Rietjens I M, Cnubben N H, van Haandel M, Tyrakowska B, Soffers A E, Vervoort J
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Chem Biol Interact. 1995 Jan;94(1):49-72. doi: 10.1016/0009-2797(94)03317-2.
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.
采用尿液样本的¹⁹F核磁共振分析，测定了2,5 - 二氟氨基苯及其硝基苯类似物2,5 - 二氟硝基苯的体内代谢物模式。所得结果表明这两种类似物的生物转化模式存在显著差异。对于氨基苯，细胞色素P450催化的芳香族羟基化是主要代谢途径。2,5 - 二氟硝基苯主要通过谷胱甘肽结合代谢，导致5 - 氟 - 2 -（N - 乙酰半胱氨酰）硝基苯和氟离子排出，在较小程度上，通过细胞色素P450催化的羟基化和硝基还原代谢。用各种已知也会影响谷胱甘肽S - 转移酶模式的细胞色素P450酶诱导剂预处理大鼠，然后使其接触2,5 - 二氟氨基苯或2,5 - 二氟硝基苯，通常导致代谢物模式仅在较小（≤12%）程度上有所变化。基于这些结果得出结论，生物转化酶模式不是决定这两种模型化合物代谢途径的主要因素。用2,5 - 二氟氨基苯和2,5 - 二氟硝基苯进行的额外体外微粒体和胞质孵育定性地证实了体内结果。仅在2,5 - 二氟氨基苯的孵育中观察到NADPH/氧气支持的微粒体细胞色素P450催化的羟基化，而胞质谷胱甘肽结合仅在以2,5 - 二氟硝基苯为底物的孵育中发生。分子轨道计算结果提供了一个可行的假设，该假设可以根据硝基苯和氨基苯衍生物的化学特性解释它们代谢途径的差异。该假设指出，硝基苯或氨基苯衍生物进入细胞色素P450或谷胱甘肽结合途径的可能性由化合物前沿轨道的相对能量水平决定。氨基苯衍生物具有相对较高能量的分子轨道，导致其最高占据分子轨道（HOMO）与细胞色素P450（FeO）³⁺中间体的单占据分子轨道有效反应，但其最低未占据分子轨道（LUMO）与谷胱甘肽的HOMO反应性较低。另一方面，硝基苯具有相对较低能量的分子轨道，这解释了其LUMO与谷胱甘肽的HOMO电子之间的有效相互作用，从而发生反应，但与细胞色素P450（FeO）³⁺反应中间体的单占据分子轨道（SOMO）电子反应性较低。
