Glutathione diminishes the anti-tumour activity of 4-hydroperoxycyclophosphamide by stabilising its spontaneous breakdown to alkylating metabolites

Evidence was obtained showing that GSH protects against the cytotoxicity of 4-hydroperoxycyclophosphamide (4-OOH-CP) by minimizing the spontaneous fission of 4-hydroxycyclophosphamide (4-OH-CP), its breakdown product, to the ultimate toxic species, phosphoramide mustard (PM). This conclusion was borne out in two series of experiments. The first demonstrated that 4-OH-CP was progressively more stable in aqueous solutions containing increasing concentrations of GSH. The second series of experiments were carried out with tumour cell lines with high (SKOV-3) and low (KHT) GSH contents. The cytotoxicity of 4-OOH-CP, a stable precursor that rapidly gives rise to 4-OH-CP spontaneously under physiological conditions, was enhanced in GSH-depleted SKOV-3 cells, but was unchanged in GSH-depleted KHT cells. It is concluded that the high GSH content of SKOV-3 cells provides a significant protection against 4-OH-CP by limiting the breakdown/activation of 4-OH-CP. Deschloro-4-hydroperoxycyclophosphamide (deschloro-4-OOH-CP), an analogue of 4-OOH-CP that generates acrolein (AC) but not PM in the spontaneous fission reaction, is essentially non-toxic when compared with 4-OOH-CP but is equally potent in depleting GSH. It is postulated that AC may promote the cytotoxicity of the parent 4-OH-CP by depleting cellular GSH. Consequently, the stabilising influence of GSH on 4-OH-CP is removed, leading to increased formation of PM, the ultimate cytotoxic agent.