Evaluación de la citotoxicidad y genotoxicidad en células V79 y HaCaT asociado a la exposición a nanopartículas de carbón de La Loma Cesar
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Fecha
2024
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Ediciones Universidad Simón Bolívar
Facultad de Ciencias Básicas y Biomédicas
Facultad de Ciencias Básicas y Biomédicas
Resumen
El carbón es un mineral que en la actualidad representa una fuente de energía clave
en la economía mundial. Durante décadas ha sido utilizado para la generación de
electricidad, calefacción y como materia prima en la industria. La extracción de este
mineral involucra la remoción de capas de tierra y roca para acceder a las capas de
carbón debajo de la superficie, proceso que deja una huella ambiental significativa,
y que ha desencadenado preocupaciones cruciales relacionadas con la salud,
especialmente a través de la generación del polvo proveniente de las actividades
de minería. En este sentido se considera que la composición de las partículas de
carbón, el tamaño y forma, juegan un papel fundamental en las afecciones
respiratorias de las poblaciones humanas. El objetivo principal de este estudio fue
analizar los efectos citotóxicos y genotóxicos in vitro de nanopartículas de carbón
en células V79 y HaCaT. Mediante el método de separación en medio ácido se
aislaron las nanopartículas. Posteriormente estas nanopartículas fueron usadas
para exponer células V79 y HaCaT a diferentes concentraciones. A través del
ensayo con rezasurina y sulforodamina se determinaron los efectos de estas
partículas en la viabilidad celular, y se seleccionaron las concentraciones 50, 150 y
300 μg/mL para realizar los ensayos de genotoxicidad, ensayo cometa y
micronúcleos. La microscopía de fuerza atómica proporcionó una visión detallada
de la topografía de las nanopartículas, destacando su propensión a la aglomeración.
Mediante SEM-EDS se evidenció la forma y diversidad química de estas
nanopartículas, constituidas principalmente por elementos como carbono (C),
oxígeno (O), hierro (Fe), calcio (Ca) y mediante cromatografía de gases acoplada a
espectrometría de masas (GC/MS), se determinaron los hidrocarburos aromáticos
policíclicos (HAP) presentes en las nanopartículas como fluoranteno, naftaleno,
antraceno, 7H-benzo[c]fluoreno, fenantreno, pireno, benzo[a]antraceno, criseno y
algunos derivados alquílicos. La evaluación de la genotoxicidad mediante
marcadores como el ensayo cometa, la formación de micronúcleos y la
inmunomarcación empleando anticuerpos anti-Gamma H2AX, mostró un efecto
dosis-respuesta evidenciando la capacidad de las nanopartículas de carbón para
inducir inestabilidad genética y muerte celular. El uso de la técnica de temperatura
melting y PCR en tiempo real permitió evidenciar una posible alteración en la
estructura y estabilidad del ADN debido a la interacción físico-química de este con
las nanopartículas de carbón. En conclusión, este estudio destaca la relación entre
las características específicas de las nanopartículas de carbón para llegar a
comprender el entendimiento de sus interacciones a nivel celular, molecular y sentar
las bases para dilucidar los mecanismos relacionados con el desarrollo de diferentes
enfermedades respiratorias.
El coal is a mineral that currently represents a key energy source in the global economy. For decades, it has been used for electricity generation, heating, and as a raw material in industry. The extraction of this mineral involves the removal of layers of soil and rock to access the coal seams beneath the surface, a process that leaves a significant environmental footprint and has triggered crucial health concerns, especially through the generation of dust from mining activities. In this regard, it is considered that the composition, size, and shape of coal particles play a fundamental role in respiratory conditions in human populations. The main objective of this study was to analyze the in vitro cytotoxic and genotoxic effects of coal nanoparticles on V79 and HaCaT cells. Nanoparticles were isolated using the acid medium separation method. Subsequently, these nanoparticles were used to expose V79 and HaCaT cells to different concentrations. The effects of these particles on cell viability were determined through resazurin and sulforhodamine assays, and concentrations of 50, 150, and 300 μg/mL were selected for genotoxicity assays, comet assay, and micronuclei assay. Atomic force microscopy provided a detailed view of the topography of the nanoparticles, highlighting their tendency to agglomerate. SEM-EDS revealed the shape and chemical diversity of these nanoparticles, primarily composed of elements such as carbon (C), oxygen (O), iron (Fe), calcium (Ca), and through gas chromatography coupled with mass spectrometry (GC/MS), polycyclic aromatic hydrocarbons (PAHs) present in the nanoparticles were determined, such as fluoranthene, naphthalene, anthracene, 7Hbenzo[c]fluorene, phenanthrene, pyrene, benzo[a]anthracene, chrysene, and some alkyl derivatives. The evaluation of genotoxicity using markers such as the comet assay, micronucleus formation, and immunostaining employing antibodies against Gamma H2AX showed a dose-response effect, demonstrating the ability of coal nanoparticles to induce genetic instability and cell death. The use of melting temperature technique and real-time PCR allowed for the evidence of a possible alteration in the structure and stability of DNA due to the physicochemical interaction with coal nanoparticles. In conclusion, this study highlights the relationship between the specific characteristics of coal nanoparticles to understand their interactions at the cellular and molecular levels and lay the groundwork for elucidating mechanisms related to the development of various respiratory diseases.
El coal is a mineral that currently represents a key energy source in the global economy. For decades, it has been used for electricity generation, heating, and as a raw material in industry. The extraction of this mineral involves the removal of layers of soil and rock to access the coal seams beneath the surface, a process that leaves a significant environmental footprint and has triggered crucial health concerns, especially through the generation of dust from mining activities. In this regard, it is considered that the composition, size, and shape of coal particles play a fundamental role in respiratory conditions in human populations. The main objective of this study was to analyze the in vitro cytotoxic and genotoxic effects of coal nanoparticles on V79 and HaCaT cells. Nanoparticles were isolated using the acid medium separation method. Subsequently, these nanoparticles were used to expose V79 and HaCaT cells to different concentrations. The effects of these particles on cell viability were determined through resazurin and sulforhodamine assays, and concentrations of 50, 150, and 300 μg/mL were selected for genotoxicity assays, comet assay, and micronuclei assay. Atomic force microscopy provided a detailed view of the topography of the nanoparticles, highlighting their tendency to agglomerate. SEM-EDS revealed the shape and chemical diversity of these nanoparticles, primarily composed of elements such as carbon (C), oxygen (O), iron (Fe), calcium (Ca), and through gas chromatography coupled with mass spectrometry (GC/MS), polycyclic aromatic hydrocarbons (PAHs) present in the nanoparticles were determined, such as fluoranthene, naphthalene, anthracene, 7Hbenzo[c]fluorene, phenanthrene, pyrene, benzo[a]anthracene, chrysene, and some alkyl derivatives. The evaluation of genotoxicity using markers such as the comet assay, micronucleus formation, and immunostaining employing antibodies against Gamma H2AX showed a dose-response effect, demonstrating the ability of coal nanoparticles to induce genetic instability and cell death. The use of melting temperature technique and real-time PCR allowed for the evidence of a possible alteration in the structure and stability of DNA due to the physicochemical interaction with coal nanoparticles. In conclusion, this study highlights the relationship between the specific characteristics of coal nanoparticles to understand their interactions at the cellular and molecular levels and lay the groundwork for elucidating mechanisms related to the development of various respiratory diseases.
Descripción
Palabras clave
Nanopartículas, Carbón, Citotoxicidad, Genotoxicidad, Daño oxidativo, HAP, Metales, Muerte celular, Nanoparticles, Coal, Cytotoxicity, Genotoxicity, Oxidative damage, PAHs, Metals, Cell death