Covalent sequestration of phosphoramide mustard by metallothionein--an in vitro study

Acquired drug resistance is one of the most important problems in cancer chemotherapy. One of the proposed mechanisms for these phenomena is the sequestration of alkylating agents by metallothionein in vivo. This research shows that metallothionein can covalently sequester phosphoramide mustard, the active form of cyclophosphamide in vitro. On-line electrospray mass spectrometry reveals that it is phosphoramide, not nornitrogen mustard that alkylates metallothionein, although the metallothionein/nornitrogen mustard adduct was isolated as the major adduct. Tandem mass spectrometric experiments were performed on an isolated drug-modified tryptic peptide. The alkylation occurred predominantly at Cys48 of metallothionein. These results provide further evidence that overexpression of metallothionein can detoxify the active form of the drugs.