Human UDP-glucuronosyltransferase 1A1 is the primary enzyme responsible for the n-glucuronidation of N-hydroxy-PhIP in vitro
Human UDP-glucuronosyltransferase 1A1 is the primary enzyme responsible for the n-glucuronidation of N-hydroxy-PhIP in vitro
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DOI：
10.1021/tx049898m
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发表时间：
2004-08-01
影响因子：
4.1
通讯作者：
Felton, JS
中科院分区：
文献类型：
--
作者：
Malfatti, MA;Felton, JS
udp -葡萄糖醛基转移酶1A蛋白（UGT1A）催化许多内源性和外源性化合物的葡萄糖醛酸化，包括杂环胺及其羟基化代谢物。研究表明，在人体中，ugt1a介导的葡萄糖醛酸化是食源性致癌物杂环胺解毒的重要途径。2-氨基-1-甲基-6-苯基咪唑[4,5-b]吡啶（PhIP）是熟肉中发现的质量最丰富的杂环胺，其生物转化高度依赖于细胞色素P4501A2的羟基化，然后是ugt催化的n -羟基PhIP反应中间体的葡萄糖醛酸化。为了确定哪些UGT1A蛋白参与了n -羟基phip的糖醛酸化，我们将杆状病毒感染的昆虫细胞中表达所有已知功能的UGT1A同工酶（UGT1A1, -1A3, -1A4, -1A6, -1A7, -1A8， -1A9和-1A10）的微体制剂暴露于n -羟基phip中，并通过HPLC分离反应产物。除UGT1A6外，所有UGT1A蛋白对n -羟基phip均有一定程度的活性。n-羟基- phip - n- 2-glucuronide和n-羟基- phip - n3 -glucuronide的形成都是时间和底物浓度依赖的。UGT1A1将n -羟基- phip转化为两种偶联物的效率最高，产生的n -2偶联物比活性第二高的UGT1A4多5倍，比活性最低的UGT1A7多286倍。表观K-m为52 muM， K-cat为114 min(-1)， UGT1A1在生成n -羟基- phip - n2 -glucuronide中的催化效率最高。与n -羟基phip - n2 -glucuronide生成的Kat值相比，UGT1A1、-1A4和-1A8对n -羟基phip - n2 -glucuronide生成的催化效率分别低8倍、10倍和6倍。这些结果清楚地表明，UGT1A1对葡萄糖醛酸化n -羟基phip具有最高的特异性。多态性表达导致UGT1A1活性降低，可导致葡萄糖醛酸化速率降低，从而改变生物活化和解毒之间的代谢比率，有利于生物活化。这种变化将增加对PhIP暴露有害影响的易感性，因为形成无毒n -羟基PhIP葡萄糖醛酸缀合物的能力将减少。
UDP-glueuronosyltransferase 1A proteins (UGT1A) catalyze the glucuronidation of many endogenous and xenobiotic compounds including heterocyclic amines and their hydroxylated metabolites. Studies have shown that in humans UGT1A-mediated glucuronidation is an important pathway in the detoxification of food-borne carcinogenic heterocyclic amines. The biotransformation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant heterocyclic amine found in cooked meats, is highly dependent on cytochrome P4501A2 hydroxylation followed by UGT-catalyzed glucuronidation of the N-hydroxy-PhIP reactive intermediate. To determine which UGT1A proteins are involved in the glucuronidation of N-hydroxy-PhIP, microsomal preparations from baculovirus-infected insect cells that express all of the known functional human UGT1A isozymes (UGT1A1, -1A3, -1A4, -1A6, -1A7, -1A8, -1A9, and -1A10) were exposed to N-hydroxy-PhIP and the reaction products were isolated by HPLC. All UGT1A proteins except UGT1A6 showed some degree of activity toward N-hydroxy-PhIP. The formation of both N-hydroxy-PhIP-N-2-glucuronide and N-hydroxy-PhIP-N3-glucuronide was both time- and substrate concentration-dependent. UGT1A1 was the most efficient in converting N-hydroxy-PhIP to both conjugates producing five times more of the N-2-conjugate than UGT1A4, the next most active UGT, and 286 times more than UGT1A7, the least active UGT. With an apparent K-m of 52 muM and a K-cat of 114 min(-1), UGT1A1 was also the most catalytically efficient in forming N-hydroxy-PhIP-N2-glucuronide. The catalytic efficiency for N-hydroxy-PhIP-N3-glueuronide formation was 8, 10, and 6 times lower for UGT1A1, -1A4, and -1A8, respectively, when compared to the Kat values for N-hydroxy-PhIP-N2-glucuronide formation. These results clearly show that UGT1A1 has the highest specificity for glucuronidating N-hydroxy-PhIP. Polymorphic expression resulting in decreased UGT1A1 activity in humans can cause reduced rates of glucuronidation, which can change the metabolic ratio between bioactivation and detoxification to favor bioactivation. This change will increase the susceptibility to the deleterious effects from PhIP exposure because the capacity to form nontoxic N-hydroxy-PhIP glucuronide conjugates will be diminished.