Enzymatic Oxidation Of Drugs To Toxic And Carcinogenic M

苯并[a]芘（BaP）和苯并[c]菲（BcPh）等致癌多环芳烃的苯环上的二醇环氧化物（DE）代谢物被认为通过共价修饰DNA而启动细胞转化。两种非对映体DE，每一种都以一对对映体的形式存在，在哺乳动物中通过代谢形成：DE-1，其中苄基羟基和环氧氧是顺式的，DE-2，其中这些取代基是反式的。这些DE在DNA中的主要靶点分别是脱氧鸟苷（dG）和脱氧腺苷（dA）的环外N-2和N-6氨基。我们正在使用特定的DE-DNA加合物作为工具，用于探测涉及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。我们的目标是阐明这些DNA加合物的物理结构和它们在完整细胞和纯化的酶中的生化加工之间的关系。过去一年的研究集中在（1）SOS诱导的大肠杆菌在M13 mp 7 L2构建体中复制这些加合物的突变后果，以及（2）单个核酸外切酶在遇到烃加合物时的反应方式，这些核酸外切酶从其5 '或3'末端水解单链寡核苷酸中的磷酸二酯键。 (1)在以前的报告中，我们描述了在E. Coli-M13系统中的8种可能的加合物，它们都是由4种光学活性的BaP 7，8-二醇9，10-环氧化物通过特定脱氧腺苷（dA）残基的N-6氨基顺式和反式开环而得到的。通过dA打开DE形成的BaP DE加合物在四氢苯并环上的C-7、-8和-9处具有三个羟基，并且在第四（C-10）位具有嘌呤N-6。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加合物研究的序列合成了16聚体寡核苷酸，但含有其中两个或所有三个羟基被氢取代的dA加合物。将这些16聚体掺入到M13 DNA中，并使其在SOS诱导的E.杆菌一般而言，减少加合物羟基的数目降低了取代突变的总频率。对于所有，但本加合物之一，总的突变频率低于任何先前报道的DE加合物在相同的序列。例外的是由dA的N-6氨基在C-10顺式打开9，10-环氧基-7，8，9，10-四氢BaP衍生的加合物的10 R非对映异构体，其在该DNA序列中迄今为止研究的所有BaP dA加合物（包括DE加合物）中产生最高的突变频率。一般来说，衍生自上述四氢环氧化物以及二醇环氧化物的环氧化物环的顺式打开的加合物比相应的反式加合物更具致突变性。与本套BaP dA加合物与一个或没有羟基的四氢苯并环，A到T的颠换占主导地位，与较少数量的A到G的转换，甚至更少的A到C的颠换。 (2)蛇毒磷酸二酯酶（VPD）和牛脾磷酸二酯酶（SPD）是以相反方向（分别从3 '-至5'-和5 '-至3'-末端）切割单链寡核苷酸的外切核酸酶。我们研究了水解的容易性由VPD和SPD的短脱氧寡核苷酸含有定义的BaP或BcPh DE加合物在N-6的DA。根据以前的报告，加合物与R构型在连接的DA的烃部分的网站一般更耐水解由VPD比它们的S-非对映异构体。此外，衍生自环氧化物环的顺式打开的加合物比相应的反式打开的加合物对VPD的抗性显著更高。虽然以前的几项研究表明，寡核苷酸含有加合物与S构型的连接dA的烃的网站是更耐切割SPD比他们的R-非对映异构体，我们目前的结果与更广泛的一组寡核苷酸表明，SPD，与VPD相反，表现出很少的歧视加合物与R和S构型。在VPD的情况下，含有R-或S-加合物非对映异构体的寡核苷酸的核酸外切水解最初产生在其3 '端含有加合的dA残基的片段。然后，酶“跳过”下一个抗性键，得到二聚体（pNpA*），其具有与加合的dA* 的完整5 '-磷酸二酯键。与几个加合物，这种二聚体最终经历较慢的裂解释放单体，p（dA*）。在SPD的情况下，酶停止以在加合物的5'端产生含有一个未修饰碱基的片段（NpA*pNpNpN.....）。尽管该中间体最终可以缓慢水解至核苷酸水平，但在SPD中未观察到抗性键“跳跃”以产生二聚体。一个惊人的观察是，对于这两种酶，最具抗性的核苷酸间键是在修饰的碱基和紧邻其5'的碱基之间，而不管加合物的构型如何。这表明，R-和S-dA加合物都被VPD和SPD“视为"定性地阻断进入修饰碱基的同一侧。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，其中抗性核苷酸间键联取决于加合物构型，并且直接位于S-dG加合物的5'端，并直接位于R-dG加合物的3'端。

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.