Work

Integrated Multi-Endpoint Analysis of Soluble Copper(II) Chloride–Induced Oxidative Stress, Mitochondrial Dysfunction, and Genotoxicity in RAW264.7 Macrophages

Year: 2026

Type: article

Abstract: Copper (Cu) is an essential trace element required for mitochondrial respiration and antioxidant defence; however, excessive copper exposure disrupts redox homeostasis via Fenton-like reactions, resulting in the overproduction of reactive oxygen species (ROS). This study investigated the cytotoxic, genotoxic, and apoptotic effects of soluble copper(II) chloride (CuCl₂) in RAW264.7 macrophages. Concentration- and time-dependent decreases in cell viability were observed following 6, 12, and 24 h exposures. The IC₅₀ values calculated from viability data were 23.44, 15.93, and 13.24 µM at 6, 12, and 24 h, respectively; notably, the 40 µM concentration was excluded from IC₅₀ determination because viability fell below 20%, representing nonspecific terminal cytotoxicity. Based on these IC₅₀ values, sub-IC₅₀ concentrations of 5–20 µM were selected, and mechanistic assays were subsequently performed after 24 h of CuCl₂ treatment. CuCl₂ exposure led to a concentration-dependent decrease in DNA strand breaks, micronucleus formation, and a reduced cytokinesis-block proliferation index. Elevated ROS levels were associated with mitochondrial depolarization, cytochrome c release, and the activation of caspase-3, -8, and − 9, indicating the involvement of both intrinsic and extrinsic apoptotic pathways. Previous copper toxicology studies typically evaluated isolated endpoints or single mechanistic pathways. In contrast, this study integrates time-course cytotoxicity with multiple mechanistically related endpoints under uniform experimental conditions, enabling a more comprehensive interpretation of Cu²⁺-induced immunotoxicity in macrophages. Collectively, these results support a model in which oxidative stress and mitochondrial impairment contribute to CuCl₂-induced immunotoxicity and genotoxic injury in macrophages.

Source: Biological Trace Element Research

DOI: 10.1007/s12011-026-05027-3