RESUMO
Research and regulatory efforts in toxicology are increasingly focused on the development of suitable non-animal methodologies for human health risk assessment. In this work we used human intestinal Caco-2 and HT29/MTX cell lines to address the potential risks of mixtures of the emerging contaminants tetrabromobisphenol A (TBBPA) and commercial polystyrene nanoparticles (PSNPs). We employed different in vitro settings to evaluate basal cytotoxicity through three complementary endpoints (metabolic activity, plasmatic, and lysosomal membrane integrity) and the induction of the oxidative stress and DNA damage responses with specific endpoints. Although no clear pattern was observed, our findings highlight the predominant impact of TBBPA in the combined exposures under subcytotoxic conditions and a differential behavior of the Caco-2 and HT29/MTX co-culture system. Distinctive outcomes detected with the mixture treatments include reactive oxygen species (ROS) increases, disturbances of mitochondrial inner membrane potential, generation of alkali-sensitive sites in DNA, as well as significant changes in the expression levels of relevant DNA and oxidative stress related genes.
Assuntos
Nanopartículas , Bifenil Polibromatos , Poliestirenos , Humanos , Poliestirenos/toxicidade , Células CACO-2 , Estresse Oxidativo , Nanopartículas/toxicidade , DNARESUMO
The potential interactions between the diverse pollutants that can be released into the environment and the resulting outcomes are a challenging issue that needs to be further examined. This in vitro study was aimed to assess potential toxic effects caused by combined exposure to tetrabromobisphenol A, a flame retardant widely used and frequently detected in aquatic matrices, and commercially available polystyrene nanoparticles as reference material to evaluate nanoplastics risks. Our results, using freshwater fish cell lines and a set of relevant cytotoxicity endpoints including cell viability, oxidative stress, and DNA damage, provide additional mechanistic insights that could help to fully characterize the toxicity profiles of tetrabromobisphenol A and polystyrene nanoparticles. Furthermore, we describe subtle changes in cell viability as well as the generation of oxidative DNA damage after coexposure to subcytotoxic concentrations of the tested pollutants.
Assuntos
Nanopartículas , Poluentes Químicos da Água , Animais , Poliestirenos/toxicidade , Microplásticos , Peixes , Linhagem Celular , Poluentes Químicos da Água/toxicidade , Nanopartículas/toxicidadeRESUMO
DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium Thermus thermophilus strain HB27 encodes a Ppol protein among the genes present in mobile element ICETh2, absent in other T. thermophilus strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every ppol mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the ppol mutants in combination with addAB mutants. The results show that knockout addAB mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas ppol mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the ppol and/or addAB mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains. All these data support that Ppol appears to keep in balance the activity of AddAB as a part of the DNA housekeeping maintenance in T. thermophilus HB27, thus, playing a key role in its genome stability.
RESUMO
Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These "life" or "death" cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.
Assuntos
Drosophila , Proteína Supressora de Tumor p53 , Animais , Apoptose/genética , Ciclo Celular/genética , Proliferação de Células/genética , Dano ao DNA , Drosophila/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
A wide range of cationic amphiphilic drugs (CADs) from different therapeutic areas are known to cause phospholipidosis both in vivo and in vitro. Although the relevance of this storage disorder for human health remains uncertain, CADs have been repeatedly associated with clinical side effects, and as a result, phospholipidosis is of major concern for drug development in the pharmaceutical industry. An important unresolved question in this field is whether phospholipidosis is really linked to cellular toxicity. This work was focused on studying cellular responses associated with CAD-induced phospholipidosis in cultured mammalian kidney cells. Dibucaine (2-butoxy-N-[2-diethylaminoethyl]quinoline-4-carboxamide), an amide-type anesthetic with poorly defined cytotoxic effects, was used to induce phospholipidosis in Vero cells. The results from several assays that measure cell viability, proliferation, and morphological changes indicated that dibucaine-induced lysosomal phospholipidosis was accompanied by cellular defense responses such as transient growth arrest and autophagy, under mild stress conditions. Conversely, when tolerance limits were exceeded treated Vero cells underwent extensive and irreparable injury, leading ultimately to cell death. Our data provide additional information that may be of considerable interest for drug safety assessment.
Assuntos
Anestésicos Locais/efeitos adversos , Dibucaína/efeitos adversos , Lipidoses/induzido quimicamente , Fosfolipídeos/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Lisossomos/efeitos dos fármacos , Células VeroRESUMO
Propyl p-hydroxybenzoate, commonly referred to as propylparaben, is the most frequently used preservative to inhibit microbial growth and extend shelf life of a range of consumer products. The objective of this study was to provide further insight into the toxicological profile of this compound, because of the current discrepancy in the literature with regard to the safety of parabens. The Vero cell line, derived from the kidney of the green monkey, was selected to evaluate the adverse effects of propylparaben by use of a set of mechanistically relevant endpoints for detecting cytotoxicity and genotoxic activities. Our results demonstrate that exposure to the compound for 24h causes changes in cell-proliferation rates rather than in cell viability. A significant and dose-dependent decline in the percentage of mitotic cells was observed at the lowest concentration tested, mainly due to cell-cycle arrest at the G0/G1 phase. Immunodetection techniques revealed that induction of DNA double-strand breaks and oxidative damage underlies the cytostatic effect observed in treated Vero cells. Additional studies are in progress to extend these findings, which define a novel mode of action of propylparaben in cultured mammalian cells.
Assuntos
Dano ao DNA , Estresse Oxidativo/genética , Parabenos/toxicidade , Animais , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Conservantes de Alimentos/toxicidade , Índice Mitótico , Conservantes Farmacêuticos/toxicidade , Células VeroRESUMO
Health and environmental risks regarding perfluorooctanoic acid, a well-known perfluorinated compound, are still a subject of great concern. Ubiquitous exposure and disparity of results make it difficult to determine the underlying mechanism of action, especially at the cellular level. This study proposes an experimental design to assess the reversibility of adverse effects after a one-time exposure to the compound, in comparison with other more conventional timings. Complementary endpoints including total protein content, neutral red uptake and MTT reduction tests along with division rates and microscopic observations were evaluated in HeLa cells. In addition, PFOA quantification inside the cells was performed. The cellular effects exerted after 24 h exposure to perfluorooctanoic acid are non-reversible after a 48 h recovery period. In addition, we describe for the first time the induction of plasma membrane blebbing and the activation of membrane repair mechanisms after recovery from non-cytotoxic treatments with the compound. This experimental design has provided relevant information regarding the toxicity of this perfluorinated compound, relating all the adverse effects detected to its interaction with the plasma membrane.
Assuntos
Caprilatos/toxicidade , Membrana Celular/efeitos dos fármacos , Fluorocarbonos/toxicidade , Células HeLa , Humanos , Testes de Toxicidade AgudaRESUMO
Perfluorooctanoic acid has been used widespread, during the last decades, in a number of consumer and industrial products. Although this compound has been subjected to extensive epidemiological and toxicological studies, limited data are available concerning its potential dermal toxicity in mammalian cells. In this study, we used a two-stage approach with relevant cytotoxicity endpoints including cell viability and proliferation, oxidative stress, DNA damage and cell senescence to assess the immediate and the long-lasting or delayed cytotoxicity caused by the compound in HaCaT keratinocytes. Our results suggest that a single exposure to perfluorooctanoic acid causes concentration-dependent changes in cell proliferation that were not restored during a 48â¯h recovery period. Furthermore, we demonstrate that a moderate treatment with this perfluorochemical causes persistent DNA damage, which ultimately leads to development of the senescence-associated secretory phenotype in HaCaT cells. This paper provides unprecedented data and insights regarding the cytotoxic effects of perfluorooctanoic acid in human cells that could be of special relevance for use in comparative in vitro-in vivo studies. Moreover, our findings highlight the importance of considering both the immediate and long-lasting or delayed cytotoxic responses caused by chemical exposure, to ensure the accurate identification of toxicity in cell-based systems.
Assuntos
Caprilatos/toxicidade , Senescência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Fluorocarbonos/toxicidade , Queratinócitos/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Células Epidérmicas , Humanos , Estresse Oxidativo/efeitos dos fármacosRESUMO
Di (2-ethylhexyl) phthalate is a high-production chemical widely used as a plasticizer for polyvinyl chloride products. Due to its ubiquitous presence in environmental compartments and the constant exposure of the general population through ingestion, inhalation, and dermal absorption, this compound has been subjected to extensive in vivo and in vitro toxicological studies. Despite the available information, research on the cytotoxicity of di (2-ethylhexyl) phthalate in mammalian cells is relatively limited.In this paper, an in vitro multi-parametric approach was used to provide further mechanistic data on the toxic activity of this chemical in Vero and HaCaT cells. Our results reveal that a 24 h exposure to di (2-ethylhexyl) phthalate causes, in both cell lines, an inhibition of cell proliferation that was linked to cell cycle delay at the G1 phase. Concomitantly, the tested compound induces mild endoplasmic reticulum stress which leads to an adaptive rather than a pro-apoptotic response in mammalian cells. These findings demonstrate that there are multiple potential cellular targets of di (2-ethylhexyl) phthalate-induced toxicity and the need to develop further experimental studies for the risk assessment of this ubiquitous plasticizer.
Assuntos
Dietilexilftalato/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Fase G1/efeitos dos fármacos , Humanos , Células VeroRESUMO
Butylated hydroxyanisole and propylparaben are phenolic preservatives commonly used in food, pharmaceutical and personal care products. Both chemicals have been subjected to extensive toxicological studies, due to the growing concern regarding their possible impacts on environmental and human health. However, the cytotoxicity and underlying mechanisms of co-exposure to these compounds have not been explored. In this study, a set of relevant cytotoxicity endpoints including cell viability and proliferation, oxidative stress, DNA damage and gene expression changes were analyzed to assess whether the antioxidant butylated hydroxyanisole could prevent the pro-oxidant effects caused by propylparaben in Vero cells. We demonstrated that binary mixtures of both chemicals induce greater cytotoxic effects than those reported after single exposureto each compound. Simultaneous treatment with butylated hydroxyanisole and propylparaben caused G0/G1 cell cycle arrest as a result of enhanced generation of oxidative stress and DNA double strand breaks. DNA microarray analysis revealed that a cross-talk between transforming growth factor beta (TGFß) and ataxia-telangiectasia mutated kinase (ATM) pathways regulates the response of Vero cells to the tested compounds in binary mixture. Our findings indicate that butylated hydroxyanisole potentiates the pro-oxidant effects of propylparaben in cultured mammalian cells and provide useful information for their safety assessment.