Glutathione conjugation of busulfan produces a hydroxyl radical-trapping dehydroalanine metabolite

Abstract

1. The Phase 2 drug metabolism of busulfan yields a glutathione conjugate that undergoes a β-elimination reaction. The elimination product is an electrophilic metabolite that is a dehydroalanine-containing tripeptide, γ-glutamyldehydroalanylglycine (EdAG). In the process, glutathione lacks thiol-related redox properties and gains a radical scavenging dehydroalanine group.

2. EdAG scavenged hydroxyl radical generated in the Fenton reaction in a concentration-dependent manner was monitored by electron paramagnetic resonance (EPR) spectroscopy. The apparent rate of hydroxyl radical scavenging was in the same range as published values for known antioxidants, including N-acyl dehydroalanines.

3. A captodatively stabilized carbon-centered radical intermediate was spin trapped in the reaction of EdAG with hydroxyl radical. The proposed structure of a stable product in the Fenton reaction with EdAG was consistent with that of a γ-glutamylserylglycyl dimer.

4. Observation of the hydroxyl trapping properties of EdAG suggests that the busulfan metabolite EdAG may contribute to or mitigate redox-related cytotoxicity associated with the therapeutic use of busulfan, and reaffirms indicators that support a role in free radical biology for dehydroalanine-containing peptides and proteins.

Keywords::

• Dehydroalanine
• busulfan
• metabolite
• glutathione
• hydroxyl radical
• carbon-centered radical
• EPR

Acknowledgments

The authors thank Heather Glover for accurate mass measurements at the WVU Mass Spectrometry Center. CP current address, Clinical Pharmacology Program, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. Some authors are affiliated with the US Food and Drug Administration, Silver Spring, Maryland or the National Cancer Institute. However, their contributions to the article were made in their private capacity. No official support or endorsement by the Food and Drug Administration or the National Institutes of Health is intended or should be inferred. Funding from WVU PSCoR is gratefully acknowledged.

Declaration of interest

The authors report no conflicts of interest.