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

Abstract

There is considerable evidence that glutathione (GSH) plays a major role in protecting tumour cells against the cytotoxicity of the oxazaphosphorines, including cyclophosphamide (CP) and its active congener, 4-hydroperoxycyclophosphamide (4-OOH-CP), both in vitro (Russo et al., 1986; Crook et al., 1986a) and in vivo (Gurtoo et al., 1981;Carmichael et al., 1986b; Ono & Shrieve, 1987). Recently, in a study of 17 tumour cell lines, we noted a close correlation between the chemosensitivity of these cell lines to 4-OOH-CP and their steady-state GSH level (Lee et al., 1990). It was further noted that whilst GSH 'detoxifies' 4-OOH-CP, 4-OOH-CP in turn depletes GSH. In fact, 4-OOH-CP is at toxic concentrations a potent depletor of cellular GSH. Most important, the tumour cell GSH depletion and the lethality produced by 4-OOH-CP appear to be linked events. Significant 4-OOH-CP cytotoxicity was invariably associated with GSH depletion, and vice versa. In the present paper, evidence is presented showing that GSH modulates the cytotoxicity of 4-OOH-CP by participating in chemical reactions at three separate locations in the metabolic pathway of 4-OH-CP, its spontaneous breakdown product. (4-OOH-CP gives rise rapidly to 4-OH-CP following dissolution without any enzymic involvement and may be regarded as equivalent to 4-OH-CP pharmacologically (Sladek, 1987). 4-OOH-CP is the preferred 'activated' cyclophosphamide for routine use only because of its higher stability in crystalline state and easier synthesis.) 4-OH-CP, sometimes called 'activated' cyclophosphamide, is formed from the hydroxylation of CP by the hepatic mixed-function oxidases (Figure 1). 4-OH-CP is in reality the 'transport' form of CP since it is in this form that active CP reaches the target tumour cells (Sladek, 1987). Intracellular 4-OH-CP is in equilibrium with its ring-opened tautomer aldophosphamide (AP). The fate of AP may follow one of three main competing metabolic pathways: (1) spontaneous fission to acrolein (AC) and phosphoramide mustard (PM), (2) ring-closure and hydroxylation to produce once again the parent 4-OH-CP. As depicted in Figure 1, glutathione (GSH) can participate in conjugative reactions at three separate locations that may have considerable influence on the eventual cytotoxicity of 4-OH-CP. (1) Reaction with AP as described above which shifts the pseudoequilibrium between 4-OH-CP and AP in favour of the former and thereby curtails the spontaneous degradation of AP to toxic metabolites. GSH also reacts irreversibly with the toxic metabolites AC and PM, but particularly the former (2 and 3) (Gurtoo et al., 1981). In the accompanying paper we demonstrated that when the combined rates of the conjugation reactions exceed the rate of GSH recovery, i.e. when GSH is being depleted, significant cytotoxicity inevitably occurs. The present findings suggest that GSH depletion impacts directly on the cytotoxic potency of 4-OH-CP by destabilising AP (see Figure 1)

DNA cell cycle distribution and glutathione (GSH) content according to circadian stage in bone marrow of cancer patients

“…This was not due to a low content of GSH in the two patients having undergone intensive chemotherapy (mean GSH content of these two patients was 2.34 nmol mg-' protein). Thus, the earlier reported low level of glutathione content in human bone marrow was confirmed A A (Smaaland et al, 1991b) Tsutsui et al, 1986;Ozols et al, 1987;Lee, 1991).…”

The effect of busulphan on the pharmacokinetics of cyclophosphamide and its 4-hydroxy metabolite: time interval influence on therapeutic efficacy and therapy-related toxicity

“…Several studies have illustrated the critical importance of GSH as a determinant of cellular sensitivity to melphalan, cisplatin and cyclophosphamide [49][50][51] where GSH was rap- idly depleted by the administration of CP. 52 Cellular overexpression of glutathione content and glutathione S-transferase was also found to contribute to acquired resistance of tumor cells to 4-OHCP.…”

ABCC2-Mediated Biliary Transport of 4-Glutathionylcyclophosphamide and Its Contribution to Elimination of 4-Hydroxycyclophosphamide in Rat

“…1) has been reported to undergo reversible conjugation with glutathione chemically and enzymatically (Kwon et al, 1987;Lee, 1991;Dirven et al, 1994a,b). The resulting conjugate, 4-glutathionylcyclophosphamide (GSCY) is a potential substrate of ABCC2, a member of the multidrug resistance protein family (subsequently designated ABCC), localized on the canalicular membrane of hepatocytes.…”