Survey of X-ray induced Cherenkov excited fluorophores with potential for human use.

X-ray induced molecular luminescence (XML) is a phenomenon that can be utilized for clinical, deep-tissue functional imaging of tailored molecular probes. In this study, a survey of common or clinically approved fluorophores was carried out for their megavoltage X-ray induced excitation and emission characteristics. We find that direct scintillation effects and Cherenkov generation are two possible ways to cause these molecules' excitation. To distinguish the contributions of each excitation mechanism, we exploited the dependency of Cherenkov radiation yield on X-ray energy. The probes were irradiated by constant dose of 6 MV and 18 MV X-ray radiation, and their relative emission intensities and spectra were quantified for each X-ray energy pair. From the ratios of XML, yield for 6 MV and 18 MV irradiation we found that the Cherenkov radiation dominated as an excitation mechanism, except for aluminum phthalocyanine, which exhibited substantial scintillation. The highest emission yields were detected from fluorescein, proflavin and aluminum phthalocyanine, in that order. XML yield was found to be affected by the emission quantum yield, overlap of the fluorescence excitation and Cherenkov emission spectra, scintillation yield. Considering all these factors and XML emission spectrum respective to tissue optical window, aluminum phthalocyanine offers the best XML yield for deep tissue use, while fluorescein and proflavine are most useful for subcutaneous or superficial use.

[Experimental study of proflavine acetate phototransformation processes].

Changes in proflavine acetate phototransformation processes upon exposure to visible-range irradiation were studied by high performance liquid chromatography. Proflavine acetate was offered as a photosensitizer during photodynamic water disinfection. Dye transformation products upon time-varying exposure to irradiation were identified. By using structure-activity relationships and information from toxicity databases, the authors evaluated the hazard of the identified products and identified the most hazardous ones.

Photo-enhancement of the mutagenicity of 9-anilinoacridine derivatives related to the antitumour agent amsacrine.

The frameshift mutagenicity of the DNA intercalating drug proflavine is known to be enhanced by photoirradiation of bacterial cultures. To determine whether this phenomenon was also present in acridine-derived antitumour drugs, cultures of Salmonella typhimurium were exposed to the antileukaemia agent amsacrine and the experimental agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide dihydrochloride (acridine carboxamide) in the presence or absence of visible light. A small increase in mutagenicity was observed with amsacrine but not with acridine carboxamide. A series of analogues of amsacrine were then tested, and a striking relationship was found between the minimum drug concentration for mutagenicity and DNA binding affinity. In each case, photoirradiation was associated with a small increase in mutagenicity. Each of the compounds showing the photo-enhancement effect was capable of reversible one-electron oxidation. It is suggested that this oxidation occurs in bacteria, and that the DNA binding constant of the resulting acridine radical species will increase because of the extra positive charge. This increased DNA binding would be sufficient to explain the photo-enhancement of mutagenicity of these drugs.

Effects of proflavin and photoactivated proflavin on the template function of single-stranded DNA.

DNA context-specific effects of the association of proflavin, single-stranded DNA and DNA polymerase on DNA polymerization reactions were examined. Frameshift mutations induced by the presence of proflavin during in vitro DNA replication of a single-stranded DNA template by the Klenow fragment of Escherichia coli DNA polymerase I were sequenced. More than 80% of the frameshifts were one base-pair deletions opposite purine bases that were immediately 3' to pyrimidines. Purines (Pu) that were not adjacent to pyrimidines (Py) were not deletion sites. The remaining deletions were opposite template pyrimidines that were also immediately 3' to another pyrimidine. All pyrimidine site deletions occurred in the context 5' PyPyPu 3'. In additional experiments, the site-specific inhibition of processive DNA polymerization by proflavin was examined. A novel inhibition of polymerization was found opposite all pyrimidines in the template when proflavin-template complexes were exposed to ten seconds of white light. This inhibition of polymerization is reversible. Longer photoactivation led to an altered pattern of DNA sequence-specific inhibition that was not reversible. The role of DNA sequence-specific interactions of proflavin with DNA in proflavin mutagenesis is discussed.

Photodynamic activity of acridine orange: peroxide radical induction in DNA and synthetic polynucleotides.

As shown by electron paramagnetic resonance, acridine orange induces the formation of peroxide radicals in DNA when dye-DNA mixtures frozen at 77 K are irradiated with visible light. The reaction is oxygen dependent and strongly reduced by the addition of an electron scavenger. Factors of the medium can modulate the reaction: an ionic strength increased up to 0.3 greatly enhances the dye efficiency whereas the presence of phosphate ions has an inhibiting influence. Acridine orange, which is slightly less efficient than proflavine on native DNA, induces an important peroxide radical formation in poly(dG).poly(dC) but has no action on the poly(dA).poly(dT)polymer.

Visible-light-induced OH radicals in DNA-proflavine complexes: an e.p.r. and spin trapping study.

Using the spin-trapping technique, irradiation with visible light of complexes between DNA and proflavine was shown to generate OH radicals. The characteristic spectra were not obtained when proflavine or DNA were irradiated alone, nor when oxygen was absent. Using DMPO as a spin trap we found that the intensity of the DMPO-OH e.p.r. signal was enhanced when the molar ratio between bound proflavine and the DNA phosphorus increased up to a value of 0.15 demonstrating the efficiency of the intercalated dye molecules. A strong decrease of the e.p.r. signal was observed in the presence of various OH. scavengers like t-butanol, isopropanol or sodium benzoate. The OH. production was also decreased when the irradiation was made in the presence of SOD, ceruloplasmin or catalase and after addition of Chelex 100 resin.

Mechanism for strand-break induction in DNA-proflavine complexes exposed to visible light.

Proflavine bound-superhelical phi XRFI DNA Molecules undergo single-strand scission upon irradiation with visible light at high fluence rate. As shown by agarose gel electrophoresis analyses, the nicking reaction is (i) oxygen-dependent, (ii) strongly inhibited by catalase and an electron scavenger such as cystamine, and (iii) totally suppressed by ceruloplasmin and radical scavengers such as t-butanol sodium benzoate. This indicates that H2O2, e-, O2 and OH, respectively, are involved in the cleavage process. NaN3, a singlet-oxygen quencher, has very little effect on strand-breakage but it prevents almost completely the alteration of guanine residues (a lesion already observed after irradiation at low fluence rate). Since, in the presence of NaN3, strand scission can occur and guanine (as the other bases) recovered intact, it follows that the radical intermediates produced during breakages are probably not involved in any permanent modification of the DNA bases.

Effect of gamma irradiation on dye-DNH binding.

This paper attempts to analyse the effect of (a) gamma radiation and (b) the presence of histones in the form of nucleohistones (DNH) on the modes of ligand (Proflavine, Pf) binding to DNH. Effects were investigated by spectrophotometry. A certain dose of radiation which causes appreciable DNA denaturation has much less effect on DNH. There is a 'threshold' dose beyond which DNH denaturation is directly proportional to dose as in the case of DNA. The histones in DNH act as a shield and at higher doses get dissociated to expose the double helix to radiations. Ligand binding has a further stabilizing effect against radiation damage to DNH.

Effect of gamma-irradiation on dye-DNA binding.

The effect of gamma-rays on the binding of proflavine and acridine orange to DNA was investigated by spectrophotometry. The effect of irradiation was observed on the buffered solutions of the free dye and free DNA. A dose of about 35 krad caused a hyperchromicity of 30-40 per cent to the DNA peak at 258 nm, while the same dose introduced a hypochromic effect to the monomer peaks of the dyes by 30 per cent. This implied that gamma-rays have an effect of decreasing the monomer concentration of free-day molecules in solution. From the results, we conclude that more dye is bound to the changed conformation of dye-bound DNA on irradiation. Scratchard-binding isotherms drawn for the unirradiated and irradiated complexes of Pf-DNA showed interesting differences. Similar isotherms could not be obtained for the acridine orange-DNA system.