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1 7 4 Vol. 7 No Journal of Food Safety and Quality Apr., 2016 Cocktail P450 李宏 1, 周培源 1, 范雯婷 1, 刘晓宇 1, 2* (1., ; 2., ) 摘要 : 目的 Cocktail P450, 方法 4,, 1, - 结果 t 1/2 (P<05) AUC(P<0.01) MRT(P<0.05); CL(P<0.05) AUC(P<0.01) MRT(P<0.05), t 1/2 (P<0.05) AUC(P<0.05) MRT(P<0.01) 3 结论 CYP1A2, CYP2C9 CYP3A4, CYP2E1 CYP2D6 CYP2C19 关键词 : ; P450; ; Approach for assessing the effect of phoxim on CYP450 enzymes activities in crucian carp by cocktail LI Hong 1, ZHOU Pei-Yuan 1, FAN Wen-Ting 1 1, 2*, LIU Xiao-Yu (1. College of Food Science and Technology, Huazhong Agricultural University, Wuhan , China; 2. Key Laboratory of Environment Correlative Dietology, Ministry of Education, Wuhan , China) ABSTRACT: Objective To establish cocktail method for elucidating the effects of phoxim on the in vivo metabolism of different isoforms enzyme system of cytochrome P450 in crucian carp, in order to lay the foundation for exploring the metabolic pathways and toxicology evaluation of phoxim residues in crucian carp body. Methods The crucian carp were randomly divided into 4 groups (phoxim group, control group, inhibitors group and revulsant group). The plasma samples obtained at different intervals were analyzed by liquid-liquid extraction-high performance liquid chromatography (LLE-HPLC) after 7 d continuous dosing. Results Phoxim could significantly increase the half-life (t 1/2 ) (P<0.05), the area under the blood drug concentration and time (AUC) (P<0.01), and the average resident time (MRT) (P<0.05) of caffeine in crucian carp, and could significantly reduce the clearance (CL) (P<0.05), AUC (P<0.01) and MRT (P<0.05) of tolbutamide in crucian carp; and significantly reduce the t 1/2 (P<0.05), the AUC (P<0.05) and the MRT (P<0.01) of dapsone in crucian carp. The pharmacokinetic parameters of 基金项目 : ( , ) Fund: Supported by the National Natural Science Foundation of China ( , ) * 通讯作者 :,,,, liuxiaoyu@mail.hzau.edu.cn *Corresponding author: LIU Xiao-Yu, Professor, College of Food Science and Technology, Huazhong Agricultural University, No.1, Shizishan Street, Hongshan District, Wuhan , China. liuxiaoyu@mail.hzau.edu.cn

2 chlorzoxazone, metoprolol, omeprazole had no significant differences between phoxim group and control group. Conclusion Phoxim could strongly inhibit the activity of CYP1A2, induce the activity of CYP2C9 and CYP3A4, and had no significant effect on the activities of CYP2E1, CYP2C19 and CYP2D6 in crucian carp. KEY WORDS: phoxim; cytochrome P450; probe drugs; metabolism in vivo 1 引言 (phoxim),,, [1],, [2], [3],, P450,, CYP1A CYP2C CYP3A 3 [4,5] CYP450 [6] ; CYP450, [7] Bozcaarmutlu [8] CYP450, CYP1A CYP3A 2 CYP450 Bhutia [9] CYP450, CYP450, CYP1A CYP2E1 CYP2B, CYP3A4 Cocktail, 2 CYP450, () CYP450,, [10-12] Cocktail [13-15],, CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP2E1 CYP3A4 6 CYP450, Cocktail CYP CYP450, CYP450, 2 材料与方法 2.1 仪器与试剂 :, 96.2, ; ( ), 98.0, ;, : (ML204), ( ); (Waters e ), Waters ; ODS-C 18 (250 mm 4.6 mm, 5 μm), ; (SB-3200DT), 2.2 实验方法溶液配制 5 mg/ml : 58 mg (86%) 10 ml,, : 40 mg 40 mg 40 mg 6 ml, 40 mg 20 mg 40 mg 3 ml,, 10 ml, 色谱条件 : ODS C 18 (250 mm 4.6 mm, 5 μm) ; : 1.0 ml/min; : 230 nm; : 25 ; : 20 µl: A: ; B: (0.02%, ph=2.5), 辛硫磷对鲫鱼体内的 CYP450 不同亚型酶活性的影响 20 ( ), 4, 5 : (1) ; (2) ; (3)

3 4, : Cocktail P ; (4), 0.33 mg/l,, 50 mg/kg 1, 7 d 8 d 0.25 ml/100 g Cocktail Cocktail h 0.3 ml 1 ml, 5000 r/min 15 min, -20, Table 1 表 1 流动相的梯度洗脱程序 Gradient elution programs of mobile phase (min) (%) (0.02%) 鲫鱼血浆样品处理方法 100 μl, 1.5 ml, 50 μg/ml 10 μl, 1 min 1 ml, 5 min r/min 15 min 800 μl,, 100 μl, 20 μl 方法学验证 (1) , 2.2.1,, (2) 6, (caffeine, CAF), (dapsone, DDS), (chlorzoxazone, CZX), (tolbutamide, TOL), (metoprolol, MET) (omeprazole, OME) Cocktail 90 μl, 1.5 ml, 10 μl, 2.2.3, 2.2.1, 6, 6,, (3) 90 μl, 1.5 ml, Cocktail 10 µl, 6, 2.2.3, 2.2.1, 3 d,,, (4) 90 μl, 1.5 ml, Cocktail 10 µl, 6, 2.2.3, 2, h, 2.2.1,,, 数理统计与分析 Mean±SD, Winolin version 7, Origin SPSS 17.0 t 3 结果与分析 3.1 方法学论证方法专属性论证 1(A)~(D) : 6, 7, : min 标准曲线的绘制 6 r 2, 6,, 方法精密度和回收率测定 : %, %~96.016%6, 6,,,

4 (a): ; (b): 6 +; (c): +6 +; (d): Fig. 1 Chromatograms (a): blank fish plasma; (b): Six mixed special probe drugs with IS; (c): blank fish plasma spiked with 6 mixed special probe drugs and IS; (d): Fish plasma sample Table 2 表 2 6 种探针药物的线性回归方程相关系数线性范围及检出限 Calibration curve, correlation coefficient, linear range and LOD of 6 kinds of probe drugs r (µg/ml) LOD(µg/mL Y=0.0691X ~ Y=0.0982X ~ Y=0.1785X ~ Y=0.1125X ~ Y=0.0460X ~ Y=0.1614X ~ 表 3 6 种探针药物的日内日间精密度及方法回收率 (Mean±SD, n=6) Table 3 Intra-day and inter-day precision and recoveries of 6 kinds of probe drugs (Mean±SD, n=6) (µg/ml) (µg/ml) RSD(%) (µg/ml) RSD(%) (%) RSD(%) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5 4, : Cocktail P 方法稳定性 h 4 : h RSD 3.47%~8.95%,10% h, 6, 3.2 辛硫磷对鲫鱼体内的 CYP450 不同亚型酶活性的影响, Winolin version 7 5: t 1/2 (P<0.05) AUC(P<0.01) MRT(P<0.05) CYP1A2, CYP1A2 CYP1A2 CL(P<0.05) AUC(P<0.01) MRT(P<0.05) CYP2C9, CYP2C9 t 1/2 (P<0.05) AUC(P<0.05) MRT(P<0.01) CYP3A4, CYP3A4, CYP2E1 CYP2D6 CYP2C19 表 4 Table 4 (µg/ml) 6 种探针药物在 h 冻融条件下稳定性考察结果 (Mean±SD, n=6) Stability of 6 kinds of probe drugs at 20 for 1, 12, 24 h (Mean±SD, n=6) 20 1 h h h (µg/ml) RSD(%) (µg/ml) RSD(%) (µg/ml) RSD(%) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6 Fig Mean plasma concentration-time curves of TOL (a), DDS (b), CAF (c), CZX (d), MET (e) and OME (f) Table 5 表 5 6 种探针药物在鲫鱼体内的药代动力学参数 (Mean±SD, n=5) Pharmacokinetic parameters of 6 kinds of probe drugs in crucian carp (Mean±SD, n=5) T 1/2 (h) ± ± * ± ± CL(L/h/kg) ± ± ± ± AUC (0-t) (mg/l*h) ± ± ** ± ± MRT (0- ) (h) ± ± * ± ± C max (µg/ml) ± ± ± ±1.5274

7 4, : Cocktail P 续表 5 T 1/2 (h) ± ± * ± ± CL(L/h/kg) ± ± ± ± AUC (0-t) (mg/l*h) ± ± * ± ± MRT (0- ) (h) ± ± ** ± ± C max (µg/ml) ± ± ± ± T 1/2 (h) ± ± * ± ± CL(L/h/kg) ± ± ± ± AUC (0-t) (mg/l*h) ± ± ** ± ± MRT (0- ) (h) ± ± * ± ± C max (µg/ml) ± ± ± ± T 1/2 (h) ± ± ± ± CL(L/h/kg) ± ± ± ± AUC (0-t) (mg/l*h) ± ± ± ± MRT (0- ) (h) ± ± ± ± C max (µg/ml) ± ± ± ± T 1/2 (h) ± ± ± ± CL(L/h/kg) ± ± ± ± AUC (0-t) (mg/l*h) ± ± ± ± MRT (0- ) (h) ± ± ± ± C max (µg/ml) ± ± ± ± T 1/2 (h) ± ± ± ± CL(L/h/kg) ± ± ± ± AUC (0-t) (mg/l*h) ± ± ± ± MRT (0- ) (h) ± ± ± ± C max (µg/ml) ± ± ± ±0.4025n 4 结论 Cocktail CYP450, CYP450, CYP1A2, CYP2C9 CYP3A4, CYP2E1 CYP2D6 CYP2C19, 参考文献 [1]. [D]. :, Li HM. Study on liver toxicity of phoxim on rats [D]. Yangzhou: Yangzhou University, [2],,. [J]., 2010, 40: Cao Y, Zhu BK, Wang JW. Acute toxicity of four kinds of fish medicine on the rare crucian carp [J]. Freshwater Fish, 2010, 40: [3]. [M]. :, Zhang YJ. Guide for the use of pesticides [M]. Beijing: China Agricultural

1606 7 Press, 2002. [4] Uno T, Ishizuka M, Itakura T. Cytochrome P450(CYP) in fish [J]. Environ Toxicol, 2012, 34(1): 1 13. [5] Nelson, DR.

Implications of cytochrome 450 isoenzymes, aryl-esterase and oxonase activity in the inhibition of the acetylcholinesterase of Chirostoma jordani treated with phosphorothionate pesticides [J].

8 Press, [4] Uno T, Ishizuka M, Itakura T. Cytochrome P450(CYP) in fish [J]. Environ Toxicol, 2012, 34(1): [5] Nelson, DR. Comparison of P450s from human and fugu:420 million years of vertebrate P450 evolution [J]. Arch Biochem Biophys, 2003, 409(1): [6] Dzul-Caamal R, Domínguez-López ML, García-Latorre E, et al. Implications of cytochrome 450 isoenzymes, aryl-esterase and oxonase activity in the inhibition of the acetylcholinesterase of Chirostoma jordani treated with phosphorothionate pesticides [J]. Ecotoxicol Environ Saf, 2012, 84: [7] Bhutia D, Rai BK, Pal J. Hepatic cytochrome P450 as biomarkers of cypermethrin toxicity in freshwater teleost, Channa punctatus (Bloch) [J]. Brazilian Arch Biol Technol, 2015, 58(1): [8] Bozcaarmutlu A, Turna S, Sapmaz C, et al. Aldrin epoxidation in flathead mullet (Mugil cephalus): possible involvement of CYP1A and CYP3A [J]. J Biochem Molecular Toxicol, 2014, 28(6): [9] Bhutia D, Rai BK, Pal J. Detection of multiple cytochrome P450 in hepatic tissue of Heteropneustes fossilis (Bloch) exposed to cypermethrin [J]. Proc Zool Soc, 2013, 66(1): [10],,,. Cocktail [J]., 2010, 12(11): Wang L, Guo J, Shi ZF, et al. The research ideas and applications of "Cocktail" probe drug method [J]. J Liaoning Univ Tradit Chine Med, 2010, 12(11): [11] Zhuang Z, Dahu G, Zhong B, et al. Effects of dimethoate on the activities of CYP450 enzymes in rats using a cocktail approach [J]. Lat Am J Pharm, 2014, 33(6): [12] Lin CH, Chou PH, Chen PJ. Two azole fungicides (carcinogenic triadimefon and non-carcinogenic myclobutanil) exhibit different hepatic cytochrome P450 activities in medaka fish [J]. J Hazard Mater, 2014, 277: [13] Hu L, Wen C, Yang S, et al. Effects of alismatis rhizome on the metabolic ability of CYP450 in rats [J]. Latin Am J Pharm, 2014, 33(8): [14] Guo J, Xu Q, Wang L, et al. Effects of transmetil on the metabolic ability of CYP450 in rats [J]. Latin Am J Pharm, 2014, 33(8): [15] Wang X, Han A, Wen C, et al. The effects of H2S on the activities of CYP2B6, CYP2D6, CYP3A4, CYP2C19 and CYP2C9 in vivo in rat [J]. Inter J Molecular Sci, 2013, 14(12): 作者简介 ( 责任编辑 : 姚菲 ) 李宏, 硕士, 主要研究方向为食品安全 @qq.com 刘晓宇, 博士, 教授, 博士生导师, 主要研究方向为食品安全 liuxiaoyu@mail.hzau.edu.cn

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min 25 4 2013 8 监测技术 5 1 2 1 ( 1. 江苏省疾病预防控制中心, 江苏南京 210009; 2. 南京医科大学第二附属医院, 江苏南京 210029) 5 Phenomenex kinetex C 18 2. 6 μm 4. 6 mm 150 mm 10 mmol /L ph 2. 5 + 60 40 DAD 280 nm 5 20. 0 mg /L ~ 100 mg /L 0.

Microsoft Word 李宏 二校%

Microsoft Word 李宏二校%