Bogaards J J, Venekamp J C, Salmon F G, van Bladeren P J
Toxicology Division, TNO Nutrition and Food Research Institute, Zeist, The Netherlands.
Chem Biol Interact. 1999 Jan 1;117(1):1-14. doi: 10.1016/s0009-2797(98)00094-5.
In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.
在本研究中，使用大鼠和人类α、μ、π和θ类纯化的谷胱甘肽S-转移酶（GSTs），研究了异戊二烯单环氧化物3,4-环氧-3-甲基-1-丁烯（EPOX-I）和3,4-环氧-2-甲基-1-丁烯（EPOX-II）与谷胱甘肽的酶促结合。EPOX-I和EPOX-II与[35S]谷胱甘肽（GSH）孵育的HPLC分析表明，每种异戊二烯单环氧化物均形成两个放射性组分。通过1H-NMR分析阐明了EPOX-I和EPOX-II谷胱甘肽共轭物的结构。正如预期的那样，两种异戊二烯环氧化物均发现了两个结合位点。EPOX-II的结合效率高于EPOX-I。此外，对于大鼠和人类，μ和θ类谷胱甘肽S-转移酶比α和π类谷胱甘肽S-转移酶效率更高。由于μ和θ类谷胱甘肽S-转移酶分别在约50%和40%-90%的人群中表达，这可能对缺乏这些酶的个体中异戊二烯单环氧化物的解毒产生重大影响。大鼠谷胱甘肽S-转移酶比人类谷胱甘肽S-转移酶更有效：大鼠GST T1-1在EPOX-I和EPOX-II的结合方面比人类GST T1-1表现出约2.1-6.5倍的更高活性，而大鼠GST M1-1和GST M2-2比人类GST M1a-1a表现出约5.2-14倍的更高活性。大多数谷胱甘肽S-转移酶在所使用的浓度范围（50-2000 microM）内呈现一级动力学。除了GST类之间的活性差异外，还发现了结合位点之间的差异。除大鼠GST M1-1在C3位也有显著结合外，所有谷胱甘肽S-转移酶几乎都将EPOX-I与谷胱甘肽在C4位结合。EPOX-II的结合未观察到这种选择性。用EPOX-I和EPOX-II与小鼠、大鼠和人类的肝脏S9组分孵育，结果显示小鼠和大鼠的GSH结合速率相似。与小鼠和大鼠相比，人类肝脏S9的GSH结合速率低25-50倍。
