Figure 1 Typical photohemolysis plot in nitrogen, air and oxygen saturated solutions of the NSAID Carprofen.

10 20 30 Irradiation time, min

Figure 1 Typical photohemolysis plot in nitrogen, air and oxygen saturated solutions of the NSAID Carprofen.

In the presence of oxygen, Type I mechanism involves the superoxide anion. Even if it is known that this species is not directly responsible for lipid peroxidation, it can promote, via Haber-Weiss reaction in the presence of traces of metal ions, formation of the hydroxyl radical, which is one of the most harmful species as regards the photoinduced damage. This is due to its ability in abstracting hydrogen from the bio-substrate. Reduction of photohemolysis, lipid peroxidation and marker efflux operated by SOD suggests the presence of 02"-, which can be evidenced also by spectroscopic determination of the reduction of cytochrome III.36 In anaerobic conditions, the same method can be useful for detecting solvated electrons produced during the irradiation.37,38

3.1.3 Protein photoinduced crosslinking. Protein and lipid components are closely related in the membrane structure, but treatment of erythrocyte ghost membranes with a surfactant permits the isolation of the protein fraction, which can be used for the study of photosensitized protein crosslinking. This process can be studied by detecting high molecular weight protein components by SDS polyacrylamide gel electrophoresis.39

3.2 DNA Targets

3.2.1 DNA-drug interaction. The study of photosensitized DNA damage is needed for a deep understanding of both phototoxicity and phototherapy. It contributes to clarify the mechanisms of degenerative skin diseases and of the cell toxicity of potential anticancer drugs. Under this respect it is very important to consider the biodistribution of the photosensitizing agent and its binding mode to DNA.40"43 In fact, the photosensitizer can associate to DNA either through a surface interaction such as hydrogen-bonding or van der Waals forces along the grooves of the helix or through non-covalent intercalation via it-stacking of aromatic heterocyclic groups between base pairs. In the latter case a site-selective photocleavage can be promoted. The study of the influence of parameters like hydrophobicity, geometry, size, shape and ability in the formation of H-bonds with the base pairs permits the assessment of the affinity between the photosensitizer and DNA. Useful information about modes of interaction is provided from induced linear and circular dichroism, fluorescence anisotropy measurements and low temperature phosphorescence44 as well as by other techniques such as microcalorimetry and topoisomerase assays.

Interesting information can be obtained by studying the emission of nucleic acid bases in the absence and in the presence of the drug at 77 K. In the latter case, the appearance of the typical phosphorescence maximum of the adduct drug-DNA indicates the occurrence of short distance energy transfer suggesting close contact between DNA bases and sensitizer.43'45 The changes in fluorescence intensity and/or UV spectra due to drug-DNA association can be used to estimate binding constant through non-linear regression analysis based on mathematic models such as that proposed by McGhee and von Hippel.46

The presence of circular dichroism signals from a non optically asymmetric drug molecule is also indicative of drug-DNA interaction. The positive band of calf thymus DNA, centered at 275 nm decreases with the addition of the increasing concentrations of the drug (such as Suprofen),43 whereas a new band with maximum corresponding to the maximum of the UV spectrum of the drug grows up. The circular dichroism signal originates mainly from the dipole-dipole interaction between the electric dipole moment of the drug transitions with the dipoles represented by the bonds of the chiral double helix of DNA. The CD spectrum is expected in this case to have the same shape as the absorption spectrum.43'44

3.2.2 DNA photosensitization. The mechanism of photoinduced DNA damage, leading to nucleic acid oxidation and single strand break, involves three main pathways: i) participation of hydroxyl radicals, known as one of the most noxious species in promoting DNA damage,47 ii) electron transfer48 and iii) oxidation via singlet oxygen.49 Analysis of nucleoside photoproducts permits a preliminary discrimination between type I and type II mechanisms. The photosensitization process efficiency is determined by measuring supercoiled plasmid linearization rates. This is obtained by monitoring the changes in conformation of supercoiled form I of DNA, which is at first converted in relaxed open circular form II via single strand break and finally to linear form III through double strand break.43'50 DNA forms are isolated via agarose gel electrophoresis.

Moreover, it is possible to follow the kinetics of photoinduced DNA cleavage in the presence of various scavengers or quenchers (also in an oxygen-modified atmosphere), with the aim of emphasizing the role of the different transient species (free radicals, superoxide anion or singlet oxygen) involved in the photocleavage process.49 If DNA photoinduced damage proceeds via a type I mechanism, a hydrogen abstraction by the photogenerated drug radicals more likely involves DNA than solvent or scavengers, if an efficient drug-DNA interaction occurs, (nevertheless, a less efficient production of hydroxyl radicals from solvent cannot be excluded). The general type I-mediated pathway can involve hydrogen abstraction from a sugar of DNA and this process leads directly to DNA breakage.51

In the case a type II mechanism is operative, the presence of singlet oxygen can be shown in experiments carried out with quenchers like sodium azide and 1.4-diazabicyclo[2.2.2]octane. If the quencher is able to reduce ssb with high efficiency in aerobic conditions, this is a strong indication of the involvement of singlet oxygen in ssb (also if it is a controversial matter). An excellent review by Piette gives a wide discussion on this point.52 Further evidence for participation of singlet oxygen can be provided by photocleavage experiments carried out in D20 (to increase the lifetime of singlet oxygen): in this case the cleavage efficiency is increased.43

The presence of efficient type II processes can give indication of the predominance of a surface binding mode of the sensitizer to DNA compared to the intercalative one because in the latter case the sensitizer cannot be available for bimolecular energy transfer with oxygen.53'54

A further contribution to the elucidation of the mechanism of drug induced DNA photodamage can be provided by using 2-deoxyguanosine as a DNA model compound. The major photo-oxidation products of this nucleoside can be identified and classified according to the formation mechanism (mediated by a radical or singlet oxygen).55

Moreover, DNA sequencing through acrylamide gel electrophoresis gives detailed information about site selectivity of sensitizer binding to specific DNA base sequences. DNA sequencing can be performed using either restriction fragments of plasmides or oligonucleotides, which can be synthesized according to a specific sequence design.56

Finally, interesting data can be provided from the photophysical and photochemical behaviour of the sensitizer in the absence and in the presence of DNA. Time resolved photochemical experiments could enlighten the role played by transient species in DNA sensitization.57

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