title: N-[5-nitro-2-furfurylidene]-3-amino-2-oxazolidinone activation by the human intestinal cell line Caco-2 monitored through noninvasive electron spin resonance spectroscopy.
abstract: The pathways participating in the metabolism of the nitrofuran antimicrobial drug N-[5-nitro-2-furfurylidene]-3-amino-2-oxazolidinone (furazolidone) in intact cells were investigated in the human intestinal cell line Caco-2. One-electron reduction of furazolidone led to the formation of a free radical intermediate that could be monitored in dense cell suspensions by noninvasive electron spin resonance spectroscopy. The effects of enzyme inhibitors on the kinetics of radical production and decay were used to estimate the relative contribution of different enzymes to the reductive activation of the drug. Although many enzymes are known to reduce nitrofurans in vitro (e.g., xanthine oxidase, aldehyde oxidase, DT-diaphorase, mitochondrial redox chain components), their contributions were insignificant in living Caco-2 cells. The first reducing equivalent required for the formation of the nitroanion derivative of furazolidone appeared to be provided essentially by the microsomal cytochrome P450 reductase. This was confirmed through studies of the NADPH-dependent radical formation by microsomes. Differentiated Caco-2 cells, an established enterocyte model, showed only modestly increased radical formation and the same enzyme-specificity pattern as undifferentiated cells. Consistently, only a small increase in P450 reductase activity was found in differentiated cells, in contrast to the 10-fold increase seen in typical differentiation marker enzymes. With the electron spin resonance method that we describe, it is possible to distinguish between sites of bioactivation of redox active drugs in intact cells.
authors.affiliation: Department of Biology, University of Rome "Tor Vergata," Italy.
authors.forename: L
authors.initials: L
authors.lastname: Rossi
authors.forename: I
authors.initials: I
authors.lastname: De Angelis
authors.forename: J Z
authors.initials: JZ
authors.lastname: Pedersen
authors.forename: E
authors.initials: E
authors.lastname: Marchese
authors.forename: A
authors.initials: A
authors.lastname: Stammati
authors.forename: G
authors.initials: G
authors.lastname: Rotilio
authors.forename: F
authors.initials: F
authors.lastname: Zucco
mesh_terms: D000818:Animals; D000891:Anti-Infective Agents, Local / Q000378:metabolism / Q000493:pharmacokinetics*; D001665:Binding Sites; D001711:Biotransformation; D018938:Caco-2 Cells / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism; D005664:Furazolidone / Q000378:metabolism / Q000493:pharmacokinetics*; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D009574:Nitro Compounds / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D013053:Spectrophotometry
chemical_list: D000891:Anti-Infective Agents, Local; D005609:Free Radicals; D009574:Nitro Compounds; D005664:Furazolidone; D009251:NADPH-Ferrihemoprotein Reductase