99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

^99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, ^99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a ^99mTc-labeled HYNIC-DAPI compound with that of ^99mTc pertechnetate (^99mTcO₄ ⁻). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by ^99mTc-HYNIC-DAPI (1.03) was twice that caused by ^99mTcO₄ ⁻ (0.51), and the number of DSBs increased fivefold in the ^99mTc-HYNIC-DAPI-treated sample compared with the ^99mTcO₄ ⁻ treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the ^99mTcO₄ ^– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the ^99mTc-HYNIC-DAPI-treated samples. These results indicated that ^99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the ^99mTc-labeled compound with DNA. In contrast to these results, ^99mTcO₄ ⁻ induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of ^99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs.     

Probing the microenvironmental of benzo[a]pyrene diol epoxide-DNA adducts by triplet excited state quenching methods

Triplet flash photolysis techniques, coupled with quenching of the triplets by molecular oxygen, are utilized as probes of the microenvironment of polycyclic aromatic molecules bound covalently and non-covalently to DNA. The triplet-oxygen quenching properties of the following adducts in aqueous solutions at 25±1°C were investigated: covalent adducts derived from the reaction of (±)-7β,8α-dihydroxy-9α,10α-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE) and of (±)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPE) with DNA, and non-covalent intercalation complexes of acridine orange (AO) and DNA. In all cases the quenching follows the Stern-Volmer quenching law with a quenching constant of K_O2^T≈10⁹ M^?1·s^?1 for the covalent BaPDE-DNA and BaPE-DNA complexes in aqueous solution. This value of K_O2^T is characteristic of free molecules (not bound to DNA) and indicates that the pyrene chromophore is totally accessible to oxygen, and is thus not located at an intercalation-type of binding site in these covalent adducts. In contrast, the AO-DNA complexes are characterized by values of K_O2^T≈10⁸ M^?1·s^?1 indicating that the intercalated AO molecules are about ten times less accessible to molecular oxygen than free AO molecules. The K_O2^T values for the covalent BaPDE-DNA and BaPE-DNA adducts decrease when the DNA concentration is increased in the 1·10^?4?3·10^?3 M range (expressed in nucleotide concentration). This effect is attributed to intermolecular DNA-DNA interactions in which segments of adjacent DNA molecules tend to cover the pyrene chromophores on other strands, thus decreasing their accessibility to oxygen. In contrast the values of K_O2^T for the non-covalent AO-DNA intercalation complexes are independent of DNA concentration, as expected for interior binding sites.     

APE1 overexpression in XRCC1-deficient cells complements the defective repair of oxidative single strand breaks but increases genomic instability

XRCC1 protein is essential for mammalian viability and is required for the efficient repair of single strand breaks (SSBs) and damaged bases in DNA. XRCC1-deficient cells are genetically unstable and sensitive to DNA damaging agents. XRCC1 has no known enzymatic activity and is thought to act as a scaffold protein for both SSB and base excision repair activities. To further define the defects leading to genetic instability in XRCC1-deficient cells, we overexpressed the AP endonuclease APE1, shown previously to interact with and be stimulated by XRCC1. Here, we report that the overexpression of APE1 can compensate for the impaired capability of XRCC1-deficient cells to repair SSBs induced by oxidative DNA damage, both in vivo and in whole-cell extracts. We show that, for this kind of damage, the repair of blocked DNA ends is rate limiting and can be performed by APE1. Conversely, APE1 overproduction resulted in a 3-fold increase in the sensitivity of XRCC1-deficient cells to an alkylating agent, most probably due to the accumulation of SSBs. Finally, the overproduction of APE1 results in increases of 40% in the frequency of micronuclei and 33% in sister chromatid exchanges of XRCC1⁻ cells. These data suggest that the spontaneous generation of AP sites could be at the origin of the SSBs responsible for the spontaneous genetic instability characteristic of XRCC1-deficient cells.     

Nuclear mutations and mitotic recombination in saccharomyces by light-activated ethidium azides

Ethidium mono- and diazide analogs have been used as photoaffinity probes to study the mechanism of the ethidium-induced petite mutation in yeast [7,10,14]. The azide moiety when exposed to light is converted to a reactive nitrine intermediate. The diradical nitrene effects a covalent attachment to sites of interaction bound reversibly by the drug. Ethidium azide photoaffinity labeling has been used to verify the prerequisite covalent attachment of ethidium to mitochondrial DNA to bring about the petite mutation in yeast [6]. Bastos has also reported a specific photoattachment of ethidium azide to a polypeptide (subunit 9) of the membrane bound ATPase in yeast mitochondria [1].Isolated DNA from yeast cells treated in vivo with [¹⁴C]ethidium monoazide plus light contained covalent adducts on both nuclear and mitochondrial DNA, although the specific radioactivity of mitochondrial DNA was more than 10 times higher than nuclear DNA [11]. Drug distribution studies of [¹⁴C]ethidium monoazide have indicated that greater than 50% of the covalent adducts in the nuclear centrifugation fraction (2000 × g) reside on nuclear proteins [3]. Nuclear damage in yeast by the photolysis of ethidium azides is apparent from the increased killing effect in cells treated with ethidium azide derivatives plus light as opposed to dark-treated cells [7,10,11]. This communication reports a concomitant increase in nuclear mutations and genetic activity from the photoactivated covalent attachment of ethidium azides to nuclear components.     

Mechanistic DNA damage simulations in Geant4-DNA Part 2: Electron and proton damage in a bacterial cell

We extended a generic Geant4 application for mechanistic DNA damage simulations to an Escherichia coli cell geometry, finding electron damage yields and proton damage yields largely in line with experimental results. Depending on the simulation of radical scavenging, electrons double strand breaks (DSBs) yields range from 0.004 to 0.010 DSB Gy⁻¹ Mbp⁻¹, while protons have yields ranging from 0.004 DSB Gy⁻¹ Mbp⁻¹ at low LETs and with strict assumptions concerning scavenging, up to 0.020 DSB Gy⁻¹ Mbp⁻¹ at high LETs and when scavenging is weakest. Mechanistic DNA damage simulations can provide important limits on the extent to which physical processes can impact biology in low background experiments. We demonstrate the utility of these studies for low dose radiation biology calculating that in E. coli, the median rate at which the radiation background induces double strand breaks is 2.8 × 10⁻⁸ DSB day⁻¹, significantly less than the mutation rate per generation measured in E. coli, which is on the order of 10⁻³.     

PARP1 suppresses homologous recombination events in mice in vivo

Recent studies suggest that PARP1 inhibitors, several of which are currently in clinical trial, may selectively kill BRCA1/2 mutant cancers cells. It is thought that the success of this therapy is based on immitigable lethal DNA damage in the cancer cells resultant from the concurrent loss or inhibition of two DNA damage repair pathways: single-strand break (SSB) repair and homologous recombination repair (HRR). Presumably, inhibition of PARP1 activity obstructs the repair of SSBs and during DNA replication, these lesions cause replication fork collapse and are transformed into substrates for HRR. In fact, several previous studies have indicated a hyper-recombinogenic phenotype in the absence of active PARP1 in vitro or in response to DNA damaging agents. In this study, we demonstrate an increased frequency of spontaneous HRR in vivo in the absence of PARP1 using the p^un assay. Furthermore, we found that the HRR events that occur in Parp1 nullizygous mice are associated with a significant increase in large, clonal events, as opposed to the usually more frequent single cell events, suggesting an effect in replicating cells. In conclusion, our data demonstrates that PARP1 inhibits spontaneous HRR events, and supports the model of DNA replication transformation of SSBs into HRR substrates.     

NBS1 cooperates with homologous recombination to counteract chromosome breakage during replication

Nijmegen breakage syndrome (NBS) is characterized by genome instability and cancer predisposition. NBS patients contain a mutation in the NBS1 gene, which encodes the NBS1 component of the DNA double-strand break (DSB) response complex MRE11/RAD50/NBS1. To investigate the NBS phenotype in more detail, we combined the mouse mimic of the most common patient mutation (Nbs1^ΔB/ΔB) with a Rad54 null mutation, which diminishes homologous recombination. Double mutant cells were particularly sensitive to treatments that cause single strand breaks (SSBs), presumably because these SSBs can be converted into detrimental DSBs upon passage of a replication fork. The persistent presence of nuclear RAD51 foci and increased levels of chromatid type breaks in metaphase spreads indicated that replication-associated DSBs are repaired inefficiently in the double mutant cells. We conclude that Nbs1 and Rad54 function cooperatively, but in separate pathways to counteract this type of DNA damage and discuss mechanistic implications of these findings.     

PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1^−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1^−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.     

A re-investigation of the reactions between superoxide anion and metal picolinate complexes

Rate constants for the reactions of superoxide with the α-picolinate ion and its complexes with copper(II), iron(III) and zinc(II), and for the reaction of α-picolinate with the hydrated electron, were measured using pulse radiolysis. The rate constant for the reaction of superoxide with copper(II)picolinate at pH 9 [(4.1 ± 0.4) × 10⁷l mol⁻¹ s⁻¹] was an order of magnitude higher than that determined previously (W. H. Bannister, J. V. Bannister, A. J. F. Searle and P. J. Thornally, Inorg. Chim. Acta, 78, 139 (1983)) using a less direct competitive inhibition method. The corresponding rate constant for iron(III)picolinate [(7.5 ± 1.5) X 10³ l mol⁻¹ s⁻] was an order of magnitude lower than a previous pulse radiolysis determination (same reference as above). We are not able to reconcile these two values for iron(III)picolinate, although a possible source of spuriously high results is contamination with the kinetically active copper(II) complex. The likely roles of iron(III)picolinate and other low molecular weight iron complexes as potential catalysts of an in vivo superoxide-driven Fenton reaction are discussed in the light of present measurements.     

Repair of lesions induced in human liver cell DNA by N-nitroso compounds as measured by the bromodeoxyuridine photolysis method

Chang human liver cells were treated with the carcinogens N-methyl-N′-nitrosoguanidine (MNNG) and nitrosomorpholine (NM). In addition, cells were exposed to the folic acid analog, 2-hydroxy-N¹⁰ nitrosofolic acid. Repair of the damage to DNA was estimated by selective photolysis of BUdR-containing repaired regions with 313 nm radiation. The influence of the co-carcinogen Arlacel A was estimated with the three compounds. Results indicated significant repair synthesis with MNNG- and NM-treated cells. 2-Hydroxy-N¹⁰ nitrosofolic acid elicited no damage to the liver DNA. Arlacel A prevented repair synthesis in cells treated with NM and MNNG.     

The major Arabidopsis thaliana apurinic/apyrimidinic endonuclease,ARP is involved in the plant nucleotide incision repair pathway

Apurinic/apyrimidinic (AP) endonucleases are important DNA repair enzymes involved in two overlapping pathways: DNA glycosylase-initiated base excision (BER) and AP endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, AP endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in NIR, the same AP endonucleases incise DNA 5' to a wide variety of oxidized bases. The flowering plant Arabidopsis thaliana contains three genes encoding homologues of major human AP endonuclease 1 (APE1): Arp, Ape1L and Ape2. It has been shown that all three proteins contain AP site cleavage and 3'-repair phosphodiesterase activities; however, it was not known whether the plant AP endonucleases contain the NIR activity. Here, we report that ARP proteins from Arabidopsis and common wheat (Triticum aestivum) contain NIR and 3' → 5' exonuclease activities in addition to their AP endonuclease and 3'-repair phosphodiesterase functions. The steady-state kinetic parameters of reactions indicate that Arabidopsis ARP cleaves oligonucleotide duplexes containing α-anomeric 2'-deoxyadenosine (αdA) and 5,6-dihydrouridine (DHU) with efficiencies (k_cat/K_M = 134 and 7.3 μM⁻¹·min⁻¹, respectively) comparable to those of the human counterpart. However, the ARP-catalyzed 3'-repair phosphodiesterase and 3' → 5' exonuclease activities (k_cat/K_M = 314 and 34 μM⁻¹·min⁻¹, respectively) were about 10-fold less efficient as compared to those of APE1. Interestingly, homozygous A. thaliana arp^–/– mutant exhibited high sensitivity to methyl methanesulfonate and tert-butyl hydroperoxide, but not to H₂O₂, suggesting that ARP is a major plant AP endonuclease that removes abasic sites and specific types of oxidative DNA base damage. Taken together, these data establish the presence of the NIR pathway in plants and suggest its possible role in the repair of DNA damage generated by oxidative stress.     

Synthesis,characterization, and evaluation of (E)-methyl 2-((2-oxonaphthalen-1(2H)-ylidene)methylamino)acetate as a biological agent and an anion sensor

An amino acid based and bidentate Schiff base, (E)-methyl 2-((2-oxonaphthalen-1(2H)-ylidene)methylamino)acetate (ligand), was synthesized from the reaction of glycine-methyl ester hydrochloride with 2-hydroxy-1-naphthaldehyde. Characterization of the ligand was carried out using theoretical quantum–mechanical calculations and experimental spectroscopic methods. The molecular structure of the compound was confirmed using X-ray single-crystal data, NMR, FTIR and UV–Visible spectroscopy, which were in good agreement with the structure predicted by the theoretical calculations using density functional theory (DFT). Antimicrobial activity of the ligand was investigated for its minimum inhibitory concentration (MIC) to several bacteria and yeast cultures. UV–Visible spectroscopy studies also shown that the ligand can bind calf thymus DNA (CT-DNA) electrostatic binding. In addition, DNA cleavage study showed that the ligand cleaved DNA without the need for external agents. Energetically most favorable docked structures were obtained from the rigid molecular docking of the compound with DNA. The compound binds at the active site of the DNA proteins by weak non-covalent interactions. The colorimetric response of the ligand in DMSO to the addition of equivalent amount of anions (F⁻, Br⁻, I⁻, CN⁻, SCN⁻, ClO₄⁻, HSO₄⁻, AcO⁻, H₂PO₄⁻, N₃⁻ and OH⁻) was investigated and the ligand was shown to be sensitive to CN⁻ anion.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Abstract

Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Photosystem I charge separation in the absence of centers A and B. II. ESR spectral characterization of center ‘X’ and correlation with optical signal ‘A2’

The Photosystem I electron acceptor complex was characterized by optical flash photolysis and electron spin resonance (ESR) spectroscopy after treatment of a subchloroplast particle with lithium dodecyl sulfate (LDS). The following properties were observed after 60 s of incubation with 1% LDS followed by rapid freezing. (i) ESR centers A and B were not observed during or after illumination of the sample at 19 K, although the P-700⁺ radical at g = 2.0026 showed a large, reversible light-minus-dark difference signal. (ii) Center ‘X’, characterized by g factors of 2.08, 1.88 and 1.78, exhibited reversible photoreduction at 8 K in the absence of reduced centers A and B. (iii) The backreaction kinetics at 8 K between P-700, observed at g = 2.0026, and center X, observed at g = 1.78, was 0.30 s. (iv) The amplitudes of the reversible g = 2.0026 radical observed at 19 K and the 1.2 ms optical 698 nm transient observed at 298 K were diminished to the same extent when treated with 1% LDS at room temperature for periods of 1 and 45 min. We interpret the strict correlation between the properties and lifetimes of the optical P-700⁺ A⁻₂ reaction pair and the ESR P-700⁺ center X⁻ reaction pair to indicate that signal A₂ and center X represent the same iron-sulfur center in Photosystem I.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a³⁺a²⁺₃-CO) complex and the mixed-valence carboxycytochrome c oxidase (a³⁺a²⁺₃-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a²⁺₃-CO complex. This would confirm that the spectral properties of cytochrome a₃ are not affected by the redox state of cytochrome a and Cu_A. When the absorbance changes after photolysis of cytochrome a²⁺₃-CO with a laser flash were followed on a time scale from 1 μs to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7·10³s^?1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a₃. Concomitantly, a decrease of the 830 nm band was observed due to reduction of Cu_A. This demonstrates that in the partially reduced enzyme a pathway is present between Cu_A and the cytochrome a₃-Cu_B pair, via which electrons are transferred rapidly.     

Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba

We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant–DSBs versus alkaline–DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). I. Undamaged covalent circular lambda DNA molecules in uvrA+ recA+ lysogenic host cells are cut following superinfection with psoralen-damaged lambda phages.

When λ bacteriophages were treated with a photosensitizing agent, psoralen or khellin, and 360 nm light, monoadducts and interstrand crosslinks were produced in the phage DNA. The DNA from the treated phages was injected normally into Escherichia coli uvrA^? (λ) cells and it was converted to the covalent circular form in yields similar to those obtained in experiments with undamaged λ phages. In excision-proficient host cells, however, there was a dose-dependent reduction in the yield of rapidly sedimenting molecules, and a corresponding increase in slow sedimenting material, the extent of this conversion corresponding to about one cut per two crosslinks. Presumably, the damaged λ DNA molecules were cut by the uvrA endonuclease of the host cell, but were not restored to the original covalent circular form.The presence of psoralen damage in λ phage DNA greatly increased the frequency of genetic exchanges in λ phage-prophage crosses in homoimmune lysogens (Lin et al., 1977). As genetic recombination is thought to depend on cutting and joining in DNA molecules, experiments were performed to test whether psoralen-damaged λ DNA would cause other λ DNA in the same cell to be cut. E. coli (λ) host cells were infected with ³²P-labeled λ phages and incubated to permit the labeled DNA to form covalent circles. When these host cells were superinfected with untreated λ phages, there was no effect upon the circular DNA. When superinfected with λ phages that had been treated with psoralen and light, however, many of the covalent circular molecules were cut. The cutting of undamaged molecules in response to the damaged DNA was referred to as “cutting in trans”. It required the uvrA⁺ and recA⁺ host gene functions, but neither recB⁺ nor any phage gene functions. It occurred normally in non-lysogenic hosts treated with chloramphenicol before infection. Cutting in trans may be one of the steps in recA-controlled recombination between psoralen crosslinked phage λ DNA and its homologs.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Abstract

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) after the incubation of 2′-deoxyguanosine (dGuo) with ChOOH and Cu²⁺. In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

Structure-dependent reactivity of oxyfunctionalized acetophenones in the photooxidation of DNA: base oxidation and strand breaks through photolytic radical formation (spin trapping, EPR spectroscopy, transient kinetics) versus photosensitization (electron transfer, hydrogen-atom abstraction)

The photooxidative damage of DNA, specifically guanine oxidation and strand-break formation, by sidechain-oxyfunctionalized acetophenones (hydroxy, methoxy, tert-butoxy and acetoxy derivatives), has been examined. The involvement of triplet-excited ketones and their reactivity towards DNA has been determined by time-resolved laser-flash spectroscopy. The generation of carbon-centered radical species upon Norrish-type I cleavage has been assessed by spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide, coupled with electron paramagnetic resonance spectroscopy. The observed DNA-base oxidation and strand-break formation is discussed in terms of the peroxyl radicals derived from the triplet-excited ketones by α cleavage and molecular oxygen trapping, as well as direct interaction of the excited states by electron transfer and hydrogen-atom abstraction. It is concluded that acetophenone derivatives, which produce radicals upon photolysis, in particular the hydroxy (AP-OH) and tert-butoxy (AP-O^tBu) derivatives, are more effective in oxidizing DNA.