Efeitos das formulações dos agroquímicos imazetapir e quincloraque em linhagem celular de fígado de peixe-zebra (ZF-L) (Danio rerio): citotoxicidade e estresse oxidativo

Abstract

Brazil, in addition to being one of the countries that uses the most agrochemicals in compliance with legal requirements, also shows evidence of the use of illicit agrochemicals. Several studies point to the harmful effects of these legal products on non-target organisms, both in vitro and in vivo. The objective of this study was to evaluate the cytotoxicity and oxidative stress, in vitro, of the seized formulations of herbicides imazethapyr (IMZT) and quinclorac (QCR) using the zebrafish liver cell line ZF-L. The active ingredient in the samples was identified by gas chromatography coupled with mass spectrometry (GC-MS). Thermogravimetric analysis (TGA) thermal stability tests and differential scanning calorimetry (DSC) analysis were also carried out. In the TGA thermal analyses, it was demonstrated that total mass loss occurred at 400 ºC for both samples, and, in the DSC analysis, the herbicides presented several physicochemical transition events. Cytotoxicity was initially assessed by hemolytic activity (AH), using defibrinated sheep blood (IMZT: 4.82 to 308.64 µg/mL and QCR: 2.63 to 84.82 µg/mL) and, subsequently, by MTT, neutral red (VN), lactate dehydrogenase (LDH) assays, using the zebrafish liver cell line (ZFL), exposed to herbicides (IMZT: 0.44 to 28.06 µg/mL and QCR: 0.27 to 7.67 µg/mL). Oxidative stress was evaluated by determining reactive oxygen species (ROS), total sulfhydryl content (SH) and antioxidant activities of superoxide dismutase (SOD), catalase (CAT) also in ZF-L cells, exposed to 1/2x, 1x and 2x of the median IC50 of cytotoxicity assays. The period of exposure of ZF-L cells to herbicides was 24 hours. In the hemolytic assay, erythrocytes were affected at the highest concentrations for both IMZT (19.29 to 308.64 µg/mL) and QCR (21.08 to 84.82 µg/mL) and presented an IC50 of 12.75 µg/mL and 19.83 µg/mL, respectively. In the MTT, NR, and LDH assays, both herbicides affected mitochondrial and lysosomal organelles and the integrity of the plasma membrane. The IC50 for IMZT was 3.01, 2.67, and 1.61 µg/mL, respectively, and for QCR theywere 1.78, 5.13, and 0.95 µg/mL, respectively. From these IC50 concentrations, the median value was estimated, corresponding to IC50 of 2.84 µg/mL (IMZT) and 3.46 µg/mL (QCR). The IC50 results of the medians were used in oxidative stress assays. Both IMZT and QCR increased the production of ROS and promoted changes in the antioxidant system in the total content of SH, SOD and CAT. To our knowledge, this is the first study that reports the in vitro cytotoxic effect and oxidative stress induced by seized formulations of herbicides containing IMZT and QCR, in the ZF-L cell line. Taken together, these results indicate the importance of in vitro tests for evaluating the toxicity ofagrochemicals, as they demonstrate the harmful effects that can affect human health and the environment.