Toxic mechanism on phenanthrene-triggered cell apoptosis, genotoxicity, immunotoxicity and activity changes of immunity protein in Eisenia fetida: Combined analysis at cellular and molecular levels

Phenanthrene (PHE) is a harmful organic contaminant and exists extensively in the soil environment. The accumulation of PHE would potentially threaten soil invertebrates, including earthworms, and the toxicity is also high. Currently, the possible mechanisms underlying apoptotic pathways induced by PHE and its immunotoxicity and genotoxicity in earthworms remain unclear. Thus, Eisenia fetida coelomocytes and immunity protein lysozyme (LYZ) were chosen as targeted receptors to reveal the apoptotic pathways, genotoxicity, and immunotoxicity triggered by PHE and its binding mechanism with LYZ, using cellular, biochemical, and molecular methods. Results indicated that PHE exposure can cause cell membrane damage, increase cell membrane permeability, and ultimately trigger mitochondria-mediated apoptosis. Increased 8-hydroxy-2-deoxyguanosine (8-OHdG) levels indicated PHE had triggered DNA oxidative damage in cells after PHE exposure. Occurrence of detrimental effects on the immune system in E. fetida coelomocytes due to decreased phagocytic efficacy and destroyed the lysosomal membrane. The LYZ activity in coelomocytes after PHE exposure was consistent with the molecular results, in which the LYZ activity was inhibited. After PHE binding, the protein structure (secondary structure and protein skeleton) and protein environment (the micro-environment of aromatic amino acids) of LYZ were destroyed, forming a larger particle size of the PHE-LYZ complex, and causing a significant sensitization effect on LYZ fluorescence. Molecular simulation indicated the key residues Glu 35, Asp 52, and Trp 62 for protein function located in the binding pocket, suggesting PHE preferentially binds to the active center of LYZ. Additionally, the primary driving forces for the binding interaction between PHE and LYZ molecule are hydrophobicity forces and hydrogen bonds. Taken together, PHE exposure can induce apoptosis by mitochondria-mediated pathway, destroy the normal immune system, and trigger DNA oxidative damage in earthworms. Besides, this study provides a comprehensive evaluation of phenanthrene toxicity to earthworms on molecular and cellular level.