Trapping of cis-2-Butene-1,4-dial to Measure Furan Metabolism in Human Liver Microsomes by Cytochrome P450 Enzymes
Trapping of cis-2-Butene-1,4-dial to Measure Furan Metabolism in Human Liver Microsomes by Cytochrome P450 Enzymes
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DOI：
10.1124/dmd.111.043679
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发表时间：
2012-03-01
影响因子：
3.9
通讯作者：
Peterson, Lisa A.
中科院分区：
文献类型：
--
作者：
Gates, Leah A.;Lu, Ding;Peterson, Lisa A.
呋喃是啮齿动物的肝脏毒物和致癌物。呋喃被列为一种可能的人类致癌物，但接触呋喃对人类健康的影响尚不清楚。细胞色素P450 （P450）酶对呋喃的氧化是呋喃毒性产生的必要条件。该反应的产物是反应性的α， β -不饱和双醛，顺-2-丁烯-1,4-dial （BDA）。为了确定人肝微粒体是否将呋喃代谢为BDA，建立了液相色谱/串联质谱法，通过n -乙酰-l -半胱氨酸（NAC）和n -乙酰-l -赖氨酸（NAL）捕获这种活性代谢物来检测和定量BDA。NAC和NAL与BDA反应生成n -乙酰- s -[1-（5-乙酰氨基-5-羧基戊基）- 1h -吡咯-3-基]- l-半胱氨酸（NAC-BDA-NAL）。测定了21种不同人肝微粒体制剂中NAC-BDA-NAL的形成。代谢水平与在F-344大鼠和B6C3F1小鼠肝微粒体中观察到的水平相当，这两个物种已知对呋喃诱导的毒性敏感。对重组人肝脏p450的研究表明，CYP2E1是最活跃的人肝脏呋喃氧化酶。对硝基酚羟化酶活性测定的CYP2E1活性与人肝微粒体中NAC-BDA-NAL的形成程度相关。CYP2E1抑制剂能阻断NAC-BDA-NAL的形成，而其他P450抑制剂则不能。这些结果表明，人类能够将呋喃氧化为其有毒代谢物BDA，其速度与对呋喃暴露敏感的物种相当。因此，人类可能容易受到呋喃的毒性影响。
Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive alpha,beta-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-L-cysteine (NAC) and N-acetyl-L-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)- 1H-pyrrol-3-yl]-L-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.