Cell-based and computational toxicity profiling of thiourea and imine derivatives in Acanthamoeba sp. as...

Acanthamoeba sp., a sensitive yet underutilised environmental bioindicator, was utilised to evaluate the toxicity of two emerging hybrid moieties, AN6C and F-2a, with potential applications in optoelectronic materials. This study highlights implications for future safety, ecological monitoring, and environmental health assessment of the two hybrid moieties. The structures were confirmed by spectroscopic analysis, namely FTIR, 1D NMR, and UV-visible analysis. The HOMO-LUMO gaps of these compounds are below 4 eV, indicating their semiconducting properties. Additionally, the presence of the (-CHN-) moiety in AN6C, as well as the presence of carbonyl (CO) and thiocarbonyl (CS) groups in F-2a, reveals a good binding capacity in inhibiting cell growth. A cytotoxicity screening was conducted using the MTT assay to examine dose-response activities based on the IC₅₀ values obtained. Thiourea (F-2a) exhibits an IC₅₀ value of 24.32 μg/mL, while imine (AN6C) shows a significantly lower IC₅₀ value of 0.4589 μg/mL. Post-treatment cell morphology and cytoplasmic biochemical changes, assessed through light and fluorescence microscopy, revealed rounded and floating encystment phases, yellowish-orange granulation within the cells, and burst-open plasma membranes, indicating the presence of autophagy and necrosis. The DNA laddering assay indicated that DNA fragmentation occurred at 1100, 900, and 100 bp after treatment with both compounds. Analysis of mitochondrial membrane potential (MMP) by flow cytometry demonstrated that F-2a and AN6C produced 20.6% and 16% disruption of the MMP, respectively. Molecular docking simulation was conducted on the profilin protein of Acanthamoeba sp., profilin II (PDB ID: 2ACG), a small cytoskeletal protein that binds to actin and is required for normal actin cytoskeleton function. F-2a exhibited a docking score of -6.2 kcal/mol, indicating high binding affinity, and interacted with 12 amino acid residues compared to AN6C, which showed a docking score of -3.4 kcal/mol and interacted with nine amino acid residues of the 2ACG protein. These findings were evaluated to generate hypotheses regarding the roles and effects of the hybrid moieties and their conjugated functional groups at cellular, molecular, and in silico levels on eukaryotic microorganisms that are sensitive to environmental change.