Murine response to the opportunistic bacterium Pseudomonas aeruginosa infection in gut dysbiosis caused by 5-fluorouracil chemotherapy-induced mucositis.

AIMS: This manuscript aims to explain the relationship between mucositis caused by 5-FU over gut bacterial species and susceptibility to opportunistic infection caused by P. aeruginosa. MAIN METHODS: BALB/c mice were intraperitoneally treated with PBS or 5-FU. Bodyweight and faecal consistency were checked daily. Mice faecal DNA was extracted, and bacterial phylogenetic groups were analysed using qPCR or high-throughput sequencing. Immunofluorescence was used to evaluate BMDM activation by mice-treated faecal content. Mice were challenged intratracheally with virulent P. aeruginosa, and the CFU and histology were analysed. Faecal microbiota were transplanted to evaluate the gut microbiota and resistance to pulmonary P. aeruginosa recovery. KEY FINDINGS: The animals treated with 5-FU presented mucositis with great weight loss, altered faecal consistency, bacterial gut dysbiosis and histological changes in the intestinal mucosa. Mice under 5-FU treatment were more susceptible to lung infection by the bacteria P. aeruginosa and had more extensive tissue damage during their lung infection with greater pro-inflammatory gene expression. It was observed that the mucositis remained in the groups with 5-FU even with the FMT. The results caused by mucositis in animals that received allogeneic FMT were reversed, however, with a decrease in P. aeruginosa susceptibility in animals treated with 5-FU and allogeneic FMT compared to animals treated with 5-FU and autologous FMT. SIGNIFICANCE: Treatment with 5-FU in a murine model makes it more susceptible to pulmonary infection by the bacterium P. aeruginosa, FMT offers an opportunity to protect against this susceptibility to infection.

Pyrene-polyethylene glycol-modified multi-walled carbon nanotubes: Genotoxicity in V79-4 fibroblast cells.

The genotoxicity of pyrene-polyethylene glycol-modified multi-walled carbon nanotubes (MWCNT-PyPEG), engineered as a nanoplatform for bioapplication, was evaluated. Toxicity was assessed in hamster lung fibroblast cells (V79-4). MTT and Cell Titer Blue methods were used to evaluate cell viability. Genotoxicity was measured by the comet assay and the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay, and fluorescence in situ hybridization (FISH) was used to test induction of structural chromosome aberrations (clastogenic activity) and/or numerical chromosome changes (aneuploidogenic activity). Exogenous metabolic activation enzymes were used in the CBMN-Cyt and FISH tests. Only with metabolic activation, the hybrids caused chromosomal damage, by both clastogenic and aneugenic processes.

Acute oral toxicity, antinociceptive and antimicrobial activities of kava dried extracts and synthetic kavain.

Piper methysticum G. Forst, popularly known as kava, is a traditional medicinal plant widely used for the treatment of anxiety and insomnia. The aim of this study was to investigate new therapeutic applications of this plant. Nociceptive response induced by heat (hot-plate) was used as pain model. Susceptibility of different strains to kava ethanolic dried extracts was evaluated by broth microdilution method. Acute oral toxicity was performed according to Organisation for Economic Cooperation and Development (OECD) guideline. Administration of kava dried extracts and kavain inhibited the nociceptive response in the hot-plate model and did not affect the time mice spent in the rota-rod apparatus. The samples showed no significant antibacterial activity, however slight antifungal activity was verified. The extracts may be considered of low oral acute toxicity. Kava extracts exhibited promising antinociceptive activity in model of nociceptive pain, which should be deeper explored as a new therapeutic application of kava.

In silico, in vitro and in vivo evaluation of natural Bignoniaceous naphthoquinones in comparison with atovaquone targeting the selection of potential antimalarial candidates.

The natural naphthoquinones lapachol, α- and ß-lapachone are found in Bignoniaceous Brazilian plant species of the Tabebuia genus (synonym Handroanthus) and are recognized for diverse bioactivities, including as antimalarial. The aim of the present work was to perform in silico, in vitro and in vivo studies to evaluating the antimalarial potential of these three naphthoquinones in comparison with atovaquone, a synthetic antimalarial. The ADMET properties of these compounds were predicted in silico by the preADMET program. The in vitro toxicity assays were experimentally determined in immortalized and tumoral cells from different organs. In vivo acute oral toxicity was also evaluated for lapachol. Several favorable pharmacokinetics data were predicted although, as expected, high cytotoxicity was experimentally determined for ß-lapachone. Lapachol was not cytotoxic or showed low cytotoxicity to all of the cells assayed (HepG2, A549, Neuro 2A, LLC-PK1, MRC-5), it was nontoxic in the acute oral test and disclosed the best parasite selectivity index in the in vitro assays against chloroquine resistant Plasmodium falciparum W2 strain. On the other hand, α- and ß-lapachone were more potent than lapachol in the antiplasmodial assays but with low parasite selectivity due to their cytotoxicity. The diversity of data here reported disclosed lapachol as a promising candidate to antimalarial drug development.

Vancomycin-induced gut dysbiosis during Pseudomonas aeruginosa pulmonary infection in a mice model.

Pseudomonas aeruginosa is one of the most common opportunistic pathogens causing respiratory infections in hospitals. Vancomycin, the antimicrobial agent usually used to treat bacterial nosocomial infections, is associated with gut dysbiosis. As a lung-gut immunologic axis has been described, this study aimed to evaluate both the immunologic and histopathologic effects on the lungs and the large intestine resulting from vancomycin-induced gut dysbiosis in the P. aeruginosa pneumonia murine model. Metagenomic analysis demonstrated that vancomycin-induced gut dysbiosis resulted in higher Proteobacteria and lower Bacteroidetes populations in feces. Given that gut dysbiosis could augment the proinflammatory status of the intestines leading to a variety of acute inflammatory diseases, bone marrow-derived macrophages were stimulated with cecal content from dysbiotic mice showing a higher expression of proinflammatory cytokines and lower expression of IL-10. Dysbiotic mice showed higher levels of viable bacteria in the lungs and spleen when acutely infected with P. aeruginosa, with more lung and cecal damage and increased IL-10 expression in bronchoalveolar lavage. The susceptible and tissue damage phenotype was reversed when dysbiotic mice received fecal microbiota transplantation. In spite of higher recruitment of CD11b+ cells in the lungs, there was no higher CD80+ expression, DC+ cell amounts or proinflammatory cytokine expression. Taken together, our results indicate that the bacterial community found in vancomycin-induced dysbiosis dysregulates the gut inflammatory status, influencing the lung-gut immunologic axis to favor increased opportunistic infections, for example, by P. aeruginosa.

Extracellular protease profile of Acanthamoeba after prolonged axenic culture and after interaction with MDCK cells.

Free-living amoebae of the genus Acanthamoeba are causative agents of Acanthamoeba keratitis and amoebic encephalitis in humans, both of which are serious infections. The ability to produce proteases is one of the factors involved in the pathogenesis of Acanthamoeba infections. The aim of this study was to evaluate the secreted proteases of six Acanthamoeba strains from distinct genotypes (T1, T2, T4 and T11) maintained in prolonged axenic culture and following three successive passages in Madin-Darby Canine Kidney (MDCK) cells. Conditioned medium was obtained from cultures before and after interaction with the MDCK monolayers, resolved in SDS-PAGE containing gelatine, then subjected to quantitative azocasein assays. Zymography profiles varied between the strains, with the predominant proteases found to be serine-type proteases from 49 to 128 kDa. A T1 genotype strain isolated from dust showed quantitatively higher protease secretion compared to the other strains. No changes were detected in the zymography profiles of MDCK-interacted cultures compared to long-term axenic cultures. Two strains presented lower proteolytic activity post-MDCK interaction, while the remaining strains presented similar values before and after MDCK passages. In conclusion, this study confirms the predominance of serine-type protease secretion by Acanthamoeba, with distinct profiles presented by the different strains and genotypes studied. Also, interaction of trophozoites with MDCK cells did not alter the zymography pattern.

In vitro study of putative genomic biomarkers of nephrotoxicity through differential gene expression using gentamicin.

Drug-induced nephrotoxicity is one of the most frequently observed effects in long-term pharmacotherapy. The effects of nephrotoxicity are commonly discovered later due to a lack of sensitivity in in vivo methods. Therefore, researchers have tried to develop in vitro alternative methods for early identification of toxicity. In this study, LLC-PK1 cells were exposed to gentamicin through MTT and trypan blue assay. Concentrations of 4 (low), 8 (medium) and 12 (high) mM, were used to evaluate differential gene expression. A panel of genes was selected based on gene expression changes. The search for sequences of mRNA encoding proteins previously associated with kidney damage was conducted in the databases of the National Center for Biotechnology Information (USA). RNA was extracted from the cells, and RT-qPCR was performed to evaluate differential expression profiles of the selected genes. Among the 11 analyzed genes, four proved to be differentially up-regulated in cells exposed to gentamicin: HAVcr1, caspase 3, ICAM-1 and EXOC6. According to this study's results, we suggest that these genes play an important role in the mechanism of in vitro nephrotoxicity caused by gentamicin and can be used as early in vitro biomarkers to identify nephrotoxicity when developing safer drugs.

Hepatobiliary transporters in drug-induced cholestasis: a perspective on the current identifying tools.

INTRODUCTION: Impaired bile formation leads to the accumulation of cytotoxic bile salts in hepatocytes and, consequently, cholestasis and severe liver disease. Knowledge of the role of hepatobiliary transporters, especially the bile salt export pump (BSEP), in the pathogenesis of cholestasis is continuously increasing. AREAS COVERED: This review provides an introduction into the role of these transport proteins in bile formation. It addresses the clinical relevance and pathophysiologic consequences of altered functions of these transporters by genetic mutations and drugs. In particular, the current practical aspects of identification and mitigation of drug candidates with liver liabilities employed during drug development, with an emphasis on preclinical screening for BSEP interaction, are discussed. EXPERT OPINION: Within the potential pathogenetic mechanisms of acquired cholestasis, the inhibition of BSEP by drugs is well established. Interference of a new compound with BSEP transport activity should raise a warning sign to conduct follow-up experiments and to monitor liver function during clinical development. A combination of in vitro screening for transport interaction, in silico predicting models, and consideration of physicochemical and metabolic properties should lead to a more efficient screening of potential liver liability.

Alternative methods in toxicity testing: the current approach

Alternative methods are being developed to reduce, refine, and replace (3Rs) animals used in experiments, aimed at protecting animal welfare. The present study reports alternative tests which are based on the principles of the 3Rs and the efforts made to validate these tests. In Europe, several methodologies have already been implemented, such as tests of irritability, cell viability, and phototoxicity as well as in vitro mathematical models together with the use of in silico tools. This is a complex process that spans from development to regulatory approval and subsequent adoption by various official entities. Within this regulatory framework is REACH, the European Community Regulation for chemicals and their safe use. In Brazil, the BraCVAM (Brazilian Center for the Validation of Alternative Methods) was recently established to validate alternative methods and stimulate incorporation of new methodologies. A new vision of toxicology is emerging for the 21st century (Tox-21), and the subsequent changes are shaping a new paradigm.

Métodos alternativos estão sendo desenvolvidos para a redução, o refinamento e a substituição (3R) do número de animais utilizados em experimentos, visando ao seu bem-estar. Esses testes alternativos baseiam-se no princípio dos 3R e esforços têm sido empregados para que sejam validados. Na Europa, diversas metodologias já foram implantadas tais como: testes de irritabilidade, testes de viabilidade celular, testes de fototoxicidade e modelos matemáticos in vitro, além do uso de ferramentas in silico. Esse é um processo complexo, que abrange desde o seu desenvolvimento até a aceitação regulatória e posterior adoção por diversas organizações oficiais. No contexto regulatório está o REACH, o Regulamento da Comunidade Européia, para produtos químicos e sua utilização segura. No Brasil, o BraCVAM (Centro Brasileiro de Validação de Métodos Alternativos) foi recentemente estabelecido para validação de métodos alternativos e estímulo à incorporação de novas metodologias. Uma nova visão de toxicologia vem surgindo para o século XXI (Tox-21) e as mudanças ocasionadas promoverão um novo paradigma.

Perfil cinético, aspectos farmacológicos e lesÝo gástrica do piroxicam-zinco em ratos / Kinetic profile, pharmacological aspects and gastri lesion of the zinc-piroxicam in rats

O perfil plasmático do piroxicam em ratos Wistar machos (180+- 20g) foi analisado, após administraçÝo do complexo piroxicam-zinco e do piroxicam livre. Os parâmetros farmacocinéticos obtidos mostraram que a complexaçÝo do piroxicam com o zinco prolonga o tempo de absorçÝo. O 'T IND. MAX' do piroxicam no grupo do complexo foi de 5,27 h e de 2,56 h para o piroxicam livre. Os demais parâmetros farmacocinéticos foram semelhantes, na comparaçÝo dos dois. Na avaliaçÝo farmacológica, utilizando modelos de inflamaçÝo experimental - edema agudo de pata induzido pela carragenina (processo agudo), formaçÝo do tecido granulomatoso pela implantaçÝo de discos de algodÝo (processo subcrônico), artrite induzida pelo adjuvante Completo de Freund (processo crônico) e inibiçÝo da dor causada por pressÝo - nÝo houve diferença estatisticamente significante entre o piroxicam livre e seu derivado. A diferença observada nesses experimentos ocorreu somente em relaçÝo ao início da atividade do piroxicam, na inibiçÝo do edema de pata e na analgesia, onde a instalaçÝo do efeito é retardada com o complexo, provavelmente, face à absorçÝo mais lenta. Com relaçÝo ao efeito irritante do piroxicam na mucosa gástrica, a administraçào do piroxicam-zinco determinou menor açÝo gastrotóxica com diferença estatísticamente significante quando comparado à do piroxicam livre

Identificaçäo de bloqueadores beta-adrenérgicos em urina por cromatografia em camada delgada para diagnóstico da intoxicaçäo / Identification of beta-adrenergic blockers in thin-layer chromatography for intoxication diagnosis

Os bloqueadores beta-adrenérgicos (BBA) säo fármacos amplamente utilizados no tratamento de algumas doenças cardiovasculares, sendo empregados, também, na ingestäo intencional de superdose, de forma isolada ou em associaçäo com outros farmacos, com a finalidade de cometer suicídio. Em nosso meio tal fato é de crescente importância. O atenolol (A), Metoprolol (M), Nadolol (N), Pindolol (Pi) e Propranolol (Pr) säo os BBA comercializados no Brasil. Com a finalidade de determinar esses BBA em material biológico, nos últimos anos, foram estabelecidas algumas metodologias analíticas. Na determinaçäo de pequenas concentraçöes (ng/m), têm sido utilizadas técnicas cromatográficas mais sofisticadas. A cromatografia em camada delgada (CCD), além de custo inferior e fácil manipulaçäo, em relaçäo a outras técnicas cromatográficas, näo necessitando de pessoal qualificado, tem sua eficiência comprovada após o uso de doses terapêuticas e subterapêuticas. O objetivo do nosso trabalho foi desenvolver uma técnica para identificaçäo dos BBA estudados, tanto para o laboratório de análises toxicológicas, quanto para laboratórios de análises clínicas, em regiöes onde näo existam laboratórios de toxicologia...