ENZYMATIC OXIDATION OF DRUGS TO TOXIC AND CARCINOGENIC METABOLITES

致癌多环芳烃的海湾地区二醇环氧化物（DE）代谢物被认为通过DNA的共价修饰启动细胞转化。在哺乳动物体内代谢形成两种非对映异构体DE-1，其中苯基羟基和环氧氧是顺式的，而DE-2中这些取代基是反式的。这些DEs在DNA中的主要靶点分别是脱氧鸟苷（dG）和脱氧腺苷（dA）的N-2和N-6氨基酸外环。我们的研究目的是阐明由特定de形成的DNA加合物的物理结构与其在纯化酶和完整细胞中的生化处理之间的关系。该实验室今年合成工作的一个亮点是改进了在dG的N-2上制备顺式和反式开放DE加合物作为其磷酰胺的方法，使用了一种新的dG的O-6的阻断/解封协议。以前，这些反式打开的dG加合磷酸酰胺需要更费力的、多步骤的路线，而且它们的顺式打开的对应物基本上是无法合成的。构建具有明确的加合物立体化学和核苷酸序列的de -加合寡核苷酸的方法的可用性，使得系统地研究其结构特征对DNA通过加合物复制引起的突变的影响成为可能。去年，我们报道了M13mp7L2噬菌体的位点特异性诱变实验结果，使用了8个16聚体寡核苷酸，这些核苷酸含有dA加合物，对应于苯并[a]芘（BaP）中每个对映体DE-1和DE-2的反式开孔。这些加合物被结合在源自supF基因的两个不同序列背景中的括号位置：Context I， 5-TTT[A]GAGTCTGCTCCC-3，和Context II， 5-CAG[A]TTTAGAGTCTGC-3。我们现在已经将这些诱变研究扩展到顺式打开的BaP DE - dA加合物，以及类似的顺式和反式打开的峡湾地区苯并[c]菲（BcPh） DE - dA加合物。在给定的序列背景下，单个BaP加合物异构体之间的总体突变性差异相对较小，并且从该碳氢化合物衍生的加合物的立体化学和突变性之间没有明确的关系。相反，每个BcPh DE异构体与dA的N-6的连接位点的构型对致突变性有显著影响，并且构成了影响这些加合物致突变性活性的主要立体化学特征。在环境1中，所有在碳氢化合物与N-6连接点上具有R构型的BcPh加合物都是非常差的诱变剂（突变频率小于1%），而在该碳原子上具有S构型的加合物在该序列中表现出10-32%的突变频率。虽然这种立体化学效应在Context II中不太明显，但值得注意的是，在这两个序列中，S结构的加合物是所有BcPh DE dA加合物中最具诱变性的（突变频率在Context I中为32%，在Context II中为57%）。拓扑异构酶是一种DNA加工酶，通过形成可逆的DNA切割复合体，使酪氨酸与3-磷酸共价结合，并在切割位点产生新的5-羟基，从而放松超旋和减轻扭转应变。某些抗癌药物，如喜树碱，通过捕获切割复合体和阻止被切割的DNA的再生，使拓扑异构酶I （top1）失活。这些药物中的许多被认为是次要的凹槽粘合剂。我们研究了BaP DE-2的(+)-和(-)-对映体的顺式和反式打开n - 2dg加合物对top1底物短双聚寡核苷酸（22聚）的影响。这些碳氢化合物加合物为研究top1抑制机制提供了独特的探针，因为与喜树碱不同，它们与DNA共价结合，并且它们在DNA中的构象可以从先前的核磁共振研究中预测：反式打开的dG加合物的芳香部分定向在小槽中，而顺式打开的dG加合物的芳香部分嵌入螺旋中。当这些BaP - DE加合物位于dG上时，该dG在破坏通常由top1切割的磷酸二酯键后具有5-羟基，抑制该键的切割并诱导序列中其他磷酸二酯键的DNA切割。反式（小槽）加合物比顺式（插层）加合物更有效地诱导了与加合物相连的磷酸二酯第三键5上新的裂解位点。在所研究的四种加合物中，由(+)-BaP DE-2衍生的反式打开dG加合物对top1的诱导裂解最活跃。据我们所知，这是第一个在分子水平上，与其他三种立体异构体BaP DE- 2dg加合物相比，最具致癌性的BaP DE- 2dg的主要加合物明显增强潜在DNA损伤的例子。

Bay-region diol epoxide (DE) metabolites of carcinogenic polycyclic aromatic hydrocarbons are believed to initiate cell transformation by covalent modification of DNA. Two diastereomeric DEs, each of which exists as a pair of enantiomers, are formed metabolically in mammals: DE-1, in which the benzylic hydroxyl group and epoxide oxygen are cis, and DE-2, in which these substituents are trans. The primary targets in DNA for these DEs are the exocyclic N-2 and N-6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively. The goal of our research is to elucidate the relationships between the physical structures of DNA adducts formed from specific DEs and their biochemical processing with purified enzymes and in intact cells. A highlight of the laboratorys synthetic work this year has been the development of improved methods for the preparation of both cis and trans opened DE adducts at N-2 of dG as their phosphoramidites, using a novel blocking/deblocking protocol for O-6 of dG. Previously, these trans opened dG adduct phosphoramidites had required a more laborious, multi-step route, and their cis opened counterparts were essentially inaccessible synthetically. Availability of methods for the construction of DE-adducted oligonucleotides of defined adduct stereochemistry and nucleotide sequence makes possible the systematic study of effects of their structural features on mutations that result from DNA replication past the adducts. Last year, we reported the results of site-specific mutagenesis experiments in M13mp7L2 bacteriophage using eight 16-mer oligonucleotides containing dA adducts corresponding to trans opening of each enantiomer of the bay-region DE-1 and DE-2 from benzo[a]pyrene (BaP). These adducts were incorporated at the bracketed positions in two different sequence contexts derived from the supF gene: Context I, 5-TTT[A]GAGTCTGCTCCC-3, and Context II, 5-CAG[A]TTTAGAGTCTGC-3. We have now extended these mutagenesis studies to the cis opened BaP DE dA adducts, as well as the analogous cis and trans opened fjord-region benzo[c]phenanthrene (BcPh) DE dA adducts in the same two sequences. In a given sequence context, differences in overall mutagenicity between individual BaP adduct isomers were relatively small, and there was no clear relationship between stereochemistry and mutagenicity for adducts derived from this hydrocarbon. In contrast, the configuration at the site of attachment of each BcPh DE isomer to N-6 of dA had marked effects on mutagenicity, and constituted the dominant stereochemical feature influencing the mutagenic activity of these adducts. All the BcPh adducts with R configuration at the point of attachment of the hydrocarbon to N-6 were very poor mutagens in Context I (mutation frequencies less than 1%), whereas the adducts with S configuration at this carbon atom exhibited mutation frequencies from 10-32% in this sequence. Although this stereochemical effect was less pronounced in Context II, it is notable that an adduct with the S configuration was the most mutagenic of all the BcPh DE dA adducts in both sequences (mutation frequency 32% in Context I and 57% in Context II). Topoisomerases are DNA processing enzymes that relax supercoiling and relieve torsional strain by forming reversible DNA cleavage complexes in which a tyrosine in the enzyme becomes covalently bonded to a 3-phosphate group, and a new 5-hydroxyl group is generated at the cleavage site. Certain anticancer drugs such as the camptothecins inactivate topoisomerase I (top1) by trapping the cleavage complex and preventing religation of the cleaved DNA. Many of these drugs are thought to be minor groove binders. We have investigated the effects of cis and trans opened N-2 dG adducts of the (+)- and (-)-enantiomers of BaP DE-2 in a short duplex oligonucleotide (22-mer) that is a substrate for top1. These hydrocarbon adducts provide unique probes for investigating the mechanism of top1 inhibition, since, unlike the camptothecins, they are covalently bound to the DNA, and their conformations in DNA are predictable from prior NMR studies: the trans opened dG adducts have the aromatic moiety oriented in the minor groove, whereas the cis opened dG adducts have the aromatic portion intercalated into the helix. These BaP DE adducts, when located on the dG that would have a 5-hydroxyl group after breaking the phosphodiester bond normally cleaved by top1, inhibit cleavage of this bond and induce DNA cleavage at other phosphodiester bonds in the sequence. The trans (minor groove) adducts were more effective than the cis (intercalated) adducts as inducers of a new cleavage site at the third phosphodiester bond 5 to the adduct. Of the four adducts studied, the trans opened dG adduct derived from (+)-BaP DE-2 was the most active in inducing cleavage by top1. To our knowledge, this constitutes the first example at the molecular level of a clear enhancement of potential DNA damage by this major adduct of the most carcinogenic BaP DE isomer as compared with the other three, stereoisomeric BaP DE-2 dG adducts.