Insights into the Mechanisms of Ifosfamide Encephalopathy: Drug Metabolites Have Agonistic Effects on α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA)/Kainate Receptors and Induce Cellular Acidification in Mouse Cortical Neurons

Institute of Physiology and Laboratory of Neurological Research, Department of Neurology, University of Lausanne Medical School, Lausanne, Switzerland.
Journal of Pharmacology and Experimental Therapeutics (Impact Factor: 3.97). 01/2002; 299(3):1161-8.
Source: PubMed


Therapeutic value of the alkylating agent ifosfamide has been limited by major side effects including encephalopathy. Although the underlying biochemical processes of the neurotoxic side effects are still unclear, they could be attributed to metabolites rather than to ifosfamide itself. In the present study, the effects of selected ifosfamide metabolites on indices of neuronal activity have been investigated, in particular for S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA). Because of structural similarities of SCMC with glutamate, the Ca(2+)(i) response of single mouse cortical neurons to SCMC and TDGA was investigated. SCMC, but not TDGA, evoked a robust increase in Ca(2+)(i) concentration that could be abolished by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but only partly diminished by the N-methyl-D-aspartate receptor antagonist 10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK=801). Cyclothiazide (CYZ), used to prevent AMPA/kainate receptor desensitization, potentiated the response to SCMC. Because activation of AMPA/kainate receptors is known to induce proton influx, the intracellular pH (pH(i)) response to SCMC was investigated. SCMC caused a concentration-dependent acidification that was amplified by CYZ. Since H(+)/monocarboxylate transporter (MCT) activity leads to similar cellular acidification, we tested its potential involvement in the pH(i) response. Application of the lactate transport inhibitor quercetin diminished the pH(i) response to SCMC and TDGA by 43 and 51%, respectively, indicating that these compounds may be substrates of MCTs. Taken together, this study indicates that hitherto apparently inert ifosfamide metabolites, in particular SCMC, activate AMPA/kainate receptors and induce cellular acidification. Both processes could provide the biochemical basis of the observed ifosfamide-associated encephalopathy.

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    • "Intracellular pH (pHi) was measured in single cells on glass coverslips after loading the cells with the pH sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM; Teflabs, Austin, TX) as described previously [21]. Cell loading was performed at room temperature for 10 min using 1 µM BCECF-AM in a HEPES-buffered balanced solution (see composition below). "
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    • "SCMC, but not TDGA, was reported to activate AMPA/kainate receptors (non-NMDA glutamate receptors) on single mouse cortical neurons in vitro and to induce cellular acidification. These properties were believed to provide a basis for ifosfamide encephalopathy (Chatton et al., 2001). Subsequently, it was reported that SCMC was formed directly in mouse brain after the administration of IFO, lending support to the SCMC theory of ifosfamide encephalopathy (Lerch et al., 2006). "
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    • "Dye emissions were observed at 580–620 nm (CR and JC-1 aggregates ) and 500–560 nm (JC-1 monomers). Intracellular pH measurement was performed using the pH-sensitive dye BCECF-AM as described previously (Chatton et al., 2001), in some cases co-loaded with CR. ATP levels were assessed indirectly by measuring intracellular free Mg 21 using Magnesium Green AM as described previously (Chatton and Magistretti, 2005). "
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