Mechanisms for the Oxygen Radical-Mediated Toxicity of Various Thiol-Containing Compounds in Cultured Mammalian Cells

When Chinese hamster V79 cells are exposed to various thiol compounds in phosphate-buffered saline (PBS), some compounds cause toxicity (loss of colony formation), although the dependence on drug concentration and the magnitude of the cell killing vary between the different thiols. For example: dithiothreitol (DTT) and WR-1065 cause a biphasic toxicity whereby cell killing occurs at about 0.2 to 1.0 mM thiol, but is not seen at higher or lower drug concentrations; N-acetylcysteine (NAC) is toxic only at concentrations ≥2 mM and shows no biphasic pattern; and glutathione (GSH) and penicillamine are only minimally toxic at all concentrations. The effect of the addition of <tex-math>$1\ \mu M\ {\rm Cu}^{2+}$</tex-math> to the thiol also depends on the particular thiol: e.g., Cu²⁺ increases cell killing in the biphasic pattern with WR-1065; it increases the toxicity of NAC only at high thiol concentrations; and it elicits a slight toxicity in the biphasic pattern by GSH and penicillamine. In all cases tested, if the thiol is toxic, the cell killing can be decreased or prevented by addition of catalase, consistent with the hypothesis that the toxicity is mediated through H₂ O₂ produced during the thiol oxidation. However, when the oxidation rates of the various thiols in PBS without and with Cu²⁺ were measured, the data did not show a simple correlation between the toxicity of the various thiols and their oxidation rates. The rate of the reaction of the various thiols with H₂ O₂ was also determined and showed a better, but still not good, correlation with toxicity. However, cell killing by the various thiols correlated better with the ratio between the half-lives for thiol oxidation and reaction of thiol with H₂ O₂ than with either reaction rate alone. This suggests that the toxicity pattern and magnitude of cell killing in V79 cells by various thiols depend on the interplay between the rate of thiol oxidation and the rate of reaction between the thiol and the H₂ O₂ produced in the thiol oxidation.