title: Different metabolic pathways of 2,5-difluoronitrobenzene and 2,5-difluoroaminobenzene compared to molecular orbital substrate characteristics.
abstract: The in vivo metabolite patterns of 2,5-difluoroaminobenzene and of its nitrobenzene analogue, 2,5-difluoronitrobenzene, were determined using 19F NMR analysis of urine samples. Results obtained demonstrate significant differences between the biotransformation patterns of these two analogues. For the aminobenzene, cytochrome P450 catalysed aromatic hydroxylation presents the main metabolic pathway. 2,5-Difluoronitrobenzene was predominantly metabolised through glutathione conjugation leading to excretion of 5-fluoro-2-(N-acetylcysteinyl)-nitrobenzene and fluoride anions, and, to a minor extent, through cytochrome P450 catalysed hydroxylation and nitroreduction. Pretreatment of the rats with various inducers of cytochrome P450 enzymes, known also to influence glutathione S-transferase enzyme patterns, followed by exposure to the 2,5-difluoroamino- or 2,5-difluoronitrobenzene, generally resulted in metabolite patterns that varied only to a small (< or = 12%) extent. Based on these results it was concluded that the biotransformation enzyme pattern is not the predominant factor in determining the metabolic route of these two model compounds. Additional in vitro microsomal and cytosolic incubations with 2,5-difluoroaminobenzene and 2,5-difluoronitrobenzene qualitatively confirmed the in vivo results. NADPH/oxygen supported microsomal cytochrome P450 catalysed hydroxylation was observed only for 2,5-difluoroaminobenzene whereas cytosolic GSH conjugation occurred only in incubations with 2,5-difluoronitrobenzene as the substrate. Outcomes from molecular orbital calculations provided a working hypothesis that can explain the difference in metabolic pathways of the nitro- and aminobenzene derivative on the basis of their chemical characteristics. This hypothesis states that the chances for a nitro- or aminobenzene derivative to enter either a cytochrome P450 or a glutathione conjugation pathway are determined by the relative energy levels of the frontier orbitals of the compounds. The aminobenzene derivative has relatively high energy molecular orbitals leading to an efficient reaction of its highest occupied molecular orbital (HOMO) with the singly occupied molecular orbital of the cytochrome P450 (FeO)3+ intermediate, but a low reactivity of its lowest unoccupied molecular orbital (LUMO) with the HOMO of glutathione. The nitrobenzene, on the other hand, has molecular orbitals of relatively low energy, explaining the efficient interaction, and, thus, reaction between its LUMO and the HOMO electrons of glutathione, but resulting in low reactivity with the SOMO electron of the cytochrome P450 (FeO)3+ reaction intermediate.
authors: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors: I M
authors: IM
authors: Rietjens
authors: N H
authors: NH
authors: Cnubben
authors: M
authors: M
authors: van Haandel
authors: B
authors: B
authors: Tyrakowska
authors: A E
authors: AE
authors: Soffers
authors: J
authors: J
authors: Vervoort
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry* / Q000493:pharmacokinetics* / Q000652:urine; D000818:Animals; D001711:Biotransformation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004790:Enzyme Induction / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D009578:Nitrobenzenes / Q000737:chemistry* / Q000493:pharmacokinetics* / Q000652:urine; D051381:Rats; D017208:Rats, Wistar; D015262:Xenobiotics
chemical_list: D000814:Aniline Compounds; D009578:Nitrobenzenes; D015262:Xenobiotics; C091455:2,5-difluoronitrobenzene; C091456:2,5-difluoroaniline; D003577:Cytochrome P-450 Enzyme System; D005982:Glutathione Transferase; D005978:Glutathione