Enzymatic Oxidation Of Drugs To Toxic And Carcinogenic M

多环芳烃（PAHs）如苯并[a]芘（BaP）是广泛存在的环境污染物，其中许多是哺乳动物的强致癌物。这些碳氢化合物通过细胞色素P450和环氧化物水解酶的作用进行代谢活化，生成高活性的海湾区二醇环氧化物（DEs）。DEs诱导细胞损伤的一种可能机制涉及DNA中嘌呤的外环氨基打开环氧环，随后修饰残基的错误复制导致突变。16个海湾地区的去嘌呤加合物对一个给定的母体烃是代谢可能的。含有这些加合物的双寡核苷酸表现出不同的结构基序，这些结构基序取决于碳氢化合物、目标嘌呤核苷（dA或dG）和加合物的立体化学，因此为探测dna加工酶的催化和识别位点提供了独特的工具。以前制备位点特异性加合寡核苷酸的方法受到产率低（在50微克范围内）以及无法将加合物放置在含有多个嘌呤核苷酸残基的生物相关序列中的限制。我们已经开发出强大的新合成方法，可以在3-5毫克的范围内相对容易地合成任何所需序列的内合核苷和位点特异性内合寡核苷酸，适用于核磁共振和x射线晶体学研究以及生化研究。目前正在研究的酶包括人类y家族DNA聚合酶eta和kappa以及Werner综合征解旋酶、HIV-1整合酶和HIV-1逆转录酶。另一个研究领域涉及确定人体代谢多环芳烃到致癌DEs和排出的解毒产物的个体差异，以及这些代谢差异与癌症风险的可能关系。

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are widespread environmental pollutants, many of which are potent carcinogens in mammals. These hydrocarbons are metabolically activated through the action of cytochrome P450 and epoxide hydrolase to give highly reactive bay-region diol epoxides (DEs). A plausible mechanism for the induction of cell damage by DEs involves opening of the epoxide ring by the exocyclic amino groups of the purines in DNA, followed by erroneous replication of the modified residues, leading to mutations. Sixteen bay-region DE-purine adducts are metabolically possible for a given parent hydrocarbon. Duplex oligonucleotides containing these adducts exhibit distinct structural motifs which depend on the hydrocarbon, the target purine nucleoside (dA or dG) and the stereochemistry of the adduct, and thus provide unique tools to probe the catalytic and recognition sites of DNA-processing enzymes. Previous methods for preparing site-specifically adducted oligonucleotides were limited by low yields (in the 50 microgram range) as well as the inability to place adducts in biologically relevant sequences containing multiple purine nucleotide residues. We have developed powerful new synthetic methods which make possible the relatively facile synthesis of adducted nucleosides and site-specifically adducted oligonucleotides in any desired sequence contest on 3-5 milligram scale, suitable for NMR and X-ray crystallographic studies as well as for biochemical investigations. Enzymes currently under study include the human Y-family DNA polymerases eta and kappa as well as Werner syndrome helicase, HIV-1 integrase and HIV-1 reverse transcriptase. An additional area of investigation involves determination of individual differences in the human metabolism of PAHs to carcinogenic DEs vs. excreted detoxification products and the possible relationship of these metabolic differences to cancer risk. Enzymology of PAH DE Adduct Processing: Replication of oligonucleotides containing site-specific N6-dA adducts of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE) by human DNA polymerase eta (pol eta) has been examined (1) and contrasted with our previous findings with N2-dG adducts, which consistently resulted in purine misincorporation by pol eta opposite the adduct site. When pol eta encounters dA adducts with S absolute configuration at the point of hydrcarbon attachment to the adenine base (formed by trans epoxide ring opening of the most carcinogenic BaP DE isomer), misincorporation of A and G and correct incorporation of T all occur with approximately equal frequency. In contrast, pol eta exhibits a 3-to 4-fold preference for correct T incorporation relative to A or G opposite dA adducts with R absolute configuration (formed by trans opening of the less-carcinogenic enantiomer of BaP DE). Adducts at dA formed by cis ring opening of these two BaP DE enantiomers favor A over T incorporation by approximately 2 to 3-fold, with G intermediate between the two. Primer extension one nucleotide beyond the adducts is generally weaker than nucleotide incorporation across from them. Extension of the A-(adducted A*) mispair is the most favored mispair extension, and is generally comparable in rate to extension of the correct T-(adducted A*). Since mutations can only occur if mispairs are extended, this observation is consistent with the occurrence of A-T to T-A transversions as common mutations in animal cells treated with BaP DEs. We have examined the electrophoretic mobility on non-denaturing gels of a series of template-primers containing dA adducts of BaP DEs and BaP tetrahydroepoxides (in which the 7- and 8- hydroxyl groups are replaced by hydrogen) located on the template strand at the template-primer junction. A correlation was observed between decreased electrophoretic mobility and increased efficiency of nucleotide incorporation by pol eta opposite these adducts. This suggests that distortions of the adducted DNA template-primer structure which retard migration on electrophoresis may enhance replication by pol eta. Studies are also in progress on the efficiency and fidelity of another human Y-family polymerase, pol kappa, on replication of templates containing BaP DE-dA adducts. Pol kappa is known to be relatively efficient as well as highly accurate in inserting C opposite BaP DE-dG adducts. In marked contrast, we observe that nucleotide insertion opposite BaP DE-dA adducts by pol kappa is both inefficient and error-prone. Biomarkers for PAH Metabolic Pathways: We have proposed the use of oxidative metabolites of the non-carcinogen phenanthrene, the simplest PAH with a bay region, as markers for metabolism of PAHs in humans. The goal of these studies is to assess individual differences in metabolic pathways (detoxification vs. metabolic activation) of PAHs. We previously reported development of a method for quantitation of the benzo-ring phenanthrene 1,2,3,4-tetraol in human urine. Since this tetraol is a spontaneous hydrolysis product of the bay-region phenanthrene 1,2-diol 3,4-epoxide, it provides an easily determined marker for the metabolic activation of phenanthrene, a pathway analogous to formation of the carcinogenic BaP DE metabolites. To normalize for different levels of PAH exposure in individuals, we chose four phenanthrols (excreted as glucuronides and sulfates) as markers for detoxification pathways. We have now developed a convenient, accurate and precise method for quantitation of 1-, 2-, 3-, and 4-phenanthrols in human urine (2). The method involves enzymatic hydrolysis of the sulfate and glucuronide conjugates to free phenanthrols, followed by solid phase extraction, silylation and GC-MS analysis, utilizing C-13 labeled 3-hydroxyphenanthrene as an internal standard. The ratio of tetraol to phenanthrols was found to vary by as much as 12-fold among different individuals, suggestive of significant differences in the relative magnitudes of activation and detoxification pathways for PAH metabolism among these subjects. We anticipate that this approach should prove useful in determining whether differences in PAH metabolism affect cancer risk in individuals with comparable levels of PAH exposure.