Avaliação de métodos analíticos para estudos em material biológico de ratos submetidos ao tratamento com nanotubos de carbono

Abstract

In this work, methods for the analysis of tissues, fluids and furs of rats submitted to the treatment with carbon nanotubes (CNTs) were evaluated, allowing to identify where they are concentrated, indirectly, by the determination of metal contaminants from CNTs. Contaminants determination was carried out by inductively coupled plasma optical emission spectrometry (ICP-OES) and/or inductively coupled plasma mass spectrometry (ICP-MS). Also, the distribution of inorganic contaminants in the tissues of animals was evaluated through bio-imaging by laser ablation (LA) combined to ICP-MS (LA-ICP-MS). Initially, it was necessary to obtain the tissues (spleen, brain, heart, liver, pancreas, lung and kidney), fluids (feces and blood) and furs of animals submitted to the treatment with CNTs, as suggested by the Comitê de Ética Animal of UFSM, with the study of acute oral toxicity and chronic toxicity. In this study, a functionalized CNT with hydroxyl groups (CNT-OH) was used. In the acute oral toxicity test, CNTs shown to be non-toxic when administered to rats at the maximum dose of 2000 mg kg-1 per body weight. According to the histological analyzes of the liver, doses of 300 mg kg-1 and 2000 mg kg-1 caused small hepatic lesions. Because of this, 1 mg kg-1, 10 mg kg-1 and 100 mg kg-1 of CNT were administered in the chronic toxicity study for 31 days. Oral administration of CNTs did not cause significant changes in biochemical parameters and at the end of the treatment, the rats were euthanized and the organs, fluids, furs and feces of the animals were collected. The need to evaluate tissues contaminated with CNTs required the development of a method that was efficient for the decomposition of this matrix, a challenge in terms of sample preparation, to avoid results underestimated by the inefficient digestion of the CNT into the tissue. In this purpose, the decomposition method in open system with digestion heating block was evaluated, as well as a method based on microwave-assisted wet digestion using a pressurized digestion cavity (MAWD-PDC). Sample preparation using the digestion block was considered unsuitable once it does not allowed the decomposition of CNTs. For MAWD-PDC, the influence of the mass of animal tissue to be decomposed and the efficiency for the digestion of CNTs contained in the tissues were investigated. The accuracy of the method was evaluated with the decomposition of a reference sample of bovine liver, added to samples of bovine liver and chicken, used to simulate the tissues. Suitable recoveries (90 to 127%) were observed. Results obtained using 275 mg of CNT for decomposition by MAWD-PDC, presented no difference (t-student test) when compared with reference values. After optimization, the MAWD-PDC was considered suitable for the decomposition of tissues, fluids and furs of animals submitted to the treatment with CNTs using 6 mL of concentrated HNO3 and 0.5 mL of 30% H2O2 as a decomposition solution and a program consisting of i) 25 min at 180 °C (10 min ramp) and ii) 40 min at 270 °C (5 min ramp). In general, differences were observed between the results obtained for Ni in the liver, heart, lung and feces of the animals in the groups treated with CNTs in relation to the control group. The bio-distribution analysis by LA-ICP-MS in the spleen, liver, lung and kidney of the animals from the control group and those from the 100 mg kg-1 CNT treated group was performed. Bio-imaging of the distribution of elements considered macronutrients, such as Ca and Mg, and micronutrients as Cu, Fe, Mn and Zn, were obtained. Differences in the distribution of some elements in the kidney, spleen, and lung were observed. In view of the results obtained using MAWD-PDC and the determination of metals by ICP-OES and/or ICP-MS and the bio-distribution analysis by LA-ICP-MS, it can be inferred that both investigated strategies were promising and can be used contributing to the assessment of the possible toxic effects of CNTs.