DNA repair capacities of cutaneous fibroblasts: effect of sun exposure, age and smoking on response to an acute oxidative stress.

BACKGROUND: Sun irradiation causes skin ageing and cancer through the accumulation of damage to cell components. Intrinsic ageing is also associated with accumulation of oxidized macromolecules. OBJECTIVES: In this study we investigated the effects of sun exposure on response to an acute in vitro oxidative stress (H(2)O(2)) using normal human fibroblasts prepared from biopsies from 10 volunteers taken from sun-protected and sun-exposed sites. METHODS: Time-course experiments measuring repair of DNA strand-breaks and formamidopyrimidine DNA N-glycosylase-sensitive sites were conducted using the single-cell gel electrophoresis (comet) assay. RESULTS: Our results demonstrated that repair of strand-breaks was slower in sun-exposed compared with sun-protected cells. Interestingly, ageing was also associated with decreased DNA repair capacities for single-strand breaks in both sun-exposed and sun-protected cells whereas for formamidopyrimidine glycosylase (Fpg)-sensitive sites, this feature was in evidence only in sun-protected cells. Smoking, associated with age, was shown to have a markedly negative impact on DNA repair. CONCLUSIONS: Taken together our data suggest that stresses like ageing, sun exposure and smoking might have an additive effect contributing to the overall heterogeneity and decrease of DNA repair capacities in human cells and so increase the danger of sun exposure for health. They also emphasize the importance of the quality of the biological samples when repair studies on skin cells are to be conducted.

Formation of modified DNA bases in cells exposed either to gamma radiation or to high-LET particles.

The aim of the present study was to measure the formation of eight base modifications in the DNA of cells exposed to either low-LET ((60)Co gamma rays) or high-LET ((12)C(6+) particles) radiation. For this purpose, a recently optimized HPLC-MS/MS method was used subsequent to DNA extraction and hydrolysis. The background level of the measured modified bases and nucleosides was shown to vary between 0.2 and 2 lesions/10(6) bases. Interestingly, thymidine glycols constitute the main radiation-induced base modifications, with an overall yield of 0.097 and 0.062 lesion/10(6) bases per gray for gamma rays and carbon heavy ions, respectively. Both types of radiations generate four other major degradation products, in the following order of decreasing importance: FapyGua > 5-HmdUrd > 5-FordUrd > 8-oxodGuo. The yields of formation of FapyAde and 8-oxoAde are one order of magnitude lower than those of the related guanine modifications, whereas the radiation-induced generation of 5-OHdUrd was below the limit of detection of the assay. The efficiency for both types of radiation to generate base damage in cellular DNA is low because the highest yield per gray was 0.097 thymine glycols per 10(6) DNA bases. As a striking observation, the yield of formation of the measured DNA lesions was found to be, on average, twofold lower after exposure to high-LET radiation ((12)C(6+)) than after exposure to low-LET gamma radiation. These studies show that the HPLC-MS/MS assay provides an accurate, reliable and sensitive method for measuring cellular DNA base damage.

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.

Formation of the main UV-induced thymine dimeric lesions within isolated and cellular DNA as measured by high performance liquid chromatography-tandem mass spectrometry.

UVB radiation-induced formation of dimeric photoproducts at bipyrimidine sites within DNA has been unambiguously associated with the lethal and mutagenic properties of sunlight. The main lesions include the cyclobutane pyrimidine dimers and the pyrimidine (6-4) pyrimidone adducts. The latter compounds have been shown in model systems to be converted into their Dewar valence isomers upon exposure to UVB light. A new direct assay, based on the use of liquid chromatography coupled to tandem mass spectrometry, is now available to simultaneously detect each of the thymine photoproducts. It was applied to the determination of the yields of formation of the thymine lesions within both isolated and cellular DNA exposed to either UVC or UVB radiation. The cis-syn cyclobutane thymine dimer was found to be the major photoproduct within cellular DNA, whereas the related (6-4) adduct was produced in an approximately 8-fold lower yield. Interestingly, the corresponding Dewar valence isomer could not be detected upon exposure of human cells to biologically relevant doses of UVB radiation.

Effects of heavy ions on nucleic acids: measurement of the damage.

In this short survey the main, available information on the molecular mechanisms of action of heavy ions on DNA is critically reviewed. Formation of single- and double-stranded DNA breaks in cells exposed to heavy particles is well established. On the other hand, base damage and, in a more general way, clustered lesions, whose formation should be increased upon exposure to heavy ions, have not yet been isolated and characterized. Efforts should be made to identify this important class of DNA damage in both isolated and cellular DNA. Sensitive and specific assays involving chemical and biochemical approaches have to be developed for such a purpose.

Simple chromatographic systems permitting both DNA purification and separation of 2'-deoxyribonucleoside 3'-monophosphates as substrates for 32P-postlabelling studies.

The 32P-postlabelling method has recently been applied to the measurement of oxidative DNA damage. The assay requires the isolation of 2'-deoxyribonucleoside 3'-monophosphates subsequent to the extraction of DNA followed by its enzymatic digestion. As an alternative to the use of toxic and oxidizing solvents such as phenol, a simple purification method is proposed, based mainly on size-exclusion chromatography carried out either with ready-made columns (NAP-10, SEC-2000) or, more conveniently, with stainless-steel laboratory-packed columns (Fractogel HW 65 F). This method was applied to the purification of the DNA extracted from seeds of Lactuca sativa. After enzymatic digestion of DNA, the 2'-deoxyribonucleoside 3'-monophosphates may be further separated in less than 30 min by high-performance liquid chromatography on a Hypersil octadecylsilylsilica gel column in the ion-suppression mode by using either ammonium formate (0.05 M, pH 6.5) or sodium succinate (0.02 M, pH 6.0). The use of these eluent systems is compatible with straightforward 32P-labelling of the 2'-deoxyribonucleoside 3'-monophosphates without any concentration and desalting steps.

Measurement of oxidative base damage to DNA by using HPLC-32P-postlabelling and GC/MS-selective ion monitoring assays.

A 32P-postlabelling assay has been developed for singling out specific oxidized base lesions. Emphasis was placed on the quantitative aspect and the accuracy of the assay, which require the use of calibration curves and microreactions, respectively. The method was successfully applied to the detection and the measurement of adenine N1-oxide and 5-hydroxymethyluracil in cells exposed to agents inducing oxidative stress including H2O2 and UV-A radiation. The sensitivity of the assay allows the detection of one lesion in 10(6) normal bases in 1 microgram of DNA. The GC/MS method when coupled to the selective ion monitoring (SIM) technique is about twenty times less sensitive, even for suitable substrates such as 5-hydroxymethyluracil and 5-hydroxyuracil, than the 32P-postlabelling assay. However, the former assay is much easier to apply, even though a derivatization step is necessary, and provides unambiguous structural information on the compound to be measured. Accurate quantitative measurements can be obtained when stable, isotopically labelled standards are available.

Chemical and biochemical postlabeling methods for singling out specific oxidative DNA lesions.

A survey of the main available chemical and biochemical postlabeling assays for measuring oxidative DNA damage is reported. Two main approaches, radio and fluorescent postlabeling, have been used in order to reach a high level of sensitivity of detection. This is required for the measurement of DNA damage within cells and tissues upon exposure to agents of oxidative stress. Most of the methods are based on liquid chromatographic separation of defined DNA modifications following either acidic hydrolysis or enzymic digestion of DNA. In a subsequent step, the isolated base or sugar damages are either radiolabeled or made fluorescent by chemical or enzymatic reactions. Emphasis is placed on the recently developed high performance liquid chromatographic 32P-postlabeling assay, which allows the specific and sensitive measurement of various base damages including adenine N-1 oxide and 5-hydroxymethyluracil at the level of one modification per 10(7) normal bases in a sample size of 1 microgram of DNA. Examples of application of radioactive postlabeling to the measurement of DNA base damage following exposure of human cells to oxidizing agents including hydrogen peroxide and UVA radiation are provided.

32P-postlabeling measurement of adenine N-1-oxide in cellular DNA exposed to hydrogen peroxide.

A 32P-postlabeling assay has been developed for monitoring the formation within DNA of adenine N-1-oxide, the specific H2O2-mediated oxidation product under nonradical conditions. This has required the chemical synthesis of both 2'-deoxyadenosine N-1-oxide 3'-monophosphate and 2'-deoxyadenosine N-1-oxide 5'-monophosphate, the substrate and the product of polynucleotide kinase mediated phosphorylation. Isolation of the substrate from the other nucleotides was found to be necessary in order to improve the rate of phosphorylation and to prevent self-radiolysis processes. [32P]-2'-deoxyadenosine N-1-oxide 5'-monophosphate was obtained after successive ion exchange and reverse-phase HPLC and was characterized by a microreaction. The sensitivity of the assay, which is close to 1 modified adenine N-1-oxide/10(6) bases, allowed the determination of this lesion within the DNA of cells exposed to nonlethal levels of H2O2.