Eco-Cito-Genotoxicidade de misturas dos fármacos metformina, valsartana e atenolol

Abstract

Pharmaceutical active compounds (PhACs) are currently classified as emerging contaminants, for which regulatory frameworks and a better understanding of their environmental impacts are still lacking. Due to their widespread use, high production, and continuous emission, these substances are frequently reported in aquatic environments. This study aimed to investigate the ecotoxicological and cytogenotoxic effects of exposure to mixtures of three widely used pharmaceuticals: metformin, valsartan, and atenolol. The concentrations applied were selected based on the PHARMS-UBA (2024) database, which compiles global records of PhAC occurrence in the environment. Treatments were prepared using the highest concentrations reported worldwide, followed by successive dilutions down to the lowest detected levels, resulting in 16 treatment groups. Cell cycle analyses were conducted through root growth assessment in Lactuca sativa and flow cytometry in both L. sativa and Allium cepa, revealing a biphasic response pattern consistent with hormesis. Treatments T5, T6, and T7 stimulated cell division, evidenced by enhanced root growth in L. sativa and increased percentages of nuclei in G2/M phase observed via flow cytometry in both plant models. In contrast, higher concentration treatments (T14, T15, and T16) inhibited root growth in L. sativa by approximately 30%. From treatment T12 onwards (T12-T16), flow cytometry revealed inhibitory effects, with significant alterations in cell cycle phases, particularly reduced proportions of cells in G1 and G2/M. Additionally, changes were observed in markers associated with adverse effects and cell death, such as increased sub-G1 population and decreased cell size (FSC) and nuclear complexity (SSC). Beyond the plant assays, ecotoxicity was also assessed through the acute immobilization test with Daphnia magna. In this assay, treatment T11 corresponded to the EC₅₀, immobilizing 50% of the organisms at concentrations of 31.26 µg/L (metformin), 11.57 µg/L (valsartan), and 36.84 µg/L (atenolol). The most concentrated treatments (T15 and T16) resulted in complete immobilization, indicating high toxicity of the mixtures at elevated doses. Cytogenotoxicity was further evaluated in A. cepa using cytogenetic analysis, comet assay, DNA electrophoresis, and the TUNEL assay. The cytogenetic analysis revealed a progressive decrease in mitotic index with increasing concentrations, highlighting the cytotoxic potential of the mixtures. Chromosomal alterations were predominantly aneugenic, suggesting mitotic spindle dysfunction. This pattern was supported by the comet assay, which showed no significant DNA strand breaks, indicating the absence of clastogenic damage. In the most concentrated treatments (T14 to T16), both flow cytometry and cytogenetic data indicated markers consistent with cell death, confirmed by the appearance of DNA laddering in electrophoresis and positive labeling in the TUNEL assay. These findings demonstrate that exposure to PhAC mixtures can adversely affect non-target organisms, causing eco-cyto-genotoxic damage and highlighting the environmental risks associated with the presence of these contaminants.