Alterações estruturais e funcionais da hemoglobina na presença de acetato de fenilmercúrio: de proteínas isoladas a modelos biológicos

Abstract

Contamination by potentially toxic metals such as mercury (Hg) and its derivatives poses a serious risk to human health and the environment. Mercury can cause neurological disorders, immune system impairment, and cardiovascular disease. Phenylmercury (PM) is an organic compound derived from mercury used in fungicides and pesticides. Erythrocytes (Ery) are blood cells rich in hemoglobin, a protein that binds to oxygen and promotes its delivery to tissues and the removal of carbon dioxide. Exposure to Hg and its derivatives can cause an increase in the generation of reactive oxygen species (ROS), causing damage to proteins, lipids, and DNA, thus contributing to the emergence of several pathologies. The scientific literature presents a significant gap on the impacts of PM exposure on cells and experimental models as a whole. Therefore, this study aims to investigate possible structural, functional and redox alterations of PM in three distinct systems regarding the complexity of the experimental models: hemoglobin (protein), human erythrocytes (cell) and Zebrafish (Danio rerio) (animal). Theoretical studies indicate that PM tends to interact with the heme group or with the cysteine residues of hemoglobin (Hb), which may impact the functionality of the protein. The hypothesis of binding/interaction with Hb was evidenced through biophysical studies, which demonstrated a binding constant (Kb) value of 1.47 (± 0.22) × 104 M-1 , inferring that the formation of the Hb-PM complex is significant and favorable. Experimentally, exposure to PM resulted in binding with cysteine residues (Ellman assay), structural alterations in the heme group of pure Hb, evidenced by the analysis of the Soret band, and reduction of electrophoretic mobility due to the formation of the Hb-PM supramolecular complex. In human erythrocytes from volunteers, exposure to PM (0.125–1.0 µM) caused a decrease of up to 34% in oxygen uptake. The generation of ROS and reactive nitrogen species (RNS) were verified by means of appropriate fluorescent probes, and a 2.2-fold increase in the generation of global reactive species was observed. Furthermore, we noted that H2O2 production increased by 11%, while NO production increased by 90%. In the evaluation of antioxidant enzymes, there was a decrease in catalase and glutathione peroxidase activity by 25 and 49%, respectively, while superoxide dismutase activity increased by 13%. Analysis of secondary markers of oxidative stress revealed a 34% reduction in the GSH/GSSG ratio and a decrease in total free sulfhydryl groups by 44%, suggesting depletion of antioxidant defense mechanisms. We also emphasize that no significant differences were detected in lipid peroxidation, but there was a 2.2-fold increase in the levels of carbonyl proteins, reflecting protein oxidation. In the membrane fragility assay, we observed structural changes in Ery exposed to PM, indicating membrane damage. In this line, the analysis by atomic force microscopy revealed changes in the morphology of these cells as well as in membrane elasticity. Finally, the toxicity tests demonstrated that PM has a teratogenic effect on zebrafish embryos, as well as causing cardiac damage (number of heartbeats) in the animals. Given our set of results, we conclude that exposure to PM impairs the structure, functionality, and morphology of Ery, due to its interaction with hemoglobin, causing changes in the redox system of these cells, and presenting a teratogenic effect in an animal model.