Unravelling Joint Cytotoxicity of Ibuprofen and Oxytetracycline on Chlamydomonas Reinhardtii Using a Programmed Cell Death-Related Biomarkers Panel

Pharmaceutical active compounds (PhACs) are emerging contaminants that pose a growing concern due to their ubiquitous presence and harmful impact on aquatic ecosystems. Among PhACs, the anti-inflammatory ibuprofen (IBU) and the antibiotic oxytetracycline (OTC) are two of the most used compounds whose presence has been reported in different aquatic environments worldwide. However, there is still scarce information about the cellular and molecular alterations provoked by IBU and OTC on aquatic photosynthetic microorganisms as microalgae, even more if we refer to their potential combined toxicity. To test the cyto- and genotoxicity provoked by IBU, OTC and their binary combination on Chlamydomonas reinhardtii, a flow cytometric panel was performed after 24 h of single and co-exposure to both contaminants. Assayed parameters were cell vitality, metabolic activity, intracellular ROS levels, and other programmed cell death (PCD)-related biomarkers as cytoplasmic and mitochondrial membrane potentials and caspase-like and endonuclease activities. In addition, a nuclear DNA fragmentation analysis by comet assay was carried out. For most of the parameters analysed (vitality, metabolic activity, cytoplasmic and mitochondrial membrane potentials, and DNA fragmentation) the most severe damages were observed in the cultures exposed to the binary mixture (IBU+OTC), showing a joint cyto- and genotoxicity effect. Both PhACs and their mixture caused a remarkable decrease in cell proliferation and metabolic activity and markedly increased intracellular ROS levels, parallel to a noticeable depolarization of cytoplasmic and mitochondrial membranes. Moreover, a strong increase in both caspase and endonuclease activities as well as a PCD-related loss of nuclear DNA integrity was observed in all treatments. Results analysis showed that the PhACs caused cell death on this non-target organism, involving mitochondrial membrane depolarization, enhanced ROS production and activation of PCD process. Thus, PCD should be an applicable toxicological target for unraveling the harmful effects of co-exposure to PhACs in aquatic organisms as microalgae.