Disruption of Copper Homeostasis by Copper Chelating Agents in Embryonic Zebrafish

Abstract

Disruption of copper homeostasis has been well-documented in cancers and neurodegenerative diseases. Recent studies highlight the potential efficacy of metal chelating compounds in the treatment of altered copper phenotypes. Disulfiram (DSF) is a dithiocarbamate that chelates copper, and DSF treatment has been shown to limit growth of cancer cells. These findings stimulated a search for other thione-containing copper chelating agents with capacity to alter abnormal cellular physiology. Thiomaltol (Htma) and dithiomaltol (Httma) are thiol derivatives of 3-hydroxy-2-methyl-4-pyrone (maltol) with high affinity for copper. In the current study, the efficacy of these compounds in eliciting copper-related phenotypes was compared to that of DSF using the embryonic zebrafish model. Embryonic viability and morphology following DSF, Htma and Httma was assessed in the presence and absence of copper supplementation. Treatment with DSF, Htma and Httma induced mortality or malformations characteristic of copper deficiency in a concentration-dependent manner (LC50 = 41.0, 23.0, 6.4 μM, Htma, Httma and DSF, respectively). Unexpectedly, co-treatment with 2.0 μM copper sulfate (CuSO4) exacerbated the effects of malformation-inducing levels of Htma, but not DSF; embryos co-treated with CuSO4 and nonlethal concentrations of Htma exhibited 100% mortality within 24 hours. Additionally, follow-up studies revealed that addition of CuSO4 to the culture water 15 minutes after treatment with high-dose DSF (but not Htma) could temporarily ‘rescue’ the embryos from lethality. The differing responses to copper supplementation of Htma versus DSF-treated embryos suggest unique interactions within the copper homeostatic pathway beyond simple direct chelation. Future studies will address the potential intracellular targets of Htma versus DSF.