Photohydrolysis of methotrexate produces pteridine,which induces poly-G-specific DNA damage through photoinduced electron transfer

Methotrexate (MTX), an antineoplastic agent, demonstrates phototoxicity. The mechanism of damage to biomacromolecules induced by photoirradiated MTX was examined using 32P-labeled DNA fragments obtained from a human gene. Photoirradiated MTX caused DNA cleavage specifically at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA only when the DNA fragments were treated with piperidine, which suggests that DNA cleavage was caused by base modification with little or no strand breakage. With denatured single-stranded DNA the damage occurred at most guanine residues. The amount of formation of 8-hydroxy-2'-deoxyguanosine (8-oxodGuo), an oxidative product of 2'-deoxyguanosine, in double-stranded DNA exceeded that in single-stranded DNA. These results suggest that photoirradiated MTX participates in 8-oxodGuo formation at the underlined G in 5'-GG and 5'-GGG sequences in double-stranded DNA through electron transfer, and then 8-oxodGuo undergoes further oxidation into piperidine-labile products. Fluorescence measurement, high-pressure liquid chromatography and mass spectrometry have demonstrated that photoexcited MTX is hydrolyzed into 2,4-diamino-6-(hydroxymethyl)pteridine (DHP). DNA damage induced by DHP was observed in a similar manner as was the damage induced by MTX. The extent of DNA damage and the formation of 8-oxodGuo by DHP were much larger than those induced by MTX. The kinetic analysis, based on the time course of DNA oxidation by photoirradiated MTX, suggests that DNA damage is caused by photoexcited DHP rather than by photoexcited MTX. In conclusion, photoexcited MTX undergoes hydrolysis through intramolecular electron transfer, resulting in the formation of DHP, which exhibits a phototoxic effect caused by oxidation of biomacromolecules through photoinduced electron transfer.     

Guanine-specific DNA oxidation photosensitized by the tetraphenylporphyrin phosphorus(V) complex via singlet oxygen generation and electron transfer

The photosensitized DNA damage caused by dihydroxoP(V)tetraphenylporphyrin (P(V)TPP), a cationic water-soluble porphyrin, was examined. The study of near-infrared emission measurements demonstrated the photosensitized singlet oxygen ((1)O(2)) generation by P(V)TPP (quantum yield: 0.28 in ethanol). The fluorescence quenching of P(V)TPP by DNA showed the electron transfer (ET) from nucleobases to photoexcited P(V)TPP. These results have shown that P(V)TPP has ability to damage DNA through dual mechanisms, (1)O(2) generation and ET. Under aerobic conditions, P(V)TPP photosensitized damage was more severe for single-stranded DNA compared to its double-stranded counterpart. Photoexcited P(V)TPP damaged every guanine residue in single-stranded DNA. HPLC measurements confirmed the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), an oxidized product of 2'-deoxyguanosine, and showed that the yield of 8-oxodGuo in single-stranded DNA is larger than that in double-stranded DNA. The guanine-specific DNA damage and the enhancement in single-stranded DNA suggest that the (1)O(2) generation mainly contributes to the mechanism of DNA photodamage by P(V)TPP. Absorption spectrum measurements suggested the interaction between P(V)TPP and DNA. This interaction is expected to enhance the (1)O(2)-mediated DNA damage since the lifetime of (1)O(2) is very short. On the other hand, for double-stranded DNA, photosensitized damage at consecutive guanines was much less pronounced. Because the consecutive guanines act as a hole trap, this DNA-damaging pattern suggests the partial involvement of photoinduced ET. However, DNA damage by ET was not a main mechanism, possibly due to the reverse ET. In conclusion, P(V)TPP induces guanine specific photooxidation mainly via (1)O(2) generation. The interaction with DNA and the energy level of the photoexcited porphyrin may be advantageous for (1)O(2)-mediated DNA damage rather than ET mechanism.     

Base oxidation at 5' site of GG sequence in double-stranded DNA induced by UVA in the presence of xanthone analogues: relationship between the DNA-damaging abilities of photosensitizers and their HOMO energies

UVA contributes to skin cancer by solar UV light. Photosensitizers are believed to play an important role in UVA carcinogenesis. We investigated the mechanism of DNA damage induced by photoexcited xanthone (XAN) analogues (XAN, thioxanthone [TXAN] and acridone [ACR]), exogenous photosensitizers, and the relationship between the DNA-damaging abilities and their highest occupied molecular orbital (HOMO) energies. DNA damage by these photosensitizers was examined using 32P-labeled DNA fragments obtained from the p53 tumor suppressor gene. Photoexcited XAN caused DNA cleavage specifically at 5'-G of the GG sequence in the double-stranded DNA only when the DNA fragments were treated with piperidine, suggesting that DNA cleavage is due to base modification with little or no strand breakage. With denatured single-stranded DNA, the extent of XAN-sensitized photodamage was decreased. An oxidative product of G, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo), was formed by photoexcited XAN, and the 8-oxo-dGuo formation was decreased in single-stranded DNA. TXAN and ACR induced DNA photodamage as did XAN, although the order of DNA-damaging ability was XAN > TXAN > ACR. These findings suggest that photoexcited XAN analogues induce nucleobase oxidation at 5'-G of GG sequence in double-stranded DNA through electron transfer. The HOMO energies of these photosensitizers, estimated from ab initio molecular orbital (MO) calculation, decreased in the following order: XAN > TXAN > ACR. Extents of DNA damage increased exponentially with the HOMO energies of XAN analogues. This study suggests that DNA-damaging abilities of photosensitizers can be estimated from their HOMO energies.     

PHOTOSENSITIZED FORMATION OF 8-HYDROXY-2'-DEOXYGUANOSINE AND DNA STRAND BREAKAGE BY A CATIONIC meso-SUBSTITUTED PORPHYRIN

Abstract Cationic porphyrins, known to have a high affinity for DNA, are useful tools with which to probe a variety of interactions with DNA. In this study we have examined both DNA strand scission and oxidative DNA base damage, measured by 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation, using a photoactivated cis-dicationic por-phyrin. The data demonstrated a dose-dependent formation for each type of DNA damage. Inhibition of strand scission and 8-OHdG formation with the singlet oxygen scavenger 1,3-diphenylisobenzofuran and with MgCl₂ and no apparent effect by D₂O suggests that a singlet oxygen mechanism generated in close proximity to the DNA may be responsible for the damage. However, a nearly complete inhibition of 8-hydroxy-2'-deoxyguanosine formation in 75% D₂O and the substantial enhancement of 8-hydroxy-2'-deoxyguanosine formation in a helium atmosphere by photoactivated porphyrin rules out singlet oxygen as a primary mechanism for this process. These data indicate that distinct mechanisms lead to 8-OHdG formation and strand scission activity.     

Photosensitized DNA damage and its protection via a novel mechanism

UVA, which accounts for approximately 95% of solar UV radiation, can cause mutations and skin cancer. Based mainly on the results of our study, this paper summarizes the mechanisms of UVA-induced DNA damage in the presence of various photosensitizers, and also proposes a new mechanism for its chemoprevention. UVA radiation induces DNA damage at the 5'-G of 5'-GG-3' sequence in double-stranded DNA through Type I mechanism, which involves electron transfer from guanine to activated photosensitizers. Endogenous sensitizers such as riboflavin and pterin derivatives and an exogenous sensitizer nalidixic acid mediate DNA photodamage via this mechanism. The major Type II mechanism involves the generation of singlet oxygen from photoactivated sensitizers, including hematoporphyrin and a fluoroquinolone antibacterial lomefloxacin, resulting in damage to guanines without preference for consecutive guanines. UVA also produces superoxide anion radical by an electron transfer from photoexcited sensitizers to oxygen (minor Type II mechanism), and DNA damage is induced by reactive species generated through the interaction of hydrogen peroxide with metal ions. The involvement of these mechanisms in UVA carcinogenesis is discussed. In addition, we found that xanthone derivatives inhibited DNA damage caused by photoexcited riboflavin via the quenching of its excited triplet state. It is thus considered that naturally occurring quenchers including xanthone derivatives may act as novel chemopreventive agents against photocarcinogenesis.     

Chemopreventive action of xanthone derivatives on photosensitized DNA damage

Photosensitized DNA damage participates in solar-UV carcinogenesis, photogenotoxicity and phototoxicity. A chemoprevention of photosensitized DNA damage is one of the most important methods for the above phototoxic effects. In this study, the chemopreventive action of xanthone (XAN) derivatives (bellidifolin [BEL], gentiacaulein [GEN], norswertianin [NOR] and swerchirin [SWE]) on DNA damage photosensitized by riboflavin was demonstrated using [32P]-5'-end-labeled DNA fragments obtained from genes relevant to human cancer. GEN and NOR effectively inhibited the formation of piperidine-labile products at consecutive G residues by photoexcited riboflavin, whereas BEL and SWE did not show significant inhibition of DNA damage. The four XAN derivatives decrease the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), an oxidative product of G, by photoexcited riboflavin. The preventive action for the 8-oxodGuo formation of these XAN derivatives increased in the following order: GEN>NOR>BEL>SWE. A fluorescence spectroscopic study and ab initio molecular orbital calculations suggested that the prevention of DNA photodamage is because of the quenching of the triplet excited state of riboflavin by XAN derivatives through electron transfer. This chemoprevention is based on neither antioxidation nor a physical sunscreen effect; rather, it is based on the quenching of a photosensitizer. In conclusion, XAN derivatives, especially GEN, may act as novel chemopreventive agents by the quenching mechanism of an excited photosensitizer.     

DNA cleavage induced by photoexcited antimalarial drugs: a photophysical and photobiological study

The interactions and the photosensitizing activity of three antimalarial drugs quinine (Q), mefloquine (MQ) and quinacrine (QC) toward DNA was studied. Evidences obtained by absorption and emission spectroscopy and by linear dichroism measurements indicate that these derivatives bind the macromolecule with a high affinity (binding constants Ka approximately 10(5) M(-1)). The absorption characteristics of the drugs changed markedly by addition of DNA and their fluorescence was quenched with rate constants higher than that of diffusion. The geometry of binding involves predominantly the intercalation into the double helix. The DNA photocleavage properties of antimalarials was investigated using plasmid DNA as a model, at different [drug]/ [DNA] ratios. The results indicate that mainly MQ and Q are able to induce significant photodamage to DNA. In particular the marked effect of the former drug is evidenced after treatment of photosensitized DNA by two base excision repair enzymes, formamydo-pyrimidine glycosilase (Fpg) and Endonuclease III (Endo III). From a mechanistic point of view, experiments carried out in different experimental conditions indicate that these drugs photoinduce DNA damage through singlet oxygen and/or radical cation production. These findings are further supported by the determination of two photoproducts of 2'-deoxyguanosine, which are diagnostic for Type I and Type II pathways, namely 2,2-diamino(2-deoxy-beta-D-erythro-pentofuranosyl)-4-amino-5(2H)-oxazolone and (R,S)4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine (4-OH-8-oxo-dGuo). Laser flash photolysis experiments carried out in the presence of DNA indicates that the excitation produces mainly the triplet state for Q and the triplet and radical cation for QC. Moreover the singlet and triplet states and radical cations of the drugs are quenched by 2'-deoxyguanosine monophosphate. The absorbances of these transients decrease with increasing DNA concentration.     

Sequence specificity of alkali-labile DNA damage photosensitized by suprofen

On irradiation at UVB wavelengths, in aerated neutral aqueous solution, the anti-inflammatory drug suprofen (SP) photosensitizes the production of alkali-labile cleavage sites in DNA much more efficiently than direct strand breaks. It is active at submillimolar concentrations despite having no significant binding affinity for DNA. Gel sequencing studies utilizing 32P-end-labeled oligonucleotides have revealed that piperidine-sensitive lesions are formed predominantly at the positions of guanine (G) bases, with the extent of modification being UV dose- and SP concentration-dependent. Quite distinct patterns of G-specific damage are observed in single-stranded and duplex DNA molecules. The uniform attack at all G residues in single-stranded DNA, which is enhanced in D2O, is compatible with a Type-II mechanism. SP is a known generator of singlet oxygen whose participation in the reaction is supported by the effects of quenchers and scavengers. In duplex DNA, piperidine-induced cleavage occurs with high selectivity at the 5'-G of GG and (less prominently) GA doublets. This behavior is characteristic of a Type-I process involving electron transfer from DNA to photoexcited SP molecules. The ability of SP to sensitize the formation of Type-I and Type-II photo-oxidation products from 2'-deoxyguanosine attests to the feasibility of competing mechanisms in DNA.     

Mechanism of DNA damage photosensitized by trisbipyrazyl ruthenium complex. Unusual role of Cu/Zn superoxide dismutase

Trisbipyrazyl ruthenium(II) (Ru[bpz]3(2+)) was examined as DNA photosensitizer. Damage resulting from the photolysis of synthetic oligonucleotides has been monitored by polyacrylamide gel electrophoresis. Photoadduct formation is found on both single- and double-stranded oligonucleotides. On oligonucleotide duplex, oxidative damage occurs selectively at the 5'G of the 5'GG3' site and to a lesser extent at the 5'G of a GA sequence. These findings suggest the involvement of electron transfer and show that this mechanism is the main DNA damaging process involved in Ru(bpz)3(2+) photosensitization. In addition, photoadducts and oxidative damage are both highly affected by an increase of salt concentration in the reaction medium, stressing the importance of direct interactions between nucleic acid bases and the excited ruthenium complex for efficient electron transfer. On single-stranded oligonucleotides, all the guanines are oxidized to the same extent. In this case, oxidative damage, which is not affected by an increase of salt in the solution, has been attributed, in part, to singlet oxygen. More importantly, Cu/Zn superoxide dismutase (SOD) strongly enhances the yield of all damage, correlated to an increase of both electron transfer and singlet oxygen production. This original activity of SOD is the first example of bioactivation of a polyazaaromatic ruthenium complex.     

PHTHALOCYANINE AND NAPHTHALOCYANINE PHOTOSENSITIZED OXIDATION OF 2'-DEOXYGUANOSINE: DISTINCT TYPE I AND TYPE II PRODUCTS

Abstract
The photodynamic properties of the di-and tetrasulfonated zinc and aluminium phthalocyanines and a tetrasulfonated aluminium napththalocyanine were studied using 2'-deoxyguanosine as a DNA model compound. The major photooxidation products of this nucleoside were identified and classified according to their formation through a radical mechanism (type I) or a singlet oxygen mediated mechanism (type II). The major type I product was obtained and identified as 2,2-diamino [(2-deoxy-β- d - erythro pentofuranosyl)-4-amino]-5( 2H )-oxazolone. Two major type II products were characterized as the 4R* and 4S* diastereomers of 9-(2-deoxy-β- d - erythro pentofuranosyl)-7,8-dihydro-4-hydroxy-8-oxoguanine. In addition a third product, also resulting from a type II photooxidation, was identified as 8-oxo-7,8-dihydro-2'-deoxyguanosine. Quantification of these products provided a means to estimate the contribution of type I and type II pathways during the phthalocyanine and naphthalocyanine mediated photooxidation of 2'-deoxyguanosine, confirming the major role of singlet oxygen in these processes.     

Oxidative DNA damage induced by autoxidation of microquantities of S(IV) in the presence of Ni(II)-Gly-Gly-His

NiIIGGH (GGH = glycylglycylhistidine) reacts rapidly with S(IV), in air-saturated solution, to produce NiIIIGGH. A mechanism is proposed where initial NiIII oxidizes SO3(2-) to SO3*-, which reacts with dissolved oxygen to produce SO5*-, initiating radical chain reactions. DNA strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine formation were observed in air-saturated solutions containing micromolar concentrations of Ni(II) and S(IV). The extent of DNA damage showed dependence on the ratio of the NiIIGGH : S(IV) concentrations and the ionic strength.     

Comparison of DNA damage photoinduced by ketoprofen, fenofibric acid and benzophenone via electron and energy transfer.

Ketoprofen (KP) and fenofibrate, respectively, anti-inflammatory and hypolipidemiant agents, promote anormal photosensitivity in patients and may induce photoallergic cross-reactions correlated to their benzophenone-like structure. Here, their ability to photosensitize the degradation of biological targets was particularly investigated in DNA. The photosensitization of DNA damage by KP and fenofibric acid (FB), the main metabolite of fenofibrate, and their parent compound, benzophenone (BZ), was examined on a 32P-end-labeled synthetic oligonucleotide in phosphate-buffered solution using gel sequencing experiments. Upon irradiation at lambda > 320 nm, piperidine-sensitive lesions were induced in single-stranded oligonucleotides by KP, FB and BZ at all G sites to the same extent. This pattern of damage, enhanced in D2O is characteristic of a Type-II mechanism. Spin trapping experiments using 2,2,6,6-tetramethyl-4-piperidone have confirmed the production of singlet oxygen during drug photolysis. On double-stranded oligonucleotides, highly specific DNA break occurred selectively at 5'-G of a 5'-GG-3' sequence, after alkali treatment. Prolonged irradiation led to the degradation of all G residues, with efficiency decreasing in the order 5'-GG > 5'-GA > 5'-GC > 5'-GT, in good agreement with the calculated lowest ionization potentials of stacked nucleobase models supporting the assumption of a Type-I mechanism involving electron transfer, also observed to a lesser extent with adenine. Cytosine sites were also affected but the action of mannitol which selectively inhibited cytosine lesions suggests, in this case, the involvement of hydroxyl radical, also detected by electronic paramagnetic resonance using 5,5-dimethyl-1-pyrrolidine-1-oxide as spin trap. On a double-stranded 32P-end-labeled 25-mer oligonucleotide containing TT and TTT sequences, the three compounds were found to photosensitize by triplet-triplet energy transfer the formation of cyclobutane thymine dimers detected using T4 endonuclease V.     

DNA Damage and Apoptosis Induced by Photosensitization of 5,10,15,20-Tetrakis (N-methyl-4-pyridyl)-21H,23H-porphyrin via Singlet Oxygen Generation

Cancer photodynamic therapy (PDT) requires photosensitizers that efficiently and selectively destroy tumor cells. We investigated 5,10,15,20-tetrakis ( N -methyl-4-pyridyl)-21 H ,23 H -porphyrin (TMPyP) as a potential cancer treatment. Confocal fluorescence microscopy showed that TMPyP was localized in the nuclei, whereas 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PPIX) was localized diffusely in the cytoplasm of human leukemia (HL-60) cells. In HL-60 cells under UVA irradiation, TMPyP effectively induced apoptosis. Moreover, 8-oxo-7,8-dihydro-2'-deoxyguanosine, an oxidative product of 2'-deoxyguanosine, was accumulated in the DNA of cells treated with photoirradiated TMPyP, whereas only small amounts were observed in ALA-treated cells in the presence of UVA light. TMPyP and UVA caused extensive damage at every guanine residue in DNA fragments obtained from the human p 53 tumor suppressor gene and the c-Ha- ras -1 proto-oncogene, whereas PPIX induced little DNA damage under these conditions. Electron spin resonance spectroscopy using a singlet oxygen (¹O₂) probe and D₂O showed that photoexcited TMPyP generated ¹O₂. These results suggest that photoexcited TMPyP reacts with oxygen to generate ¹O₂, which in turn, oxidizes guanine residues. Taken together, the results demonstrated that TMPyP was localized in the nucleus where it was photosensitized to induce DNA damage, suggesting that TMPyP may have clinical utility as a nucleus-targeted PDT.     

PHTHALOCYANINE AND NAPHTHALOCYANINE PHOTOSENSITIZED OXIDATION OF 2'-DEOXYGUANOSINE

Abstract— The photodynamic properties of the di-and tetrasulfonated zinc and aluminium phthalocyanines and a tetrasulfonated aluminium napththalocyanine were studied using 2'-deoxyguanosine as a DNA model compound. The major photooxidation products of this nucleoside were identified and classified according to their formation through a radical mechanism (type I) or a singlet oxygen mediated mechanism (type II). The major type I product was obtained and identified as 2,2-diamino [(2-deoxy-β- d - erythro pentofuranosyl)-4-amino]-5( 2H )-oxazolone. Two major type II products were characterized as the 4R* and 4S* diastereomers of 9-(2-deoxy-β- d - erythro pentofuranosyl)-7,8-dihydro-4-hydroxy-8-oxoguanine. In addition a third product, also resulting from a type II photooxidation, was identified as 8-oxo-7,8-dihydro-2'-deoxyguanosine. Quantification of these products provided a means to estimate the contribution of type I and type II pathways during the phthalocyanine and naphthalocyanine mediated photooxidation of 2'-deoxyguanosine, confirming the major role of singlet oxygen in these processes.     

Supramolecular Cationic Tetraruthenated Porphyrin Induces Single-Strand Breaks and 8-Oxo-7,8-dihydro-2'-deoxyguanosine Formation in DNA in the Presence of Light

Abstract— The aim of this investigation is the evaluation of DNA interaction of with tetraruthenated porphyrin (TRP) and of DNA damage in the presence of light. Direct-fluorescence and electronic absorption measurements after incubation of DNA with TRP indicate strong binding between pBR322 DNA or calf thymus DNA with the modified porphyrin. Exposure of pBR322 DNA to TRP (up to 3 μ M ) and light leads to single-strand break formation as determined by the conversion of the supercoiled form (form I) of the plasmid into the nicked circular form (form II). Oxidative DNA base damage was evaluated by the detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after irradiation of calf thymus DNA in the presence of the TRP. The data demonstrated a dose and time dependence with each type of DNA damage. These data indicate (1) a specificity of the binding mode and (2) type I and II photoinduced mechanisms leading to strand scission activity and 8-oxodGuo formation. Accordingly, singlet molecular oxygen formation, after TRP excitation, was confirmed by near-infrared emission. From these investigations a potential application of TRP in photodynamic therapy is proposed.     

Mechanisms of photosensitized DNA cleavage

Photosensitization may promote DNA damages such as nucleic acid oxidation or single strand breaks via three main pathways: hydroxyl radicals attack, electron transfer process or oxidation by singlet oxygen. While direct production of OH^. by photosensitization is rarely observed, the mechanism of DNA attack by OH^. is now well established on the basis of informations provided by water radiolysis experiments. Some dyes may also induce single strand breaks via an electron transfer occurring from a nucleobase to the sensitizer in the excited state. This process generates base radical cations identical to those arising from DNA photoionisation. These radicals may undergo deprotonation or dehydration to form the same neutral radicals as those produced by OH^. but with a slightly different pattern. In contrast, while many sensitizers produce singlet oxygen, the mechanism of DNA damages induced by this way is still unclear. In this case the guanine moiety in nucleosides or in DNA is selectively altered leading to the formation of 8 oxoG or 8 oxodG and FapyGua. The mechanism of single strand breaks formation by singlet oxygen is discussed in this overview.     

Analysis of fluoroquinolone-mediated photosensitization of 2'-deoxyguanosine, calf thymus and cellular DNA: determination of type-I, type-II and triplet-triplet energy transfer mechanism contribution

Fluoroquinolone (FQ) antibacterials are known to exhibit photosensitization properties leading to the formation of oxidative damage to DNA. In addition, photoexcited lomefloxacin (Lome) was recently shown to induce the formation of cyclobutane pyrimidine dimers via triplet-triplet energy transfer. The present study is aimed at gaining further insights into the photosensitization mechanisms of several FQ including enoxacin (Enox), Lome, norfloxacin (Norflo) and ofloxacin (Oflo). This was achieved by monitoring the formation of DNA base degradation products upon UVA-mediated photosensitization of 2'-deoxyguanosine, isolated and cellular DNA. Oflo and Norflo act mainly via a Type-II mechanism whereas Lome and, to a lesser extent, Enox behave more like Type-I photosensitizers. However, the extent of oxidative damage was found to be relatively low. In contrast, it was found that cyclobutane thymine dimers represent the major class of damage induced by Enox, Lome and Norflo within isolated and cellular DNA upon UVA irradiation. This striking observation confirms that FQ are able to promote efficient triplet energy transfer to DNA. The levels of photosensitized formation of strand breaks, alkali-labile sites and oxidative damage to cellular DNA, as measured by the comet assay, were confirmed to be rather low. Therefore, we propose that the phototoxic effects of FQ are mostly accounted for energy transfer mechanism rather than by Type-I or -II photosensitization processes.     

Pyrimidine dimer formation and oxidative damage in M13 bacteriophage inactivation by ultraviolet C irradiation

The mechanism by which UV-C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South-Western blotting for pyrimidine dimers. The involvement of singlet oxygen (1O2) was also investigated using azide and deuterium oxide and under deoxygenated conditions. With a decrease in M13 infectivity on irradiation, single-stranded circular genomic DNA (sc-DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc-DNA and linear-form DNA. However, the amount of sc-DNA remaining was not correlated with the survival of M13. The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts ((6-4)PP) increased as a function of irradiation dose. The decrease in M13 infectivity was highly correlated with the increase in CPD and (6-4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 8-Oxo-7,8-dihydro-2'-deoxyguanosine did not form in the M13 genome after UV-C irradiation. Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide. Deoxygenation of the M13 suspension did not affect the inactivation, indicating that 1O2 did not participate in the inactivation of M13 by UV-C irradiation under these conditions. These results indicated that UV-C irradiation induced not only CPD and (6-4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity. The indirect effect of UV-C irradiation such as 1O2 production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV-C.     

A comparative photomechanistic study (spin trapping, EPR spectroscopy, transient kinetics, photoproducts) of nucleoside oxidation (dG and 8-oxodG) by triplet-excited acetophenones and by the radicals generated from alpha-oxy-substituted derivatives through Norrish-type I cleavage

The photooxidation of 2'-deoxyguanosine (dG) and its derivative 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) by a series of acetophenones (AP-X) and benzophenone (BP) has been studied.The favorable absorption characteristics of the benzoyl chromophore enables time-resolved spectroscopy of the triplet ketones to assess their quenching kinetics by dG and 8-oxodG. Whereas the photolysis of acetophenone (AP), 2-acetoxyacetophenone (AP-OAc), and benzophenone (BP) does not produce radicals (group A ketones), the oxymethyl-substituted derivatives 2-hydroxyacetophenone (AP-OH) and 2-tert-butoxyacetophenone (AP-O(t)Bu) lead to carbon-centered radicals by alpha cleavage (group B ketones). For the latter ketones, this was confirmed by EPR studies with the spin trap 5,5-dimethylpyrroline N-oxide (DMPO) and by their triplet lifetimes that were shorter than those for the unsubstituted acetophenone. Both groups of ketones photooxidize dG and 8-oxodG; the oxidation products are spiroiminodihydantoin and guanidine-releasing products (GRP) in the case of dG and AP-OH also 8-oxodG. In the presence of O(2), the photooxidation by the group A ketones is efficient at high dG or 8-oxodG concentrations, whereas the group B ketones photooxidize dG and 8-oxodG also at low substrate concentrations. These results imply that peroxyl radicals are responsible for the photooxidation by the group B ketones, which are formed by alpha cleavage of the triplet ketone and subsequent O(2) trapping of the carbon-centered radicals. At higher dG concentrations, direct electron transfer from dG to the triplet ketone, as observed for the group A ketones, competes with the radical activity.     

An improved liquid chromatography/tandem mass spectrometry method for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA samples using immunoaffinity column purification

The analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents an important biomarker of oxidative stress. A sensitive method for the detection of 8-oxodG in DNA samples has been developed that utilizes immunoaffinity column purification of 8-oxodG followed by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) multiple reaction monitoring (MRM) mode analysis. An internal standard of stable-isotopically labelled 8-oxodG containing [(15)N(5)] was added prior to the enzymatic digestion of DNA to deoxynucleosides, which was then subjected to immunoaffinity column purification followed by microbore positive ion LC/MS/MS MRM. The 8-oxo-7,8-dihydroguanine (8-oxoG) base product ion at m/z 168 was monitored following cleavage of the glycosidic bond of the 8-oxodG [M+H](+) ion at m/z 284. Similar determinations were made for [(15)N(5)]8-oxodG by monitoring the [(15)N(5)]8-oxoG base product ion at m/z 173 formed from the [M+H](+) ion at m/z 289. The introduction of the immunoaffinity column purification step into the method represents a significant improvement for the accurate determination of 8-oxodG since all artefactual peaks that are observed following the direct injection of digested DNA onto the LC/MS/MS system are removed. The identity of these artefactual peaks has been confirmed to be 2'-deoxyguanosine (dG), thymidine (dT) and 2'-deoxyadenosine (dA). The presence of these artefactual peaks in MRM mode analysis can be explained as a consequence of a concentration effect due to their considerably higher relative abundance in DNA compared to 8-oxodG. The highest signal intensity was observed for the artefactual peak for dA due to the fact that the adenine base formed an adduct with methanol, which is a constituent of the mobile phase. The resulting [M+H](+) ion at m/z 284 (dA m/z 252 + CH(3)OH m/z 32) gave rise to a product ion at m/z 168 following the loss of deoxyribose in MRM mode analysis. Control calf thymus DNA was digested to deoxynucleosides and unmodfied deoxynucleosides were removed by immunoaffinity column purification; the enriched 8-oxodG was determined by LC/MS/MS MRM. The level of 8-oxodG in control calf thymus DNA was determined to be 28.8 +/- 1.2 8-oxodG per 10(6) unmodified nucleotides (n = 5) using 5 microg of digested DNA. The limit of detection of the microbore LC/MS/MS MRM for 8-oxodG was determined to be 25 fmol on-column with a signal-to-noise ratio of 3.5.