Enzymatic Oxidation Of Drugs To Toxic And Carcinogenic M

致癌多环芳烃（如苯并[a]芘（BaP）和苯并[c]菲（BcPh））的苯环上的二醇环氧化物（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。我们正在使用特定的DE-DNA加合物作为工具来探索涉及DNA加工的基本细胞功能所需的酶的结构生物学和机制酶学。目的是阐明这些DNA加合物的物理结构及其在完整细胞和纯化酶中的生化处理之间的关系。过去一年的研究主要集中在(1)由sos诱导的大肠杆菌在M13mp7L2构建体中复制这些加合物的突变后果，以及(2)单个外切酶在遇到碳氢化合物加合物时的反应方式，这些外切酶从单链寡核苷酸的5‘或3’端水解磷酸二酯键。(1)在之前的报道中，我们描述了四种具有光学活性的BaP 7,8-二醇9,10-环氧化物的顺式和反式开环所衍生的八种可能的加合物中的每一种在大肠杆菌- m13体系中由特定脱氧腺苷（dA）残基的N-6氨基引起的突变。由dA打开DE形成的BaP DE加合物在四氢苯甲酸环的C-7、-8和-9上有三个羟基，嘌呤N-6在第四个（C-10）位置。观察到的突变频率部分取决于三个羟基的相对空间取向。为了确定这些羟基的存在或不存在如何影响突变反应，用先前用BaP DE-dA加合物研究过的序列合成了16聚寡核苷酸，但含有dA加合物的两个或三个羟基被一个氢取代。这些16-mers被整合到M13 DNA中，并允许在sos诱导的大肠杆菌中复制。总的来说，减少加合羟基的数量会降低取代突变的总频率。除了一种加合物外，所有加合物的总突变频率都低于先前报道的相同序列的DE加合物。唯一的例外是由dA的N-6氨基在C-10上顺式打开9,10-环氧-7,8,9,10-四氢BaP的10R非对映体，这是迄今为止在该DNA序列中研究的所有BaP dA加合物中突变频率最高的，包括DE加合物。总的来说，上述四氢环氧化物和二醇环氧化物的环氧环顺式开孔衍生的加合物比相应的反式加合物更具有诱变性。在四氢苯甲酸环上有一个或没有羟基的BaP - dA加合物中，A到T的转变占主导地位，A到G的转变较少，A到C的转变更少。(2)蛇毒磷酸二酯酶（VPD）和牛脾磷酸二酯酶（SPD）是沿相反方向（分别从3′-端到5′-端和5′-端到3′-端）切割单链寡核苷酸的外切酶。我们研究了VPD和SPD对在dA的N-6上含有确定的BaP或BcPh DE加合物的短脱氧寡核苷酸的水解难易程度。根据先前的报道，在dA与碳氢化合物部分的连接部位具有R构型的加合物通常比它们的s -非对映体更耐VPD水解。此外，由环氧化物环顺式打开的加合物比相应的反式打开的加合物更能抵抗VPD。虽然之前的一些研究表明，在dA与碳氢化合物的连接部位含有S构型加合物的寡核苷酸比它们的R-非对映体更能抵抗SPD的裂解，但我们目前对更广泛的寡核苷酸的研究结果表明，与VPD相比，SPD在R和S构型加合物之间几乎没有区别。在VPD的情况下，含有R-或s -加合物非对映体的寡核苷酸的外核水解最初产生一个片段，在其3'端含有加合的dA残基。然后，酶“跳过”下一个耐药键，将具有完整5'-磷酸二酯键的二聚体（pNpA*）与内合的dA*结合。在几种加合物的作用下，二聚体最终发生较慢的裂解，释放单体p（dA*）。在SPD的情况下，酶将含有一个未修饰的碱基5'的片段提供给加合物（NpA*pNpNpN.....）。虽然这种中间体最终可以缓慢地水解到核苷酸水平，但SPD没有观察到抗性键的“跳过”以产生二聚体。一个引人注目的观察结果是，对于这两种酶来说，最具抗性的核苷酸间连锁是在修饰的碱基和与它紧邻5'的碱基之间，而不管加合物的结构如何。这表明R-和S-dA加合物都被VPD和SPD“看到”定性地阻断了进入修饰碱基同一侧的途径。BaP和BcPh DE-dA加合物的这些结果与其他研究人员对BaP DE-dG加合物的观察结果形成对比，其中耐药的核苷酸间连锁依赖于加合物的构型，并且立即是5‘到S-dG加合物，立即是3’到R-dG加合物。

Diol epoxide (DE) metabolites on benzo-rings of carcinogenic polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) and benzo[c]phenanthrene (BcPh) 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. We are using specific DE-DNA adducts as tools for probing the structural biology and mechanistic enzymology of enzymes required for essential cellular functions involving DNA processing. The goal is to elucidate the relationships between the physical structures of these DNA adducts and their biochemical processing in intact cells and with purified enzymes. Studies in the past year have focused on (1) the mutational consequences of replication of these adducts in M13mp7L2 constructs by SOS-induced Escherichia coli, and (2) the ways in which individual exonucleases, which hydrolyze the phosphodiester bonds in single-stranded oligonucleotides from either their 5'- or the 3'-ends, respond upon encountering hydrocarbon adducts. (1) In previous Reports we described the mutations induced in an E. Coli-M13 system by each of the eight possible adducts derived from cis and trans ring opening of the four optically active BaP 7,8-diol 9,10-epoxides by the N-6 amino group of a specific deoxyadenosine (dA) residue. BaP DE adducts formed by opening of the DEs by dA have three hydroxyl groups at C-7, -8, and -9 on the tetrahydrobenzo-ring and the purine N-6 at the fourth (C-10) position. Observed mutational frequencies depended in part on the relative spatial orientations of the three hydroxyl groups. To determine how the presence or absence of these hydroxyl groups affects mutational response, 16-mer oligonucleotides were synthesized with a sequence previously studied with the BaP DE-dA adducts, but containing dA adducts in which two or all three of the hydroxyl groups were replaced by a hydrogen. These 16-mers were incorporated into the M13 DNA and allowed to replicate in SOS-induced E. coli. In general, decreasing the number of adduct hydroxyl groups decreased the total frequency of substitution mutations. For all but one of the present adducts, the total mutational frequency was lower than that for any of the previously reported DE adducts in the same sequence. The exception was the 10R diastereomer of the adduct derived from cis opening of 9,10-epoxy-7,8,9,10-tetrahydro BaP at C-10 by the N-6 amino group of dA, which gave the highest mutational frequency of all the BaP dA adducts studied to date in this DNA sequence, including the DE adducts. In general, adducts derived from cis opening of the epoxide ring of the above tetrahydroepoxide as well as of the diol epoxides were more mutagenic than the corresponding trans adducts. With the present set of BaP dA adducts with one or no hydroxyl groups on the tetrahydrobenzo-ring, A to T transversions predominated, with smaller numbers of A to G transitions and even fewer A to C transversions. (2) Snake venom phosphodiesterase (VPD) and bovine spleen phosphodiesterase (SPD) are exonucleases that cleave single-stranded oligonucleotides in opposite directions (from the 3'- to 5'- and the 5'- to 3'-ends, respectively). We investigated the ease of hydrolysis by VPD and SPD of short deoxyoligonucleotides containing defined BaP or BcPh DE adducts at N-6 of dA. In accordance with earlier reports, adducts with R configuration at the site of attachment of dA to the hydrocarbon moiety are generally more resistant to hydrolysis by VPD than their S-diastereomers. Furthermore, adducts derived from cis opening of the epoxide ring are considerably more resistant to VPD than the corresponding trans-opened adducts. Although several previous investigations had suggested that oligonucleotides containing adducts with S configuration at the site of attachment of dA to the hydrocarbon are more resistant to cleavage by SPD than are their R-diastereomers, our present results with a more extensive set of oligonucleotides indicate that SPD, in contrast to VPD, exhibits little discrimination between adducts with R and S configuration. In the case of VPD, the exonucleolytic hydrolysis of oligonucleotides containing either R- or S-adduct diastereomers initially yields a fragment containing the adducted dA residue at its 3'-end. The enzyme then "skips" over the next, resistant bond to give a dimer (pNpA*) with an intact 5'-phosphodiester bond to the adducted dA*. With several of the adducts, this dimer eventually undergoes slower cleavage to release the monomer, p(dA*). In the case of SPD, the enzyme stalls to give a fragment containing one unmodified base 5' to the adduct (NpA*pNpNpN.....). Although this intermediate can eventually be slowly hydrolyzed to the nucleotide level, "skipping" of the resistant bond to give a dimer is not observed with SPD. A striking observation is that for both enzymes, the most resistant internucleotide linkage is between the modified base and the base immediately 5' to it, regardless of the configuration of the adduct. This suggests that both R- and S-dA adducts are "seen" by VPD and SPD as qualitatively blocking access to the same side of the modified base. These results with BaP and BcPh DE-dA adducts contrast with observations by other investigators with BaP DE-dG adducts, in which the resistant internucleotide linkage is dependent on adduct configuration, and is immediately 5' to S-dG adducts and immediately 3' to R-dG adducts.