A cautionary note on the use of N-acetylcysteine as a reactive oxygen species antagonist to assess copper mediated cell death.

A new form of cell death has recently been proposed involving copper-induced cell death, termed cuproptosis. This new form of cell death has been widely studied in relation to a novel class of copper ionophores, including elesclomol and disulfiram. However, the exact mechanism leading to cell death remains contentious. The oldest and most widely accepted biological mechanism is that the accumulated intracellular copper leads to excessive build-up of reactive oxygen species and that this is what ultimately leads to cell death. Most of this evidence is largely based on studies using N-acetylcysteine (NAC), an antioxidant, to relieve the oxidative stress and prevent cell death. However, here we have demonstrated using inductively coupled mass-spectrometry, that NAC pretreatment significantly reduces intracellular copper uptake triggered by the ionophores, elesclomol and disulfiram, suggesting that reduction in copper uptake, rather than the antioxidant activity of NAC, is responsible for the diminished cell death. We present further data showing that key mediators of reactive oxygen species are not upregulated in response to elesclomol treatment, and further that sensitivity of cancer cell lines to reactive oxygen species does not correlate with sensitivity to these copper ionophores. Our findings are in line with several recent studies proposing the mechanism of cuproptosis is instead via copper mediated aggregation of proteins, resulting in proteotoxic stress leading to cell death. Overall, it is vital to disseminate this key piece of information regarding NAC's activity on copper uptake since new research attributing the effect of NAC on copper ionophore activity to quenching of reactive oxygen species is being published regularly and our studies suggest their conclusions may be misleading.