DIFFERENT METABOLIC PATHWAYS OF 2,5-DIFLUORONITROBENZENE AND 2,5-DIFLUOROAMINOBENZENE COMPARED TO MOLECULAR-ORBITAL SUBSTRATE CHARACTERISTICS

The in vivo metabolite patterns of 2,5-difluoroaminobenzene and of its nitrobenzene analogue, 2,5-difluoronitrobenzene, were determined usin g F-19 NMR analysis of urine samples. Results obtained demonstrate sig nificant differences between the biotransformation patterns of these t wo analogues. For the aminobenzene, cytochrome P450 catalysed aromatic hydroxylation presents the main metabolic pathway. 2,5-Difluoronitrob enzene was predominantly metabolised through glutathione conjugation l eading to excretion of 5-fluoro-2-(N-acetytcysteinyl)-nitrobenzene and fluoride anions, and, to a minor extent, through cytochrome P450 cata lysed hydroxylation and nitroreduction. Pretreatment of the rats with various inducers of cytochrome P450 enzymes, known also to influence g lutathione 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 (less than or equal to 12%) extent. Based on these results it was concluded that the biotran sformation 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 an d 2,5-difluoronitrobenzene qualitatively confirmed the in vivo results . NADPH/oxygen supported microsomal cytochrome P450 catalysed hydroxyl ation was observed only for 2,5-difluoroaminobenzene whereas cytosolic GSH conjugation occurred only in incubations with 2,5-difluoronitrobe nzene as the substrate. Outcomes from molecular orbital calculations p rovided a working hypothesis that can explain the difference in metabo lic pathways of the nitro- and aminobenzene derivative on the basis of their chemical characteristics. This hypothesis states that the chanc es for a nitro- or aminobenzene derivative to enter either a cytochrom e P450 or a glutathione conjugation pathway are determined by the rela tive energy levels of the frontier orbitals of the compounds. The amin obenzene derivative has relatively high energy molecular orbitals lead ing to an efficient reaction of its highest occupied molecular orbital (HOMO) with the singly occupied molecular orbital of the cytochrome P4 50 (FeO)(3+) intermediate, but a low reactivity of its lowest unoccupi ed molecular orbital (LUMO) with the HOMO of glutathione. The nitroben zene, on the other hand, has molecular orbitals of relatively low ener gy, explaining the efficient interaction, and, thus, reaction between its LUMO and the HOMO electrons of glutathione, but resulting in low r eactivity with the SOMO electron of the cytochrome P450 (FeO)(3+) reac tion intermediate.