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.

Predominance of the 1,N2-propano 2'-deoxyguanosine adduct among 4-hydroxy-2-nonenal-induced DNA lesions.

4-Hydroxy-2-nonenal (HNE), one of the main aldehydic compounds released during lipid peroxidation, has been proposed to react with DNA bases in cells. Several classes of DNA lesions involving addition of either HNE or its 2,3-epoxide (epox-HNE) have been identified. In the present work, HPLC associated with tandem mass spectrometry was used to determine the pattern of HNE-induced DNA lesions.

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.

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).

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.

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.

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).

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.

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.

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.