Copper exposure damages neurons and induces paralysis in the nematode, Caenorhabditis elegans, far below lethal concentrations

This study examined the effects of copper exposure on neurons and the excretory cell in the nematode, Caenorhabditis elegans, using transgenic strains with specific green fluorescent protein (GFP)-tagged cells. Exposure to CuSO₄ damaged particular neurons in a time- and concentration-dependent manner and induced paralysis at sublethal concentrations. Neurons expressing dopamine and γ-aminobutyric acid (GABA) suffered similar copper-induced damage, while many unidentified neurons in a pan-neuronal GFP strain seemed intact. However, the excretory cell resisted damage by a factor of seven compared to neurons. The concentrations at which 50% of the animals had excretory cell damage (EC₅₀) were 7.02, 4.28, and 3.74 mM CuSO₄ after exposure for 10, 12, and 14 hours; EC₅₀ values for dopaminergic neurons were 0.94, 0.67, and 0.49 mM. Animals paralyzed by copper were misidentified as dead by previous researchers. Live/dead testing with the cell-impermeant nucleic acid stain, SYTOX Orange, identified dead animals more accurately. Since cations interfere with dye fluorescence, SYTOX Orange was validated in animals treated with 2–32 mM CuSO₄ before heat-killing; the highest usable copper concentration was 16 mM. Subsequent live-dead testing found fewer than 5% of animals died after copper treatment up to 16 mM for 14 hours. Therefore, the LC₅₀ for CuSO₄ is greater than 16 mM, approximately 25 times the EC₅₀ for toxicity in neurons. The difference in toxicity for neurons versus the excretory cell refutes the claim that copper is not neurotoxic in C. elegans because the LC₅₀ and EC₅₀ are too similar for copper to be specifically neurotoxic.