脂氧合酶介导的杀虫剂N-位脱甲基作用的体外实验

目的　探讨细胞色素P450加单氧酶系以外的代谢途径对杀虫剂的脱烷基作用 ,检测脂氧合酶 (LOX)介导的灭害威等的N 位脱甲基作用。方法　通过Nash反应检测大豆脂氧合酶 (SLO)对灭害威作用产生甲醛的量 ,反映其介导杀虫剂的N 位脱甲基反应速率。结果　SLO对灭害威的脱甲基作用与反应时间以及SLO浓度、灭害威和过氧化氢的浓度呈依赖关系。在最佳反应条件下 ,SLO产生甲醛的最大反应速度 (Vmax)为 1 8nmol·min- 1 ·nmol- 1 。SLO对灭害威的米氏常数 (Km)值为 3 .4mmol/L ,对过氧化氢的Km值为 2 35μmol/L。去甲二氢愈创木酸 (NDGA)、棉子醇 (gossypol)和芬宁东(phenidone)对脱甲基反应的强烈抑制作用提示 ,SLO起催化作用。还原型谷胱甘肽 (GSH)或二硫苏糖醇参与反应时 ,甲醛的累积量明显降低 ,表明SLO对灭害威脱甲基作用时 ,产生自由基。丁羟基甲苯(BHT)和丁羧基苯甲醚 (BHA)对甲醛的产生的抑制作用进一步支持这一论点。除灭害威外 ,发现其他的杀虫剂也发生N 位脱甲基作用 ,只是相对反应速率较低。结论　一些杀虫剂在动植物体内可能通过LOX介导发生脱烷基作用

Lipoxygenase-mediated N-demethylation of pesticides in vitro

OBJECTIVE: In order to explore the pathway of dealkylation of pesticides other than cytochrome P450 monocoxygenases, lipoxygenase (LOX)-mediated demethylation of aminocarb and some other pesticides were measured. METHOD: Formaldehyde generated in the reaction was estimated by Nash reaction to express the rate of demethylation of pesticides mediated by soy lipoxygenase (SLO). RESULTS: N-demethylation of aminocarb mediated by SLO was found to depend on the incubation time, concentration of the enzyme, concentration of aminocarb and hydrogen peroxide. Under optimal conditions, Vmax value of 18 nmol of formaldehyde.min-1.nmol-1 of lipoxygenase was observed. The reaction exhibited Km values of 3.4 mmol/L for aminocarb and 235 mumol/L for hydrogen peroxide. A strong inhibition of the reaction by nordihydroguaiaretic acid, gossypol, and phenidone clearly implicated the lipoxygenase involvement as the protein catalyst. A significant decline in the formaldehyde accumulation in the presence of either reduced glutathione or dithiothreitol suggested generation of a free radical species as an initial oxidation intermediate during the demethylation of aminocarb by SLO. The inhibition of formaldehyde generation by butylated hydroxyanisole(BHT) and butylated hydroxy toluene(BHA) further supported this contention. In addition to aminocarb, seven other pesticides were also found to undergo N-demethylation, albeit at relatively low rates. CONCLUSION: Certain pesticides may oxidatively undergo dealkylation via the lipoxygenase pathway in animals and plants.