BENZO A PYRENE MUTAGENIC MECHANISMS

苯并芘诱变机制

• 批准号: 6055891
• 负责人: EDWARD L LOECHLER
• 金额: $ 26.98万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-06-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6055891
• 关键词: DNA damage; Escherichia coli; adduct; benzopyrenediol epoxide; chemical carcinogen; conformation; embryo /fetus tissue /cell culture; human tissue; mutagen testing; mutagens; nucleic acid sequence; plasmids; point mutation; site directed mutagenesis; transfection /expression vector

Benzo[a]pyrene is a potent mutagen/carcinogen that is metabolically activated inside cells, including to its (+)-anti-7,8-diol-9,10-epoxide [(+)-anti-B[a]PDE], which gives DNA adducts, principally at N2-Gua. We showed that (+)-anti-B[a]PDE induces base substitution, frameshifts, insertion and deletions (supF gene of plasmid pUB3 in E. coli). The question we are addressing is: how is (+)-anti-B[a]PDE able to induce such a diverse array of mutations? Our working hypothesis is that adduct mutational complexity is due to adduct conformational complexity, and that the major adduct [(+)-trans-anti-B[a]P-N2-Gua] is able to induce the majority of these mutations. Adduct conformation--and, thereby, mutation-- may be controlled by various factors, notably, DNA sequence context. (1) We showed that little of mutagenesis can be attributed to AP sites formation from (+)-anti-B[a]PDE adducts. We showed that trans-(+)-anti- B[a]P-N2-Gua can be unstable in ds (but not ss) DNA and gives its corresponding tetraols. Although this unexpected reaction is interesting, it is unlikely to be at the root of any important mutational observations. (2) Using adduct site-specific methods, we showed previously that (+)- trans-anti-B[a]P-N2-Gua induced G->T mutations in a 5'-TG-3' sequence context, which correlated with our studies showing that (+)-anti-B[a]PDE itself induced principally G->T mutations in 5'-TG-3' sequence contexts suggesting a mechanistic link. 3. In supF (+)-anti-B[a]PDE induced mostly G->A in one sequence context (5'-CGT-3'). In preliminary experiments we show that (+)-trans-anti- B[a]P-N2-Gua also induces G->A mutations in this sequence context. 4. In supF we identified a sequence (5'-CGG-3') where the mutagenic specificity seemed to be changeable, being G->T in some cases and G->T, A & C in others. If our hypothesis is correct, then this might be a conformationally sensitive site. We now show that trans-(+)-anti-B[a]P- N2-Gua indeed can change its pattern of mutations in this sequence context; for example, the mutational pattern following PEG treatment (predominantly G->T) is different than following ethanol precipitation (a mixture of G->T, A & C mutations). Herein we propose to gather further evidence that adduct conformational complexity is at the root of adduct mutational complexity. We also seek to establish sequence contexts where (+)-trans-anti-B[a]P-N2-Gua can induce each individual mutation (e.g., G->T only, G->A only, and G->C only), as a prelude to doing structural studies to determine what conformation is responsible for what mutation and why. The initiation of parallel studies with shuttle vectors in human cells is also proposed.

苯并[a]芘是一种强诱变剂/致癌物， 在细胞内活化，包括其（+）-抗-7，8-二醇-9，10-环氧化物 [（+）-抗-B [a]PDE]，主要在N2-Gua处产生DNA加合物。 我们 显示（+）-抗B [a]PDE诱导碱基取代，移码， 插入和缺失（质粒pUB 3的supF基因在E.大肠杆菌）。 的 我们要解决的问题是：（+）-抗B [a]PDE如何能够诱导 如此多样化的突变 我们的假设是加合物 突变复杂性是由于加合物构象复杂性， 主要加合物[（+）-trans-anti-B[a]P-N2-Gua]能够诱导 这些突变的大部分。 加合物构象，因此 突变--可能受多种因素控制，特别是DNA序列 上下文 (1)我们发现很少的突变可以归因于AP位点 由（+）-抗-B [a]PDE加合物形成。 我们发现反式-（+）-抗- B[a]P-N2-Gua在ds（而不是ss）DNA中可能是不稳定的，并给出其 相应的四醇。 虽然这种意外的反应是 有趣的是，它不太可能是任何重要突变的根源。 意见。 (2)使用加合物位点特异性方法，我们先前表明（+）- 反式抗B[a]P-N2-Gua诱导5 '-TG-3'序列中的G->T突变 背景，这与我们的研究相关，表明（+）-抗B [a]PDE 其本身在5 '-TG-3'序列背景下主要诱导G->T突变 暗示了一种机械联系 3.在supF（+）-抗B [a]PDE中，在一个序列背景下主要诱导G->A （5 '-CGT-3'）。 在初步的实验中，我们表明，（+）-反式抗- B[a]P-N2-Gua也在该序列背景下诱导G->A突变。 4.在supF中，我们鉴定了一个序列（5 '-CGG-3'），其中诱变剂 特异性似乎是可变的，在某些情况下为G->T和G->T， A & C在其他 如果我们的假设是正确的， 构象敏感位点 我们现在表明，反式-（+）-抗B [a]P- N2-Gua确实可以改变其在该序列中的突变模式 背景;例如，PEG处理后的突变模式 （主要是G->T）与乙醇沉淀后不同 （G->T、A和C突变的混合物）。 在此，我们建议收集进一步的证据，加合物构象 复杂性是加合突变复杂性的根源。 我们还寻求 以建立序列背景，其中（+）-反式-抗B[a]P-N2-Gua可以 诱导每个单独的突变（例如，仅G->T，仅G->A和G->C 仅），作为进行结构研究以确定 构象决定了什么样的突变和为什么突变。 启动 还提出了在人类细胞中使用穿梭载体的平行研究。