Jean-Luc Ravanat

Light-Induced Acclimation of the Arabidopsis chlorina1 Mutant to Singlet Oxygen

This work shows that, similarly to green algae, vascular plants can acclimate to singlet oxygen (1O2). The Arabidopsis ch1 mutant is highly photosensitive due to increased release of 1O2 by photosystem II, but preexposure of ch1 plants to moderately elevated light intensities eliminated oxidative damage in high light without suppressing 1O2 formation. Regulation of the jasmonate biosynthesis pathway is a key factor in this acclimation process. Singlet oxygen (1O2) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, 1O2-induced transcriptomic changes result in acclimation to 1O2. Here, using a chlorophyll b–less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of 1O2 by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to 1O2 reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing 1O2 formation, indicating acclimation to 1O2. Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in 1O2-induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonate-treated ch1 plants and by the increased tolerance of the jasmonate-deficient mutant delayed-dehiscence2.

Inter-laboratory validation of procedures for measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2 '-deoxyguanosine in DNA

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CV of less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10 6 Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.

Singlet oxygen-mediated damage to cellular DNA determined by the comet assay associated with DNA repair enzymes.

Abstract The damage profile produced by the reaction of singlet molecular oxygen with cellular DNA was determined using the comet assay associated with DNA repair enzymes. Singlet oxygen was produced intracellularly by thermal decomposition of a watersoluble endoperoxide of a naphthalene derivative which is able to penetrate through the membrane into mammalian cells. We found that the DNA modifications produced by singlet oxygen were almost exclusively oxidised purines recognised by the formamidopyrimidine DNA N-glycosylase. In contrast, significant amounts of direct strand breaks and alkalilabile sites or oxidised pyrimidines, detectable by the bacterial endonuclease III, were not produced.

Minor contribution of direct ionization to DNA base damage inducedby heavy ions

Purpose: The deleterious processes triggered by heavy ions on DNA were studied through the determination of the yield of a series of oxidized bases. Emphasis was placed on the estimation of the respective contribution of direct ionization and indirect effects, mostly by comparison with low linear energy transfer (LET) γ-rays. Material and Methods: DNA samples and human monocytes were exposed either to γ-rays emitted by a 60Co source or to 12C6+ or 36Ar18+ ions. The levels of thymidine and 2′-deoxyguanosine oxidation products were determined by liquid chromatography coupled to tandem mass spectrometry subsequently to DNA digestion into nucleosides. Results: The yields of thymidine lesions were similar to those of 8-oxo-7,8-dihydro-2′-deoxyguanosine within isolated DNA exposed either to γ-rays or argon ions. Addition of spermine and Tris aimed at minimizing the indirect effect modified this ratio to the same extent with both types of radiation. In cells, the level of radiation-induced base damage was found to be correlated with the radiolytic yield of °OH that depends on the LET of the particle. In addition, radiation-induced thymidine and 2′-deoxyguanosine lesions were produced in similar amounts. In contrast, oxidation of 2′-deoxyguanosine was the main process when ionization was triggered in cellular DNA by ultraviolet laser-induced biphotonic processes. Conclusions: Predominant oxidation of 2′-deoxyguanosine is expected to be the hallmark of direct DNA ionization. The observation that thymidine and 2′-deoxyguanosine are equally damaged rules out a major contribution of the direct ionization in radiation-induced base damage to both isolated and cellular DNA by heavy ions. Dependence of the yield of lesions on the LET provides further support for this conclusion.

[18O]-labeled singlet oxygen as a tool for mechanistic studies of 8-oxo-7,8-dihydroguanine oxidative damage: detection of spiroiminodihydantoin, imidazolone and oxazolone derivatives.

Abstract A watersoluble [18O]labeled endoperoxide derived from N,Ndi(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide (DHPN18O2) has been shown to act as a clean chemical source of [18O]labeled molecular singlet oxygen. This allows the assessment of the singlet oxygen (1O2) reactivity toward biological targets such as DNA. The present work focuses on the qualitative identification of the main 1O2-oxidation products of 8-oxo-7,8-dihydro-2deoxyguanosine, which was achieved using high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLCESIMS/MS). Thus, the [18O]labeled and unlabeled imidazolone and oxazolone, together with the diastereoisomeric spiroiminodihydantoin nucleosides, were detected as the main degradation products. In addition, a modified nucleoside that exhibits similar features as those of the oxidized guanidinohydantoin molecule was detected. Our data strongly suggest that the imidazolone and oxazolone nucleosides are generated via the rearrangement of an unstable 5-hydroperoxide intermediate. Interestingly, the combined use of appropriate tools, including isotopically labeled singlet oxygen and the high resolution HPLCESIMS/MS technique, has allowed to shed new light on the 1O2- mediated oxidation reactions of guanine DNA components.

UVB and UVA radiation-mediated damage to isolated and cellular DNA*

The effects of solar light on cellular DNA are mostly explained by both direct excitation of nucleobases and photosensitized reactions that are mediated by UVB and UVA radiation, respectively. A large body of information is now available on the main photodynamic reactions to DNA, which involve guanine as the preferential target of both one-electron oxidation and singlet oxygen oxidation, as the result of type I and type II mechanisms. Most of the final products of the photosensitized reactions of guanine base in isolated DNA have been characterized, and comprehensive mechanisms for their formation have been proposed. Further insights into the mechanisms of solar radiation-induced modifications within cellular DNA have been gained from accurate measurement of the main photoproducts using recently designed sensitive chromatographic and biochemical methods. Thus, the distribution pattern of the 12 possible bipyrimidine photoproducts has been shown to be similar in the DNA of UVB-irradiated rodent and human cells. Cyclobutyl pyrimidine dimers are also generated at di-thymine and thymine-cytosine sites within nuclear DNA upon exposure to UVA radiation as the likely result of triplet energy transfer. In addition, oxidative reactions that involved mostly singlet oxygen, and to a lesser extent, .OH radicals are also implicated, although less efficiently.

A microarray to measure repair of damaged plasmids by cell lysates

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.

Urinary levels of oxidative DNA and RNA damage among workers exposed to polycyclic aromatic hydrocarbons in silicon production: Comparison with 1-hydroxypyrene

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous occupational and environmental pollutants and the urinary excretion of 1‐hydroxypyrene (1‐OHP) is classically measured for the determination of PAH exposure internal dose. Some of PAH are tumorigenic due to their metabolites ability to generate DNA adducts and oxidative DNA damage through the production of reactive oxygen species during metabolism. 8‐hydroxy‐7,8‐dihydro‐2′‐deoxyguanosine (8‐OHdGuo) is one of the major oxidative DNA lesions and its use as a potential biomarker of genotoxic PAH occupational exposure should be evaluated. Indeed conflicting results are frequently reported in occupational studies in terms of correlation between 8‐OHdGuo urinary levels and PAH exposure. The aim of our study was therefore to determine the potential for PAH occupational exposure to increase urinary oxidative DNA damage. The population consisted of 68 male workers employed in silicon production. The urinary concentrations of 8‐OHdGuo and its homologue in RNA, 8‐hydroxy‐7,8‐dihydroguanosine (8‐OHGuo) were determined using high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry, whereas those of 1‐OHP were measured using HPLC with fluorescence detection. Individual variation rates were calculated on a working day and a working week. The results indicated that, while 1‐OHP levels strongly increased on a working day and even more on a working week, 8‐OHdGuo and 8‐OHGuo urinary levels did not show similar significant increases. Moreover, no correlation between 1‐OHP and oxidative DNA and RNA lesions was found. Consequently, urinary 8‐OHdGuo and 8‐OHGuo did not seem to be relevant biomarkers of genotoxic PAH exposure in the case of the silicon plant studied. Environ. Mol. Mutagen., 2009.

Oxidatively Generated Damage to Cellular DNA: Mechanistic Aspects

In this chapter emphasis is placed on recent aspects of the oxidative formation of several classes of modified bases in cellular DNA that arise from the reaction of the hydroxyl radical (•OH), singlet oxygen and hypochlorous acid. Degradation compounds are detected quantitatively and specifically after suitable DNA hydrolysis into either nucleosides or bases by HPLC-tandem mass spectrometry. Thus, 6 oxidized nucleosides including: the four cis and trans diastereomers of 5,6-dihydroxy-5,6-dihydrothymidine, 5-(hydroxymethyl)-2′-deoxyuridine and 5-formyl-2′-deoxyuridine are found to be formed as the result of •OH radical mediated oxidation of thymidine. In addition, γ-irradiation of cellular DNA was found to generate 8-oxo-7,8-dihydropurine derivatives and related formamidopyrimidine compounds resulting from •OH radical oxidation of the guanine and adenine bases. Furthermore, singlet oxygen oxidation of guanine was found to give rise exclusively to 8-oxo-7,8-dihydro-2′-deoxyguanosine while HOCl reaction with cytosine, adenine and guanine led to the formation of 5-chlorocytosine, 8-chloroadenine and 8-chloroguanine nucleosides respectively in the DNA and RNA of human white blood cells. Interestingly, formation of these various degradation products has been rationalized in terms of existing mechanisms that were proposed previously from model studies, mostly involving free nucleosides.

Chapter 20:Reactions of Singlet Oxygen with Nucleic Acids

The chapter is aimed at providing an updated overview of the main available data on the oxidation of nucleic acids by singlet oxygen (1O2). Mechanistic insights into the selective oxidative pathways of guanine, the predominant DNA and RNA target, were gained from detailed model studies. These have also allowed identification of spiroiminodihydantoin and minor 8-oxo-7,8-dihydroguanine as the main decomposition products of nucleosides and nucleotides. Evidence has been provided that the latter guanine oxidation product and thionucleobases are also highly reactive to singlet oxygen. The 1O2 oxidation of isolated and cellular DNA and RNA is much more specific giving rise almost exclusively to 8-oxo-7,8-dihydroguanine though the intermediacy of 4,8-endoperoxide that subsequently rearranges into easily reducible 8-hydroperoxyguanine. It was also shown that 1O2 is not able to induce significant levels of strand breaks and/or alkali-labile sites in cellular DNA upon exposure to a chemical source of singlet oxygen. UVA irradiation of cells and human skin is able to oxidatively damage nuclear DNA as inferred from the measurement of 8-oxo-7,8-dihydroguanine. This has been rationalized in terms of the predominant implication of 1O2 produced by type-II photosensitization mechanism over Fenton-type reactions.