Desenvolvimento de método de cromatografia a gás bidimensional para abordagem metabolômica em células derivadas da pele humana expostas ao material particulado atmosférico e avaliação de citotoxicidade

Abstract

Particulate matter (PM) is a complex mixture of pollutants composed of particles of various sizes and different compositions present in the air that are associated with various types of environmental and public health disturbances. However, research relating the effects of these materials on the skin is still in its early stages. In this study, were investigated the profile of amino acids, a class of metabolites derived from fibroblast cells, after exposure to particulate matter collected at a point with intense car traffic and another close to an ecological station, both points within the Federal University of Minas Gerais-UFMG, Belo Horizonte, Minas Gerais, Brazil. A sample preparation method that encompassed quenching, extraction and derivatization of analytes was developed and evaluated using a one-dimensional gas chromatograph. The results showed a repeatability ranging from 4.83 to 8.88, values close to those found in the literature, demonstrating that the proposed method for analysis of amino acids derived from fibroblast cells was successfully achieved. After this step, the particulate material exposure process was optimized, for which cytotoxicity assays were used. Subsequently, a high selectivity and resolution method for amino acid analysis was developed and validated based on comprehensive two-dimensional gas chromatography coupled with mass spectrometry (GC×GC-Q-TOFMS/MS). R2 values above 0.9011 were obtained for all amino acids, RSDs between 5.01 -24.56%, limit of detection between 0.08-4.09 mg L-1, limit of quantification between 0.24 – 12 ,42 mg L-1 and recoveries in the range of 46.29 – 95.46% satisfactory results in view of the complexity of the steps. The derivatization steps, oven temperature, first and second dimension column types, injector temperature, transfer line and hot jet temperature, and finally the modulation period were optimized in this step. With all parameters adjusted, analyses of amino acids derived from fibroblast cells exposed to MP2.5 were carried out by GCxGC-Q-TOFMS/MS. The impacts of PM2.5 exposure on cells have been shown to be related to particle concentration. A total of four amino acids were down-regulated in exposed cells, including alanine, glycine, proline, and phenylalanine. By pathway analysis, aminoacyl-tRNA biosynthesis and phenylalanine, tyrosine, and tryptophan biosynthesis were highlighted as the main metabolic pathways disturbed by PM exposure. Overall, the results obtained help to improve the understanding of particulate matter toxicity in contact with the skin and demonstrate that the developed method of sample preparation together with GC×GC-Q-TOFMS/MS analysis is suitable for metabolomics research in fibroblast cells. Furthermore, the results of this work open new avenues for further studies to elucidate the association of more classes of skin metabolites associated with atmospheric exposure.