ames test, with the Salmonella typhimurium tester strains TA100, TA98, TA97a and TA102, with (+S9) and without (-S9) metabolization, by the preincubation method.Results: Only B. coccolobifolia and B. ligustrifolia showed mutagenic activity. However, the extracts of B. verbascifolia, B. correifolia, B. fagifolia and B. intermedia were found to be strongly antimutagenic against at least one of the mutagens tested.Conclusions: These results contribute to valuable data on the safe use of medicinal plants and their potential chemopreventive effects. Considering the excellent antimutagenic activities extracted from B. verbascifolia, B. correifolia, B. fagifolia and B. intermedia, these extracts are good candidate sources of chemopreventive agents. However, B. coccolobifolia and B. ligustrifolia showed mutagenic activity, suggesting caution in their use