Dr. Antonio ALEGRIA

Researcher: Dr. Antonio Alegria

Institution: Department of Chemistry, University of Puerto Rico in Humacao

Project Title: Photosensitized reduction and DNA alkylation of alkylating quinones and nitroarenes

AABRE Cluster: Drug Design & Delivery

Mentor: Dr. Nancy Oleinick, Case Western School of Medicine

Collaborators and Consultants:

• Dr. Fernando Gonzalez, University of Puerto Rico - Rio Piedras
• Dr. Beatriz Zayas, Universidad Metropolitana
• Dr. Rafael Arce, University of Puerto Rico - Rio Piedras
• Dr. Carmelo Garcia, University of Puerto Rico in Humacao

Abstract:

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.