Jin L, Baillie T A
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle 98195, USA.
Chem Res Toxicol. 1997 Mar;10(3):318-27. doi: 10.1021/tx9601768.
The chemoprotective effects of diallyl sulfide (DAS), a flavor component of garlic, have been attributed to its inhibitory effects on CYP2E1-mediated bioactivation of certain carcinogenic chemicals. In addition to being a competitive inhibitor of CYP2E1 in vitro, DAS is known to cause irreversible inhibition of CYP2E1 in rats in vivo. The latter property is believed to be mediated by the DAS metabolite diallyl sulfone (DASO2), which is thought to be a mechanism-based inhibitor of CYP2E1, although the underlying mechanism remains unknown. In order to investigate the nature of the reactive intermediate(s) responsible for the inactivation of CYP2E1 by DAS and its immediate metabolites, the present studies were carried out to detect and identify potential glutathione (GSH) conjugates of DAS and its metabolites diallyl sulfoxide (DASO) and DASO2. By means of ionspray LC-MS/MS, ten GSH conjugates were identified in bile collected from rats dosed with DAS, namely: S-[3-(S'-allyl-S'-dioxomercapto)-2-hydroxypropyl]glutathione (M1, M2; diastereomers), S-[3-(S'-allyl-S'-dioxomercapto)-2-hydroxypropyl]-glutathione (M5), S-[2-(S'-allyl-S'-dioxomercapto)-1-(hydroxymethyl)ethyl]glutathion e (M3, M4; diastereomers), S-[3-(S'-allylmercapto)-2-hydroxypropyl]glutathione (M6), S-(3-hydroxypropyl)-glutathione (M7), S-(2-carboxyethyl)glutathione (M8), allyl glutathionyl disulfide (M9), and S-allylglutathione (M10). With the exception of M6, all of the above GSH conjugates were detected in the bile of rats treated with DASO, while only M3, M4, M5, M7, M8, and M10 were found in the bile of rats treated with DASO2. Experiments conducted in vitro showed that GSH reacted spontaneously with DASO to form conjugates M9 and M10, and with DASO2 to form M10. In the presence of NADPH and GSH, incubation of DAS with cDNA-expressed rat CYP2E1 resulted in the formation of metabolites M6, M9, and M10, while incubation with DASO led to the formation of M3, M4, M5, M9, and M10. When DASO2 acted as substrate, CYP2E1 generated only conjugates M3, M4, M5, and M10. These results indicate that while DAS and DASO undergo extensive oxidation in vivo at the sulfur atom, the allylic carbon, and the terminal double bonds, CYP2E1 preferentially catalyzes oxidation of the sulfur atom to form the sulfoxide and the sulfone (DASO and DASO2). However, it appears that the end product of this sequence, namely, DASO2, undergoes further CYP2E1-mediated activation of the olefinic pi-bond, a reaction which transforms many terminal olefins to potent mechanism-based P450 inhibitors. We hypothesize, therefore, that it is this final metabolic event with DASO2 which leads to autocatalytic destruction of CYP2E1 and which is mainly responsible for the chemoprotective effects of DAS in vivo.
二烯丙基硫醚（DAS）是大蒜的一种风味成分，其化学保护作用归因于它对细胞色素P450 2E1（CYP2E1）介导的某些致癌化学物质生物活化的抑制作用。除了在体外是CYP2E1的竞争性抑制剂外，已知DAS在大鼠体内会导致CYP2E1的不可逆抑制。据信后一种特性是由DAS代谢物二烯丙基亚砜（DASO）介导的，DASO被认为是一种基于机制的CYP2E1抑制剂，尽管其潜在机制尚不清楚。为了研究导致CYP2E1被DAS及其直接代谢物灭活的反应中间体的性质，开展了本研究以检测和鉴定DAS及其代谢物二烯丙基亚砜（DASO）和二烯丙基砜（DASO2）的潜在谷胱甘肽（GSH）缀合物。通过离子喷雾液相色谱-串联质谱法，在给DAS的大鼠胆汁中鉴定出10种GSH缀合物，即：S-[3-(S'-烯丙基-S'-二氧代巯基)-2-羟丙基]谷胱甘肽（M1、M2；非对映异构体）、S-[3-(S'-烯丙基-S'-二氧代巯基)-2-羟丙基]谷胱甘肽（M5）、S-[2-(S'-烯丙基-S'-二氧代巯基)-1-(羟甲基)乙基]谷胱甘肽（M3、M4；非对映异构体）、S-[3-(S'-烯丙基巯基)-2-羟丙基]谷胱甘肽（M6）、S-(3-羟丙基)谷胱甘肽（M7）、S-(2-羧乙基)谷胱甘肽（M8）、烯丙基谷胱甘肽二硫化物（M9）和S-烯丙基谷胱甘肽（M10）。除M6外，上述所有GSH缀合物在给DASO的大鼠胆汁中均被检测到，而在给DASO2的大鼠胆汁中仅发现M3、M4、M5、M7、M8和M10。体外实验表明，GSH与DASO自发反应形成缀合物M9和M10，与DASO2反应形成M10。在存在烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和GSH的情况下，DAS与cDNA表达的大鼠CYP2E1孵育导致代谢物M6、M9和M10的形成，而与DASO孵育导致M3、M4、M5、M9和M10的形成。当DASO2作为底物时，CYP2E1仅产生缀合物M3、M4、M5和M10。这些结果表明，虽然DAS和DASO在体内硫原子、烯丙基碳和末端双键处发生广泛氧化，但CYP2E1优先催化硫原子氧化形成亚砜和砜（DASO和DASO2）。然而，似乎该序列的终产物，即DASO2，会经历CYP2E1介导的烯烃π键进一步活化，该反应将许多末端烯烃转化为有效的基于机制的细胞色素P450抑制剂。因此我们推测，正是DASO2的这一最终代谢事件导致CYP2E1的自催化破坏，并且这主要是DAS在体内化学保护作用的原因。
