Günther N. Grimm

DNA Cleavage Induced by Alkoxyl Radicals Generated in the Photolysis of N-Alkoxypyridinethiones

The photolysis of N-isopropoxypyridine-2-thione (1b) and of N-tert-butoxypyridine-2-thione (1c) generated alkoxyl radicals as confirmed by trapping experiments with DMPO and subsequent EPR spectroscopy. Upon UVA irradiation, the alkoxyl-radical sources induce strand breaks in supercoiled pBR 322 DNA, which was analyzed by gel electrophoresis. The participation of type I (electron transfer, H abstraction) or type II (1O2) photosensitization in the DNA cleavage by the oxyl-radical sources 1a-d or their photoproducts could be excluded. The present study establishes unequivocally that alkoxyl and benzoyloxyl, as well as hydroxyl radicals, cause strand breaks in DNA and, thus, may play a significant role in the DNA cleavage by peroxides.

tert-Butoxyl radicals generate mainly 7,8-dihydro-8-oxoguanine in DNA.

Like hydroxyl radicals, alkoxyl radicals have been implicated in the generation of cellular oxidative DNA damage under physiological conditions; however, their genotoxic potential has not yet been established. We have analyzed the DNA damage induced by a photochemical source of tert-butoxyl radicals, the water soluble peroxy ester [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), using various repair endonucleases as probes. The irradiation (UV(360)) of BCBT in the presence of bacteriophage PM2 DNA was found to generate a DNA damage profile that consisted mostly of base modifications sensitive to the repair endonuclease Fpg protein. Approximately 90% of the modifications were identified as 7,8-dihydro-8-oxoguanine (8-oxoGua) residues by HPLC/ECD analysis. Oxidative pyrimidine modifications (sensitive to endonuclease III), sites of base loss (AP sites) and single-strand breaks were only minor modifications. Experiments with various scavengers and quenchers indicated that the DNA damage by BCBT+UV(360) was caused by tert-butoxyl radicals as the ultimate reactive species. The mutagenicity associated with the induced damage was analyzed in the gpt gene of plasmid pSV2gpt, which was exposed to BCBT+UV(360) and subsequently transfected into Escherichia coli. The results were in agreement with the specific generation of 8-oxoGua. Nearly all point mutations (20 out of 21) were found to be GC-->TA transversions known to be characteristic for 8-oxoGua. In conclusion, alkoxyl radicals generated from BCBT+UV(360) induce 8-oxoGua in DNA with a higher selectivity than any other reactive oxygen species analyzed so far.

Photochemical reactions of triplet state of N-hydroxyacridine-9-thione studied by laser-flash photolysis

By laser-flash photolysis of N-hydroxyacridine-9-thione (HOAT) in organic solvents, the transient absorption bands observed at 410 and 530 nm, are assigned to the triplet–triplet absorption of 3(HOAT)*. The lowest triplet energy (ET1), the intrinsic triplet lifetime (τT°) and the quantum yield (ΦT) of intersystem crossing have been determined. A rather large self-quenching rate constant (ksq=1.6±0.1×109 mol-1 d s-1 in THF) was observed. In the photoinduced electron-transfer reactions, 3(HOAT)* acts as electron acceptor for tetramethylbenzidine and triphenylamine, while for dinitrobenzene and the methylviologen dication it acts as electron donor in polar solvents. In the addition reactions of 3(HOAT)* with various alkenes, the electrophilic character of 3(HOAT)* has been established. By comparison of the experimental results with MO calculations, the lowest triplet electronic-configuration of 3(HOAT)* is revealed.

4-tert-butylperoxymethyl-9-methoxypsoralen as intercalating photochemical alkoxyl-radical source for oxidative DNA damage.

We describe the synthesis of a novel psoralen peroxide 1 that generates on irradiation (350 nml alkoxyl radicals, namely tert‐butoxyl radicals, as confirmed by electron spin resonance studies with the spin trap 5,5‐dimethyl‐pyrroline‐N‐oxide. The radical source intercalates into the DNA, which has been demonstrated by linear‐flow‐dichroism measurements. Thus, the alkoxyl radicals are formed advantageously directly in the DNA matrix. In supercoiled pBR322 DNA, the generation of strand breaks by the photochemically or metal‐catalyzed generated alkoxyl radicals is demonstrated. Photosensitization by the psoralen chromophore was excluded because similar substances that do not release radicals caused no DNA damage, nor were the photoproducts of the peroxide 1 active. With calf thymus DNA, 8‐oxoGua and small amounts of guanidine‐releasing products, e.g. oxazolone, were observed. However, in these reactions the photoproduct also displayed some DNA‐oxidizing capacity.

Oxidative DNA Damage Induced by Dioxetanes, Photosensitizing Ketones, and Photo-Fenton Reagents

In the last decade, the importance of oxidative DNA damage in mutagenesis, carcinogenesis, aging, and various diseases has prompted intensive investigations of chemical, biochemical, and biological aspects of DNA oxidation caused by reactive species, which are involved in oxidative stress (Sies 1991). Oxidative degradation of DNA causes mutations predominantly at GC base pairs to yield single base substitutions and G to T transversions (Piette 1991). Consequently, the DNA transformation efficiency is diminished and replication is inhibited.