蚯蚓细胞色素P450生物标记物方法研究

以蚯蚓(Eisenia fetida)为试验动物,通过强化预处理(盐水洗脱)、改变离心力和溶解微粒体膜等实验,建立了蚯蚓细胞色素P450含量的定性、定量测定方法.在此基础上,以多环芳烃(菲)为供试外源污染物,分别以滤纸接触法和土壤投加法,进行了蚯蚓细胞色素P450含量与菲污染诱导的量-效关系试验.结果表明,增加和调整离心力,加入增溶剂等方法,可有效去除干扰,使蚯蚓微粒体样品的450峰值由完全被干扰掩盖(在455～457nm处滞后出现),转变为在450nm±1nm达到最大值.滤纸接触试验(10^-6,10^-5,10^-4,10^-3和10^-2mg·mL^-1)和土壤染毒试验(1,2,4,8 mg·kg^-1)均表明,蚯蚓体内P450含量与菲浓度存在量-效关系.对土壤染毒不同诱导时间(1,3,7,14,28d)的测定结果表明,不同浓度的菲对蚯蚓毒性为诱导刺激(7d)效应和诱导抑制(14d和28d)效应,导致蚯蚓体内P450总量分别为对照组的0.99～1.41倍及0.77～0.88倍(p<0.05).蚯蚓细胞色素P450含量测定方法具有简单、快速、低耗、敏感的优点,是适合于土壤低剂量污染暴露毒性诊断的生物标记物指标.

Establishment of Method for Cytochrome P450 of Earthworms(Eisenia fetida) as a Biomarker

Earthworm (Eisenia fetida) was chosen as test soil fauna to establish the method for determination of cytochrome P450 content. By means of thoroughly washing with salt solution, changing centrifugal acceleration and dissolving microsomal membranes of earthworms with sodium cholate, the determination of cytochrome P450 was performed. Base on the method, dynamic relationships of dose-response for cytochrome P450 contents in earthworm and phenanthrene concentrations were conducted by contact filter paper and soil contaminated with phenanthrene. Results indicate that cytochrome P450 are covered up by interferential material in earthworm, which makes the classic peak lag and appear at 455 - 457nm in the CO difference spectrum with a spectrophotometer. Through increasing centrifugal acceleration and adding reagent of solubilization, the interference is removed mostly and the classic peak of P450 returns to appear at 450nm +/- 1nm. The exposure tests of phenanthrene with contact filter paper (10(-6), 10(-5), 10(-4), 10(-3) and 10(-2) mg x mL(-1)) and soil (1, 2, 4 and 8 mg x kg(-1)) reveal that the dose-response relationships exist between the pollutant and cytochrome P450. Soil tests of different exposure durations (1, 3, 7, 14 and 28d) show the effects of different phenanthrene concentration on earthworm cytochrome P450 contents are to activate (7d) and to inhibit (14d and 28d), and the content of P450 is 0.99 - 1.41 and 0.77 - 0.88 (p < 0.05) times of control respectively. From this study, cytochrome P450 content of earthworm has the merit of simpleness, speediness and economy for determination, and it could be used as a sensitive biomarker for monitoring the exposure of sublethal pollution in terrestrial ecosystem.