PHOTOSENSITIZED REDUCTION AND DNA ALKYLATION OF ALKYLATING QUINONES AND NITROAR

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。光动力疗法（PDT）是一种利用红色激光、光敏剂和分子氧的组合来达到治疗效果的癌症治疗方法。PDT在治疗中空器官癌（如食管癌）方面尤其有前景。这是因为激光现在可以通过薄的柔性光纤和内窥镜以非常精确的方式传输到身体的几乎任何部位，并且对覆盖的健康组织的损害最小。卟啉（porphyrin， POR）、酞菁（phthalocyanines， PC）、氯啉（chlorins， CHL）等因其在500 ~ 800 nm范围内具有较大的吸收系数而被广泛用于肿瘤的光动力治疗（PDT）。在空气存在的情况下，它们将光敏化产生单线态氧和超氧化物。单线态氧气产生，即所谓的II型途径，被认为是杀死肿瘤细胞最重要的过程。然而，I型途径，即涉及底物的光还原或光氧化的途径。也被认为是PDT中的光细胞毒性事件，特别是在缺氧环境中。此外，在许多情况下，PDT在细胞中产生凋亡和坏死。实体瘤通常是缺氧的。因此，如果光敏剂存在于这些肿瘤中，单线态氧就不是最终杀死这些肿瘤细胞的活性物质。由于这些染料能够光还原氧，那么，在缺氧/缺氧细胞中，这些染料也应该具有与氧几乎相等或更高的正氧化还原电位的光还原分子。如果这个底物是DNA烷基化醌或硝基arene，通过还原激活，它可以作为烷基化物质，然后DNA烷基化应该是预期的，随之而来的细胞死亡。这种激活应该发生在DNA位点附近，以避免其他不太重要的大分子的烷基化而浪费醌或硝基芳烃。硝基芳烃被还原为亚硝基芳烃（2个电子），对硫醇具有高度反应性，或进一步还原为羟胺（4个电子），对鸟嘌呤具有反应性。相反，作为烷基化剂，偶氮基醌需要激活1或2个电子。在这方面，由于醌类化合物的烷基化活性所需的电子更少，因此氮基苯基醌类化合物更容易被光活化成dna烷基化物质。光敏剂在厌氧和缺氧条件下被硝基咪唑光氧化或其三重态淬灭。这已被证明使用闪光光解方法，甚至对硝基咪唑具有比氧更负的氧化还原电位。例如，使用血卟啉和尿卟啉作为光敏剂，在甲硝唑存在下观察到，E = -485 mV，而氧的氧化还原电位为-330 mV。然而，据我们所知，染料光还原硝基芳烃或醌的直接检测和表征尚未发生。据我们所知，以前涉及光敏剂和硝基芳烃的反应(a)没有处理缺氧条件下的细胞，(b)没有考虑硝基芳烃氧化还原电位在光还原或细胞毒性的产率或量子产率中的重要性，(c)没有在他们的研究中使用烷基化醌代替硝基芳烃。(d)未考虑在非均相介质中还原醌/硝基芳烃的产率或量子产率与水相介质的产率；(e)未探索DNA结合（或游离）敏化剂与烷基化醌-硝基芳烃在产生DNA加合物中的结合。在这项工作中，吡啶锌酞菁（PPC）和5,10,15,20-四（1-甲基-4-吡啶）卟啉四（对甲苯磺酸）（TMPyP）作为阳离子，应该结合DNA磷酸盐，将始终包括在以下特定目标的发展中。其他光敏剂（亲水或亲脂和带负电荷）将包括在内以供比较。只要有可能，就会确定pH值的作用。虽然将使用常氧条件进行比较，但也将特别强调缺氧或缺氧条件。因此，下列具体目标旨在填补上述的一些空白：测定POR、PC、CHL、醌类和硝基芳烃与DNA或二miristoyl磷脂酰胆碱（DMPC）的寡核苷酸和多层囊泡（MLVs）的结合或分布常数，以确定光敏剂和醌类/硝基芳烃与DNA或脂质膜结合的相对疏水性和量。2. 检测POR、PC和CHL光反应中醌/硝基芳烃存在或不存在以及核苷（鸟苷、腺苷）存在或不存在的中间体。3. 检测dna结合（或寡核苷酸结合）、脂质SUVs（小单层囊泡）结合和未结合的POR、PC和CHL与醌/硝基芳烃的光反应中的中间体。4. 测量这些中间体的光物理性质以及这些性质与光敏剂和底物的物理性质（底物的氧化还原电位、光敏剂的三重态能、DNA结合、脂质分配）的相互依赖性。5. 鉴定和定量上述光反应中醌/硝基芳烃的光产物，无论是否存在suv或DNA，不包括DNA共价加合物。6. 鉴定在上述光反应中形成的核苷和DNA共价加合物，包括交联。7. 探讨这些光敏剂与烷基化醌/硝基芳烃联用在低氧/缺氧与常氧条件下诱导肿瘤细胞毒性的作用。特定目标# 7将测试在特定目标2至6中测量的缺氧或缺氧条件下成功生产中间体或光产物的敏化剂和醌/硝基芳烃对。一些不成功的配对也将作为阴性对照。具体目标1和2将在头几年进行。其余年份将主要用于具体目标3至7。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Photodynamic therapy (PDT) is a cancer treatment that uses a combination of red laser light, a photosensitizing agent and molecular oxygen to bring about a therapeutic effect. PDT is particularly promising for treating hollow-organ cancers, for example oesophageal cancers. This is because laser light can now be delivered with great accuracy, via thin flexible optical fibers and endoscopy, to almost any site in the body and with minimal damage to overlying healthy tissue. Porphyrins (POR), phthalocyanines (PC), chlorins (CHL) and others are currently being used in photodynamic treatment (PDT) of tumors due to their large absorption coefficients in the 500-800 nm range. In the presence of air these will photosensitize the production of singlet oxygen and superoxide. Singlet oxygen production, the so-called Type II pathway, is claimed as the most important process which kills tumor cells. However, Type I pathways, those involving photoreduction or photooxidation of substrates, have.also been proposed as photocytotoxic events in PDT, especially in hypoxic environments. In addition, PDT produces in many instances, in cells, apoptosis and necrosis. Solid tumors are often hypoxic. Thus, if photosensitizers are localized inside these tumors, singlet oxygen would not be the reactive species which should eventually kill these tumor cells. Since these dyes are able to photoreduce oxygen, then, these should also photoreduce molecules with nearly equal or more positive redox potentials than oxygen in anoxic/hypoxic cells. If this substrate is a DNA alkylating quinone or nitroarene, which is activated by reduction, it could act as an alkylating species and then DNA alkylation should be expected, with the consequent cell death. Such activation should occur near the DNA site to avoid wasting quinones or nitroarenes by alkylation of other less critical macromolecules. Nitroarenes are reduced to nitrosoarenes (2 electrons), which are highly reactive towards thiols, or further to hydroxylamines (4 electrons), which are reactive species towards Lguanine. In contrast, the aziridinyl-quinones require either 1 or 2 electrons to be activated as alkylating agents. In this regard, since fewer electrons are needed by quinones for alkylating activity aziridinyl-quinones could be more easily photoactivated to a DNA-alkylating species. Photosensitizers are photooxidized, or their triplet states quenched, by nitroimidazoles under anaerobic and hypoxic conditions. This has been demonstrated using flash photolysis methods, even for a nitroimidazole with a much more negative redox potential than oxygen. For example, this was observed using hematoporphyrin and uroporphyrin as photosenzitizers in the presence of metronidazole, with E = -485 mV, while the redox potential of oxygen is -330 mV. However, to our best knowledge, direct detection and characterization of a dye-photoreduced nitroarene or quinone has not occurred. To our best knowledge, previous work on reactions involving photosensitizers and nitroarenes (a) have not dealt with cells under hypoxic conditions, (b) have not considered the importance of the nitroarene redox potential in the yield or quantum yields of photoreduction or cytotoxicity, (c) have not used alkylating quinones instead of nitroaranes in their studies, (d) have not considered the yields or quantum yields of reduced quinone/nitroarene in heterogeneous media vs. aqueous media and (e) have not explored the combination of a DNA-bound (or free) sensitizer with an alkylating quinonelnitroarene in producing DNA adducts. In this work, pyridinium zinc phthalocyanine (PPC) and 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), which are cations and should bind DNA phosphates, will always be included in the development of the following specific aims. Other photosensitizers (hydrophilic or lipophylic and negatively charged) will be included for comparative purposes. Whenever possible, the role of pH will be determined. Special emphasis will also be made on hypoxic or anoxic conditions, although normoxic conditions will be used for comparison. Thus, the following specific aims are designed to fill some of the gaps stated above: 1. To measure binding or distribution constants of POR, PC, CHL and quinones and nitroarenes to DNA or oligonucleotides and multilamellar vesicles (MLVs) of dimiristoylphosphatidylcholine (DMPC) in order to determine the relative hydrophobicity and amount of the photosensitizer and quinone/nitroarene bound to DNA or lipid membrane. 2. To detect intermediates in the photoreactions of POR, PC and CHL in the presence or absence of quinone/nitroarenes and in the presence or absence of nucleosides (guanosine, adenosine). 3. To detect intermediates in the photoreactions of DNA-bound (or oligonucletide-bound), lipid SUVs (small unilamellar vesicles)-bound, and unbound POR, PC and CHL with quinone/nitroarenes. 4. To measure photophysical properties of these intermediates and the interdependence of these properties on the physical properties of the photosensitizer and substrate (redox potentials of substrates, triplet energy of the sensitizer, DNA binding, lipid partition). 5. To identify and quantify photoproducts derived from the quinone/nitroarenes in the photoreactions stated above, in the presence and absence of SUVs or DNA, not including DNA covalent adducts. 6. To identify nucleoside and DNA covalent adducts, including cross-linking, formed in the photoreactions described above. 7. To determine the role of the combination of these photosensitizers with alkylating quinones/nitroarenes on inducing cytotoxicity in tumor cells under hypoxia/anoxia vs.normoxia. Specific aim # 7 will test pairs of sensitizers and quinone/nitroarenes which are successful in the production of intermediates or photoproducts under anoxia or hypoxia measured in Specific Aims 2 to 6. A few of the unsuccessful pairs will also be included as negative controls. Specific Aims 1 and 2 will be worked on during the first years. The rest of the years will essentially be devoted to specific Aims 3 to 7.