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Interaction of Oxaliplatin, Cisplatin, and Carboplatin with Hemoglobin and the Resulting Release of a Heme Group Rupasri Mandal, Robyn Kalke, and Xing-Fang Li* Environmental Health Sciences, Department of Public Health Sciences, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, Alberta T6G 2G3, Canada Received May 13, 2004
Oxaliplatin, carboplatin, and cisplatin are widely used to treat a number of cancers. While their DNA adducts are believed to cause cell death, the involvement of their protein adducts in the toxicity and action of these drugs is unclear. Here, we report the interactions of hemoglobin (Hb) with the three platinum (Pt) drugs, demonstrating the formation of Hb-Pt complexes and the release of a heme group from Hb. Oxaliplatin (0.05 µM) was able to form three major complexes with Hb (3-10 µM) after 1 h of incubation at room temperature, and these complexes accounted for ∼60% of the total oxaliplatin. Cisplatin and carboplatin formed one major and two minor complexes only after 24 and 96 h of incubation, respectively. Incubation of these Pt drugs (0.05-10 µM) with whole blood of healthy volunteers and the analysis of red blood cells confirmed the relative ability of these Pt drugs binding to Hb. For the whole blood samples incubated with oxaliplatin and cisplatin for 24 h, only protein complexes were detected in red blood cells, indicating a complete binding of oxaliplatin and cisplatin to the protein. In contrast, carboplatin was partially bound; both the free and the protein-bound carboplatin species were detected in red blood cells. The binding of the Pt drugs to Hb was accompanied by the release of a heme group from Hb, which was monitored by size fractionation, chromatographic separation, and selective detection of both Pt- and iron (Fe)containing molecular species. The released heme was further identified by size fractionation and nanospray mass spectrometry. The findings of the Pt drug interaction with Hb and the dissociation of heme from Hb are potentially useful for a better understanding of the toxicity and side effects of these chemotherapeutic drugs.
Introduction Cisplatin [cis-diamminedichloro platinum(II)] is widely used in the chemotherapy of testicular and ovarian cancer (1), although it has several side effects, including nephrotoxicity, vomiting, ototoxicity, and peripheral neurotoxicity (2). Chronic cisplatin intake has been shown to cause anemia in cancer patients (3, 4). To improve activity and to reduce toxicity, new platinum (Pt)containing drugs have been developed, which were primarily based on systematic modifications of the ligand sphere around the Pt atom. One such drug, carboplatin [cis-diammine-1,1-cyclobutanedicarboxylatoplatinum(II)] (5, 6), has shown equivalent or better antitumor activity than cisplatin but less nephrotoxicity (7). However, carboplatin is more myelotoxic than cisplatin (5, 8). Because myelosuppression is a major contributing factor to anemia, carboplatin causes anemia in many patients undergoing carboplatin therapy (9). Oxaliplatin, a Pt complex with a 1,2-diaminocyclohexane ligand, has shown in vitro and in vivo efficacy against many tumor cell lines and tumors, including some that are resistant to cisplatin and carboplatin (10-15). Unfortunately, oxaliplatin also has several toxic effects, including progressive peripheral sensory neuropathy (16, 17), mild acute neuropathy that * To whom correspondence should be addressed. Tel: 1-780-4925094. Fax: 1-780-492-7800. E-mail:
[email protected].
may be reversed (16, 17), and symptoms of diarrhea, vomiting, and hematological suppression (17-19). Nonetheless, cisplatin, carboplatin, and oxaliplatin (Scheme 1) are useful drugs for cancer chemotherapy. To understand the mechanisms of action and toxicity of these Pt drugs, the formation of DNA adducts with the Pt drugs has been extensively studied (20-22). However, little attention has been paid to the interaction of Pt drugs with specific proteins, although it has been suggested that the formation of protein adducts with Pt drugs may play an important role in the drugs’ activity and side effects (23). Furthermore, most data concerning the pharmacokinetics of the Pt drugs in patients have been obtained by determining total Pt concentration in blood, plasma, and urine without discriminating between the drugs themselves, their metabolites, or their specific protein adducts. The objective of this study was to investigate the interactions of human hemoglobin (Hb) with the three Pt drugs, cisplatin, carboplatin, and oxaliplatin. We report here a comparison of the binding behavior of the three Pt-containing chemotherapy drugs to Hb, first in a model system and then in whole blood samples. We further show the release of the heme group(s) from Hb as a result of the binding of Pt drugs to Hb. Conducted with the clinically relevant concentrations of the Pt drugs and Hb and confirmed with several techniques, this study
10.1021/tx049868j CCC: $27.50 © 2004 American Chemical Society Published on Web 09/30/2004
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Chem. Res. Toxicol., Vol. 17, No. 10, 2004 Scheme 1
provides a useful strategy for evaluating drug-protein interactions.
Experimental Section Instrumentation. HPLC-Inductively Coupled Plasma Mass Spectrometry (ICPMS). An Elan 6100 DRCPlus ICP-MS (Perkin-Elmer/Sciex, Concord, ON, Canada) and a Perkin-Elmer 200 series HPLC system (PE instruments, Toronto, ON, Canada), equipped with a pump and an autosampler, as previously described, were used in this study (24-26). A BioSep-SEC 2000 column (300 mm × 4.6 mm, Phenomenex, Torrance, CA) was used as an analytical column for size exclusion chromatography. The separations were carried out using a mobile phase of 10 mM ammonium bicarbonate (pH 7.4) at a flow rate of 0.8 mL min-1. Nanospray Mass Spectrometry. An Applied Biosystem/ MDS Sciex QSTAR Pulsar i mass spectrometer (Concord, ON, Canada), equipped with a nanospray ionization source, was used to identify the iron (Fe)-containing species in the small molecular mass fraction (
