Smith W A, Gupta R C
Graduate Center for Toxicology and Preventive Medicine, University of Kentucky Medical Center, Lexington, KY 40536-0305, USA.
Carcinogenesis. 1996 Jun;17(6):1285-90. doi: 10.1093/carcin/17.6.1285.
There is a growing need for short-term assays which can assess the mechanisms and efficacy of cancer chemopreventive agents. In the present study we have employed a microsome-mediated test system concomitantly with DNA adduct detection to assess the efficacy of five chemopreventive agents, N-acetylcysteine, butylated hydroxytoluene (BHT), curcumin, oltipraz, and ellagic acid. 32P-Postlabeling analysis of DNA incubated with benzo[a]pyrene (BP) in the presence of Aroclor 1254-induced microsomes produced two major adducts: one derived from the interaction of benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) with deoxyguanosine (dG) and the other from further activation of 9-OH-BP (309 and 34 adducts/10(7) nucleotides, respectively). With the exception of N-acetylcysteine, all test agents significantly altered BP-DNA adduct levels: Intervention with ellagic acid and oltipraz substantially (64-94%) inhibited both BPDE-dG and 9-OH-BP adducts, while intervention with curcumin and BHT inhibited the BPDE-dG adduct (57% and 38%, respectively) and enhanced the 9-OH-BP adduct (230% and 650%, respectively). Furthermore, ellagic acid was the only test agent observed to inhibit the anti BPDE-dG adduct in the absence of microsomal enzymes, which is consistent with the known conjugation of ellagic acid with BPDE. These results suggest that oltipraz may be acting as an inhibitor of P4501A1, the isozyme involved in activation of BP to BPDE, or by conjugation of the electrophilic species by a metabolite of oltipraz. A plausible mechanism for inhibition of the BPDE-dG adduct and enhancement of the 9-OH-BP adduct by curcumin and BHT includes inhibition of epoxide hydrolase. Our results also indicate that N-acetylcysteine does not act as an electrophilic trapping agent of BP metabolites but may exert its protective effect in vivo by various other means, including modulation of detoxification enzymes and altering DNA repair processes. These data suggest that this cell-free system in conjunction with the sensitive 32P-postlabeling DNA adduct analysis may prove a viable test system for assessing the mechanisms and efficacy of chemopreventive agents.
对于能够评估癌症化学预防剂作用机制和功效的短期检测方法的需求日益增长。在本研究中，我们采用了微粒体介导的检测系统并结合DNA加合物检测，以评估五种化学预防剂——N-乙酰半胱氨酸、丁基羟基甲苯（BHT）、姜黄素、奥替普拉和鞣花酸的功效。在存在Aroclor 1254诱导的微粒体的情况下，用苯并[a]芘（BP）孵育的DNA进行32P后标记分析产生了两种主要加合物：一种源自苯并[a]芘-7,8-二醇-9,10-环氧化物（BPDE）与脱氧鸟苷（dG）的相互作用，另一种源自9-羟基-BP的进一步活化（分别为309和34个加合物/10(7)个核苷酸）。除N-乙酰半胱氨酸外，所有测试剂均显著改变了BP-DNA加合物水平：鞣花酸和奥替普拉的干预大幅（64 - 94%）抑制了BPDE-dG和9-羟基-BP加合物，而姜黄素和BHT的干预抑制了BPDE-dG加合物（分别为57%和38%）并增强了9-羟基-BP加合物（分别为230%和650%）。此外，鞣花酸是唯一在不存在微粒体酶的情况下观察到能抑制抗BPDE-dG加合物的测试剂，这与鞣花酸与BPDE的已知共轭作用一致。这些结果表明，奥替普拉可能作为P4501A1的抑制剂起作用，P4501A1是参与将BP活化为BPDE的同工酶，或者是通过奥替普拉的一种代谢产物与亲电物质共轭起作用。姜黄素和BHT抑制BPDE-dG加合物并增强9-羟基-BP加合物的一种合理机制包括抑制环氧水解酶。我们的结果还表明，N-乙酰半胱氨酸不是BP代谢产物的亲电捕获剂，但可能通过各种其他方式在体内发挥其保护作用，包括调节解毒酶和改变DNA修复过程。这些数据表明，这种无细胞系统结合灵敏的3P后标记DNA加合物分析可能是一种可行的检测系统，用于评估化学预防剂的作用机制和功效。
