Lakshmi V M, Hsu F F, Davis B B, Zenser T V
VA Medical Center, Division of Geriatric Medicine, Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63125-4199, USA.
Chem Res Toxicol. 2000 Feb;13(2):96-102. doi: 10.1021/tx990031b.
Arylamine-hemoglobin conjugates identified as sulfinamides are considered dosimeters for the bioavailability of metabolically formed N-oxidation products. This report considers peroxidation as an alternative pathway for aromatic amine metabolism and examines horseradish peroxidase metabolism of N-acetylbenzidine (ABZ) in the presence of glutathione. When 0.06 mM [(3)H]ABZ was incubated with 1 mM glutathione, a decrease in the total extent of metabolism was observed along with detection of a new metabolite (ABZ-SG), representing 12% of the total radioactivity. Optimum ABZ-SG formation occurred at 0.3 mM glutathione with higher concentrations (10 mM) being inhibitory. In the absence of glutathione, a molar ratio of H(2)O(2) to ABZ of 1:1 resulted in complete metabolism of ABZ. This ratio increased to >2:1 in the presence of 0.3 mM glutathione. N-Oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected using a variety of incubation conditions. ABZ-SG was sensitive to gamma-glutamyltranspeptidase, and completely hydrolyzed by 0.1 N HC1 or 0.1 N NaOH in 10 min at room temperature. ABZ-SG was identified by mass spectrometry and NMR to be N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide. ABZ-SG formation, but not total ABZ metabolism, was prevented by 0.3 mM NaN(3), 50 mM DMPO, 1.0 mM thiourea, and 1.0 mM histidine. Cyanide (50 mM) and ascorbic acid (0.1 mM) completely inhibited ABZ metabolism. The lack of effect of 50 mM mannitol and 2 microgram of superoxide dismutase suggests that neither hydroxyl radical nor superoxide is involved in the reaction. Studies also indicated that molecular oxygen is not a source of the sulfinamide oxygen. Formation of an ABZ sulfinamide conjugate with hemoglobin was demonstrated. The proposed mechanism for sulfinamide formation, involving two consecutive one-electron oxidations with subsequent rearrangement to a sulfur-stabilized nitrenium ion, suggests that oxygen may be derived from water. The results demonstrate that while arylamine-hemoglobin conjugates serve as useful biomarkers of exposure, their mechanism of formation may be complex, perhaps involving peroxidation as in the case of N'-(glutathion-S-yl)-N-acetylbenzidine S-oxide.
被鉴定为亚磺酰胺的芳胺 - 血红蛋白共轭物被认为是代谢形成的N - 氧化产物生物利用度的剂量计。本报告认为过氧化是芳香胺代谢的另一种途径，并研究了在谷胱甘肽存在下辣根过氧化物酶对N - 乙酰联苯胺（ABZ）的代谢。当0.06 mM [³H]ABZ与1 mM谷胱甘肽一起孵育时，观察到代谢的总程度降低，同时检测到一种新的代谢物（ABZ - SG），占总放射性的12％。在0.3 mM谷胱甘肽时ABZ - SG形成最佳，更高浓度（10 mM）具有抑制作用。在没有谷胱甘肽的情况下，H₂O₂与ABZ的摩尔比为1:1导致ABZ完全代谢。在0.3 mM谷胱甘肽存在下，该比例增加到>2:1。在各种孵育条件下均未检测到ABZ代谢的N - 氧化产物，如N'-羟基 - N - 乙酰联苯胺。ABZ - SG对γ-谷氨酰转肽酶敏感，并在室温下10分钟内被0.1 N HCl或0.1 N NaOH完全水解。通过质谱和核磁共振鉴定ABZ - SG为N' - （谷胱甘肽 - S - 基） - N - 乙酰联苯胺S - 氧化物。0.3 mM NaN₃、50 mM DMPO、1.0 mM硫脲和1.0 mM组氨酸可阻止ABZ - SG的形成，但不影响ABZ的总代谢。氰化物（50 mM）和抗坏血酸（0.1 mM）完全抑制ABZ代谢。50 mM甘露醇和2微克超氧化物歧化酶没有作用，这表明羟基自由基和超氧阴离子均不参与该反应。研究还表明分子氧不是亚磺酰胺氧的来源。证明了ABZ与血红蛋白形成亚磺酰胺共轭物。提出的亚磺酰胺形成机制涉及两个连续的单电子氧化，随后重排为硫稳定的氮鎓离子，这表明氧可能来自水。结果表明，虽然芳胺 - 血红蛋白共轭物是有用的暴露生物标志物，但其形成机制可能很复杂，可能像N' - （谷胱甘肽 - S - 基） - N - 乙酰联苯胺S - 氧化物的情况一样涉及过氧化。
