乙醇对CHSE-1细胞中细胞色素P450 2E1的诱导研究

Induced effect of ethanol on cytochrome P450 2E1 in CHSE-1 cell line

以乙醇为细胞色素P450 2E1的诱导剂,在CHSE-1细胞中研究了该诱导剂与酶活性之间的时间效应关系和剂量效应关系。细胞传代后培养48 h,加入含有不同浓度乙醇的新鲜培养基,孵育诱导24 h,之后加入底物苯胺反应30 min。以苯胺的代谢产物4-氨基酚的生成量来反映CYP2E1活性。苯胺浓度为10～16 mmol/L时CYP2E1活性最大,可达(0.152±0.095)nmol.min-1.mg-1,该浓度范围的苯胺适合用于指示CYP2E1的活性。以Gentox模型对诱导的剂量效应进行拟合,得到一个先升高后降低的曲线,说明乙醇对CHSE-1细胞中CYP2E1有先诱导后抑制作用。拟合方程F(x)=(p0+kx)/(1+〔x/EC50〕nH)参数P0为0.140,nH为2.456,k为0.024,EC50为34.938。CYP2E1酶活性随着诱导时间的增加逐渐增强,24 h内酶活性随时间的增加而增强,可达(0.446±0.092)nmol/(min.mg),呈典型的酶诱导现象,建立了乙醇对CHSE-1中CYP2E1的诱导模型。

The dose- and time-dependent induction of P450 2El by ethanol in CHSE-lcell was investigated in the present study. 48 h after inoculation, cells were induced with ethanol of different concentrations for 24 h, and then aniline was added. The cells were incubated at 28 ℃ for 30 min. CYP2E1 activity was measured using the product 4- aminophenol as an index. The optimal concentration of aniline was 10- 16 mmol/L,and basal activity of cells grew into confluence for aniline hydroxylase was （0.152 ± 0.095） nmol· min^-1 ·mg^-1. The dose-dependent effect was fitted by Gentox module, which generated a curve that at first rose and then fell down, indicating that it was firstly induced and inhibited afterward by ethanol. The values of P0, nil, k and EC50 were 0.140, 2.458, 0.025 and 34.938 respectively. It was also demonstrated that the activity of CYP2E1 induced by ethanol increased with time with the highest value up to （0.446 ± 0.092） nmol·min^- 1 ·mg^- 1after 24 h,which was a typical enzyme induction exanple. Through this study, the model of CYP2E1 induction by ethanol in CHSE-1 was established. [Journal of Fishery Sciences of China,2007,14（5） ：856 - 859]