Iron increases busulfan toxicity in vivo and in vitro Free

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Busulfan is an alkylating agent widely used in combination high-dose chemotherapy regimens followed by allogeneic or autologous haematopoietic stem cell transplantation (HSCT). The effect of iron on busulfan toxicity was investigated since we proposed that iron-generated pro-oxidant state may explain busulfan toxicity during HSCT.

Toxicology and metabolism of busulfan (16.5 mg/kg bid over four days) combined with melphalan (20 mg/kg) and syngeneic bone marrow transplantation (BMT) were investigated in iron overloaded mice (C57Bl6 male). Mice were supplemented during one month with a special diet with Renutryl^® in which were added high concentrations of FeSO₄ (almost 500 fold the content in the control diet). Iron overload was evidenced by histology in the liver of supplemented mice compared to control animals. Liver accumulation was associated with a significant increase in plasma concentrations from 19 ± 4 µM to 39 ± 4 µM. No difference was observed in busulfan plasma clearance in between control animals and supplemented animals. However in both cases, a significant induction of clearance through the glutathione pathway during repeated injections was observed. In spite of equivalent busulfan exposure, we observed a dramatic increase of the toxicity after a combined treatment with busulfan and melphalan in iron supplemented mice with a median survival of 2 days after BMT. We have shown previously that busulfan metabolism through the glutathione pathway induces an increase of cysteine liver content in order to sustain glutathione levels. Consequently iron may be reduced from the ferric to the ferrous form. This latter is known to be responsible of the pro-oxidant state through the Fenton reaction.

This hypothesis was investigated in vitro. Prior cell cycle analyses have shown the time-dependent toxicity of busulfan. These investigations allowed to define optimal conditions to investigate busulfan toxicity in vitro against two cell lines; IGR-N91, a cell line derived from a neuroblastoma and HepG2, a cell line derived from an hepatocarcinoma. Busulfan was combined with iron and cysteine and the cytotoxicity was investigated after one week of treatment with the MTT assay. In vitro experiments have shown that iron enhanced significantly the cytotoxicity of busulfan. This enhancement of busulfan cytoxicity was maximal when iron (1 mM) was combined with cysteine (2 mM) to favour the pro-oxidant ferrous state. In these conditions, the CI50 of busulfan decreased from 15.5 ± 4.2 µM to 4.8 ± 1.1 µM in IGR-N91 and from 21.7 ± 7.2 µM to 13.0 ± 0.9 µM in Hep-G2.

In conclusion, iron increases busulfan toxicity. Mechanisms need to be elucidated, especially through the investigation of the metabolism of busulfan by the glutathione/glutathione S-transferase pathway.