Desvendando os efeitos genotóxicos de pesticidas : ensaios in vitro e ex vivo como ferramentas de avaliação

As acções antropogénicas são uma das principais fontes de contaminantes aquáticos presentes no meio ambiente, muitas vezes comprometendo o ecossistema e, consequentemente, os organismos presentes. Os efeitos destes compostos na biota e nos seres humanos devem ser avaliados, adoptando metodologias de confiança. Os métodos comumente usados, como as abordagens in vivo, apresentam várias desvantagens. Além disso, a implementação da política dos 3R (Redução, Refinamento e Substituição) foi considerada uma prioridade, reforçando a necessidade de encontrar métodos alternativos. Tendo isto em conta, este trabalho teve como objectivos (i) validar a abordagem ex vivo, como uma alternativa na pesquisa animal, (ii) avaliar o potencial genotóxico de três pesticidas químicos, um insecticida (dimetoato), um fungicida (imazalil) e um herbicida (penoxsulam), e do bioinsecticida Turex® nas células de brânquias de lagostim (Procambarus clarkii), usando a abordagem ex vivo, e também (iii) determinar a citotoxicidade e genotoxicidade, in vitro, do Turex®, na linha celular HepG2. A viabilidade celular das células de brânquias e da linha celular HepG2 foi avaliada às 2, 4 e 8 horas e às 24 e 48 horas, respectivamente. A integridade do ADN foi avaliada usando o ensaio do cometa com a incubação usando enzimas de reparação específicas do DNA, nomeadamente a formamidopirimidina DNAglicosilase (FPG) e a endonuclease III (EndoIII), para avaliar a oxidação de purinas e pirimidinas, respectivamente. Relativamente à abordagem ex vivo, as células de brânquias de lagostim demonstraram ser adequadas apenas durante 2 horas, quando a viabilidade e a integridade do ADN foram consideradas em conjunto. Tendo em conta esta informação, as células das brânquias foram expostas durante 2 horas a concentrações ambientalmente realistas de inseticida dimetoato (20 μg L-1), do fungicida imazalil (160 μg L-1), e do herbicida penoxsulam (23 μg L-1). Adicionalmente, e relativamente à exposição ao bioinsecticida Turex®, duas abordagens distintas foram consideradas: (1) uma exposição ex vivo de células de brânquias do lagostim durante 2 horas a cinco concentrações (25, 50, 100, 200 e 400 μg L-1), onde a genotoxicidade foi avaliada usando o ensaio do cometa, e (2) uma exposição in vitro da linha celular HepG2 a outras cinco concentrações (250, 500, 1000, 1500 e 2000 μg L-1), durante 24 e 48 horas, onde a citotoxicidade e a genotoxicidade foram avaliadas, usando o teste MTT e o ensaio do cometa, respectivamente. O dimetoato, o imazalil e o penoxsulam demostraram ser genotóxicos para as células de brânquias de lagostim, apesar de não induzirem dano oxidativo no ADN. Por outro lado, o Turex® não foi capaz de exercer efeitos genotóxicos nas células de brânquias de lagostim, apesar de apresentar genotoxicidade na linha celular HepG2 (apesar de ser apenas sem activação do insecticida e após 48 h). Além disso, este biopesticida demonstrou ser citotóxico (principalmente quando activado e após 48 h) para a linha celular testada. Em conclusão, a abordagem ex vivo demostrou ser adequada, juntamente com o ensaio do cometa, para exposições de 2 horas, quando aplicada a células de brânquias de lagostim. Deste modo, e considerando esta abordagem, a genotoxicidade dos pesticidas dimetoato, imazalil e penoxsulam foi comprovada. Este estudo demonstrou ainda os possíveis efeitos perigosos do Turex® para a linha celular humana (HepG2), direccionando a atenção para a alegada segurança de biopesticidas baseados em Bacillus thuringiensis. Consequentemente, é de todo o interesse que estes grupos de biopesticidas sejam investigados mais profundamente, de forma a determinar os possíveis efeitos em sistemas biológicos. No geral, os resultados obtidos apresentam-se como uma contribuição relevante para o aprimoramento das estratégias de triagem dos efeitos perniciosos de contaminantes, no sentido de as tornar mais rápidas e eficazes. Este trabalho pretende ainda contribuir para a (re)formulação de procedimentos regulatórios, tanto na aplicação de pesticidas como também para o controlo dos possíveis efeitos negativos dos mesmos, de forma a proteger a saúde ambiental e pública.Anthropogenic actions are one of the main sources of waterborne contaminants in the environment, often compromising the ecosystem and, consequently, inhabiting organisms. Real effects of these compounds to biota and humans must be assessed, adopting reliable approaches. Commonly used methods, such as in vivo approaches, come with several disadvantages. Moreover, the implementation of the 3R’s politic (Reduction, Refinement and Replacement) has been considered as a priority, reinforcing the need of finding alternative methods. Bearing this in mind, this study intended (i) to validate the ex vivo technique, as an alternative in animal research, (ii) to assess the genotoxicity of three chemically-based pesticides, an insecticide (dimethoate), a fungicide (imazalil) and a herbicide (penoxsulam) and the bioinsecticide Turex® to gill cells of Procambarus clarkii, using an ex vivo approach, and also (iii) to disclose the cytotoxicity and genotoxicity, in vitro, of Turex® to the cell line HepG2. Cell viability of crayfish gills and HepG2 cell line was evaluated for 2, 4 and 8 hours and 24 and 48 hours, respectively. DNA integrity was evaluated using the comet assay, improved with DNA lesion-specific repair enzymes, namely formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (EndoIII), to assess purines and pyrimidines oxidation, respectively. Concerning the ex vivo approach, crayfish gill cells only showed to be suitable considering exposures of 2 hours, when the viability and the DNA integrity were jointly considered. Accordingly, gill cells were exposed for 2 hours, to environmentally realistic concentrations of the insecticide dimethoate (20 ìg L- 1), the fungicide imazalil (160 ìg L-1), and the herbicide penoxsulam (23 ìg L-1). Additionally, and concerning the exposure to the bioinsecticide Turex®, two distinct approaches were considered: (1) an ex vivo exposure of crayfish gill cells for 2 hours to five concentrations (25, 50, 100, 200 and 400 ìg L-1), where its genotoxicity was evaluated using the comet assay, and (2) an in vitro exposure of the HepG2 cell line to other five concentrations (250, 500, 1000, 1500 and 2000 ìg L-1) for 24 and 48 hours, after which cytotoxicity and genotoxicity was evaluated using the MTT and the comet assays, respectively. Dimethoate, imazalil and penoxsulam demonstrated to be genotoxic to crayfish gill cells, despite not inducing oxidative DNA damage. On the other hand, Turex® was not able to exert genotoxic effects in crayfish gill cells, despite presenting genotoxicity to the HepG2 cell line (despite only without activation and after 48 h). Moreover, this biopesticide showed to be cytotoxic (mainly with activation and after 48 h) to the tested cell line. In conclusion, the ex vivo approach, when applied to crayfish gill cells, showed its suitability for exposures of 2 hours, when the comet assay was used. Thus, and considering this approach, the genotoxicity of the pesticides dimethoate, imazalil and penoxsulam was proved. This study also demonstrated the possible dangerous effects of Turex® to a human cell line (HepG2), pointing attention to the alleged safety of a Bacillus thuringiensis-based biopesticide. Consequently, this type of biopesticides should be further investigated to determine their possible negative effects on biological systems. Overall, the obtained results might be assumed as a relevant contribution towards the improvement of strategies for a rapid and effective screening of the pernicious effects of contaminants. This study also intended to contribute to (re)formulate regulatory procedures, both for the application of pesticides, as well as for the control of the possible negative effects, protecting the environmental and public health.Mestrado em Biologia Aplicad

Stability, dissolution, and cytotoxicity of NaYF4-upconversion nanoparticles with different coatings

Upconversion nanoparticles (UCNPs) have attracted considerable attention owing to their unique photophysical properties. Their utilization in biomedical applications depends on the understanding of their transformations under physiological conditions and their potential toxicity. In this study, NaYF4:Yb,Er UCNPs, widely used for luminescence and photophysical studies, were modified with a set of four different coordinatively bound surface ligands, i.e., citrate, alendronate (AA), ethylendiamine tetra(methylene phosphonate) (EDTMP), and poly(maleic anhydride-alt-1-octadecene) (PMAO), as well as silica coatings with two different thicknesses. Subsequently, the aging-induced release of fluoride ions in water and cell culture media and their cytotoxic profile to human keratinocytes were assessed in parallel to the cytotoxic evaluation of the ligands, sodium fluoride and the lanthanide ions. The cytotoxicity studies of UCNPs with different surface modifications demonstrated the good biocompatibility of EDTMP-UCNPs and PMAO-UCNPs, which is in line with the low amount of fluoride ions released from these samples. An efficient prevention of UCNP dissolution and release of cytotoxic ions, as well as low cytotoxicity was also observed for UCNPs with a sufficiently thick silica shell. Overall, our results provide new insights into the understanding of the contribution of surface chemistry to the stability, dissolution behavior, and cytotoxicity of UCNPs. Altogether, the results obtained are highly important for future applications of UCNPs in the life sciences and bioimaging studies

Boosting antibiotics performance by new formulations with deep eutectic solvents

The critical scenario of antimicrobial resistance to antibiotics highlights the need for improved therapeutics and/or formulations. Herein, we demonstrate that deep eutectic solvents (DES) formulations are very promising to remarkably improve the solubility, stability and therapeutic efficacy of antibiotics, such as ciprofloxacin. DES aqueous solutions enhance the solubility of ciprofloxacin up to 430-fold while extending the antibiotic stability. The developed formulations can improve, by 2 to 4-fold, the susceptibility of Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria to the antibiotic. They also improve the therapeutic efficacy at concentrations where bacteria present resistance, without promoting tolerance development to ciprofloxacin. Furthermore, the incorporation of DES decreases the toxicity of ciprofloxacin towards immortalized human epidermal keratinocytes (HaCat cells). The results herein reveal the pioneering use of DES in fluoroquinolone-based formulations and their impact on the antibiotic's characteristics and on its therapeutic action.publishe

Kekäläinen et al_Data

This file contains original data of the articl

Sperm pre-fertilization thermal environment shapes offspring phenotype and performance

201