Sylvain Caillat

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¶

Abstract 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.

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.

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.

Direct inhibition of excision/synthesis DNA repair activities by cadmium: Analysis on dedicated biochips

The well established toxicity of cadmium and cadmium compounds results from their additive effects on several key cellular processes, including DNA repair. Mammalian cells have evolved several biochemical pathways to repair DNA lesions and maintain genomic integrity. By interfering with the homeostasis of redox metals and antioxidant systems, cadmium promotes the development of an intracellular environment that results in oxidative DNA damage which can be mutagenic if unrepaired. Small base lesions are recognised by specialized glycosylases and excised from the DNA molecule. The resulting abasic sites are incised, and the correct sequences restored by DNA polymerases using the opposite strands as template. Bulky lesions are recognised by a different set of proteins and excised from DNA as part of an oligonucleotide. As in base repair, the resulting gaps are filled by DNA polymerases using the opposite strands as template. Thus, these two repair pathways consist in excision of the lesion followed by DNA synthesis. In this study, we analysed in vitro the direct effects of cadmium exposure on the functionality of base and nucleotide DNA repair pathways. To this end, we used recently described dedicated microarrays that allow the parallel monitoring in cell extracts of the repair activities directed against several model base and/or nucleotide lesions. Both base and nucleotide excision/repair pathways are inhibited by CdCl₂, with different sensitivities. The inhibitory effects of cadmium affect mainly the recognition and excision stages of these processes. Furthermore, our data indicate that the repair activities directed against different damaged bases also exhibit distinct sensitivities, and the direct comparison of cadmium effects on the excision of uracile in different sequences even allows us to propose a hierarchy of cadmium sensibility within the glycosylases removing U from DNA. These results indicate that, in our experimental conditions, cadmium is a very potent DNA repair poison.

Effect of Aging on DNA Excision/Synthesis Repair Capacities of Human Skin Fibroblasts

TO THE EDITOR A key factor in the skin aging process is the cumulative effects of chronological aging and environmental-based assaults. Endogenous cellular oxidative processes generate reactive oxygen species and reactive polyunsaturated fatty acid derivatives (Lindahl, 1993; Marnett and Plastaras, 2001). These attacks on DNA cause substantial base and sugar damage, and the persistence of such lesions leads to mutations and genome instability. Skin may also suffer from chronic exposure to sun; UV radiation causes oxidative DNA damage and induces photoproducts (mainly cyclobutane pyrimidine dimers and 6-4 photoproducts (Moriwaki and Takahashi, 2008)). Age-related accumulation of somatic damage can thus be worsened by sun exposure, leading to an increased incidence of skin disorders and dramatic acceleration of skin aging (Niedernhofer, 2008). Mammalian cells have evolved several DNA-repair pathways to remove all the categories of DNA base lesions, relying in particular on DNA excision mechanisms. One of these, nucleotide excision repair, removes bulky adducts and is thus an essential mechanism for correcting UV-induced DNA damage (Sarasin, 1999). The base excision repair pathway corrects small base modifications such as oxidized and alkylated bases (Almeida and Sobol, 2007). The importance of repair mechanisms is demonstrated by the hazardous consequences of genetic defects in DNA repair (Friedberg, 2001), but investigating DNA repair with respect to aging remains a challenge. This is due to the complexity of the underlying repair mechanisms as well as to the varying approaches in terms of assays and end points measured (Vijg, 2008). To better understand the relationship between aging and DNA repair, we took advantage of our newly developed multiplexed excision/synthesis assay (Millau et al., 2008) to examine simultaneously, using nuclear extracts, the base excision repair and nucleotide excision repair capacities of human primary fibroblasts derived from healthy donors of different ages. In addition, we investigated changes in DNA repair attributed to chronic sun exposure. A total of 33 healthy Caucasian women were recruited by the Dermscan Group (Lyon, France). Biopsy removal was performed in accordance with the Declaration of Helsinki Principles Guidelines after approval for the study had been given by a medical ethics committee and written consent obtained from the donors. The volunteers were classified into three groups by age (group 1: mean age1⁄425 years, range 20–33, n1⁄49; group 2: mean age1⁄446 years, range 40–50, n1⁄4 9; group 3: mean age1⁄4 65 years, range 61–68, n1⁄415). All subjects were nonsmokers, had phototype II or III skin, declared no excessive exposure to sun or UVA, had no cutaneous pathology, and were not receiving medical treatment. Fibroblast cultures were established from outgrowth of two 3 mm punches taken on the volar forearm (photoexposed area) and the upper inner arm (photoprotected area). Cells were harvested during the exponential phase of growth and stored frozen in liquid nitrogen at passage 5. Nuclear extracts were prepared as described by Millau et al. (2008). For each sample, excision/ synthesis repair reactions were run for 2.5 hours at 30 1C at a final protein concentration of 0.15 mg ml , along with 1 mM adenosine triphosphate and 1.25 mM dCTP-Cy5 (Amersham, Little Chalfont, England), on damaged plasmid microarrays prepared as described by Millau et al. (2008) (see Supplementary Data and Supplementary Figures online for experimental details and nuclear extract features). The microarrays were prepared using Hydrogel slides (Perkin Elmer, Courtaboeuf, France) spotted with unmodified control plasmids together with five plasmid families containing serial amounts of typical lesions (three dilutions per plasmid family). Lesions present on the support were formed by specific physical and chemical treatments: photoproducts (cyclobutane pyrimidine dimers and (6–4)photoproducts), 8-oxoguanine, alkylated bases, abasic sites, and pyrimidine glycols. Total fluorescence intensity related to the fluorescence incorporated into plasmids was the parameter used for calculations (Genepix 4200A scanner, Axon GenePix, Molecular Devices, Sunnyvale, CA). A total fluorescence intensity value was calculated for each lesion type by adding the values for the corresponding replicates. Hence, each microarray generated one value per lesion type. The mean of the two values obtained per sample and per lesion type were used for statistical purposes. Assessment of the mean fluorescence intensity (shown with the corresponding standard error for each lesion type in Figure 1) revealed a decrease in excision/ synthesis activity with age, irrespective of the repair pathway considered. Statistical analysis (general linear model (Minitab V14 software, The MathWorks, Natick, MA) was performed on these latter data from 31 samples: photoprotected and photoexposed cells from the same subject. The general linear model was used to estimate the influence of age

Functional DNA Repair Signature of Cancer Cell Lines Exposed to a Set of Cytotoxic Anticancer Drugs Using a Multiplexed Enzymatic Repair Assay on Biochip

The development of resistances to conventional anticancer drugs compromises the efficacy of cancer treatments. In the case of DNA-targeting chemotherapeutic agents, cancer cells may display tolerance to the drug-induced DNA lesions and/or enhanced DNA repair. However, the role of DNA damage response (DDR) and DNA repair in this chemoresistance has yet to be defined. To provide insights in this challenging area, we analyzed the DNA repair signature of 7 cancer cell lines treated by 5 cytotoxic drugs using a recently developed multiplexed functional DNA repair assay. This comprehensive approach considered the complexity and redundancy of the different DNA repair pathways. Data was analyzed using clustering methods and statistical tests. This DNA repair profiling method defined relevant groups based on similarities between different drugs, thus providing information relating to their dominant mechanism of action at the DNA level. Similarly, similarities between different cell lines presumably identified identical functional DDR despite a high level of genetic heterogeneity between cell lines. Our strategy has shed new light on the contribution of specific repair sub-pathways to drug-induced cytotoxicity. Although further molecular characterisations are needed to fully unravel the mechanisms underlying our findings, our approach proved to be very promising to interrogate the complexity of the DNA repair response. Indeed, it could be used to predict the efficacy of a given drug and the chemosensitivity of individual patients, and thus to choose the right treatment for individualised cancer care.

Impact of topical application of sulfur mustard on mice skin and distant organs DNA repair enzyme signature

Sulfur mustard (SM) is a chemical warfare agent that, upon topical application, damages skin and reaches internal organs through diffusion in blood. Two major toxic consequences of SM exposure are inflammation, associated with oxidative stress, and the formation of alkylated DNA bases. In the present study, we investigated the impact of exposure to SM on DNA repair, using two different functional DNA repair assays which provide information on several Base Excision Repair (BER) and Excision/Synthesis Repair (ESR) activities. BER activities were reduced in all organs as early as 4h after exposure, with the exception of the defense systems against 8-oxo-guanine and hypoxanthine which were stimulated. Interestingly, the resulting BER intermediates could activate inflammation signals, aggravating the inflammation triggered by SM exposure and leading to increased oxidative stress. ESR activities were found to be mostly inhibited in skin, brain and kidneys. In contrast, in the lung there was a general increase in ESR activities. In summary, exposure to SM leads to a significant decrease in DNA repair in most organs, concomitant with the formation of DNA damage. These synergistic genotoxic effects are likely to participate in the high toxicity of this alkylating agent. Lungs, possibly better equipped with repair enzymes to handle exogenous exposure, are the exception.

A comprehensive approach to determining BER capacities and their change with aging in Drosophila melanogaster mitochondria by oligonucleotide microarray

DNA repair mechanisms are key components for the maintenance of the essential mitochondrial genome. Among them, base excision repair (BER) processes, dedicated in part to oxidative DNA damage, are individually well known in mitochondria. However, no large view of these systems in differential physiological conditions is available yet. Combining the use of pure mitochondrial fractions and a multiplexed oligonucleotide cleavage assay on a microarray, we demonstrated that a large range of glycosylase activities were present in Drosophila mitochondria. Most of them were quantitatively different from their nuclear counterpart. Moreover, these activities were modified during aging.

6-Formylindolo[3,2-b]carbazole (FICZ) is a Very Minor Photoproduct of Tryptophan at Biologically Relevant Doses of UVB and Simulated Sunlight

Exposure to solar UV is at the origin of numerous photodegradation pathways in biomolecules. Tryptophan is readily modified by UVB radiation into ring‐opened and oxidized photoproducts. One of them, 6‐formylindolo[3,2‐b]carbazole (FICZ), has been extensively studied in the recent years because it very efficiently binds to AhR, a major factor in numerous biologic processes, such as metabolism of xenobiotics. Unfortunately, little information is available on the actual yield of FICZ upon exposure to low and biologically relevant doses of UV radiation. In the present work, we used a sensitive and specific HPLC‐tandem mass spectrometry assay to quantify a series of photoproducts induced by UVB and simulated sunlight (SSL) in solutions of tryptophan. FICZ represented only a minute amount of the photoproducts (0.02 and 0.03%, respectively). Experiments were repeated in culture medium where the yield of FICZ was also found to be very low, even when Trp was added. Last, no FICZ could be detected in cytosolic fractions of cultured cells exposed to SSL. Altogether, the present results show that FICZ is a very minor photoproduct and that it cannot be considered the only endogenous photoproduct responsible for the induction of AhR‐dependent responses in UV‐irradiated cells.

Natural variability and modulation by environmental stressors of global genomic cytosine methylation levels in a freshwater crustacean, Gammarus fossarum

To improve the assessment of aquatic organism responses to environmental stressors, there is an interest in studying epigenetic marks in addition to other validated biomarkers. Indeed, the epigenetic marks may be influenced by the surrounding environment. Non-model invertebrates such as gammarids are sentinel organisms representative of the diversity of natural stream communities. Despite their ecologically relevance, the epigenetic responses have been to date poorly documented in these species. The present study explores the measurement of the global cytosine methylation level in the genome of the freshwater crustacean Gammarus fossarum. In a first step, natural variability of global cytosine methylation level (basal level) was assessed by studying the effect of sex, age and sampling site of organisms. Results showed a significant effect of age and sampling site. In a second step, effects of water temperature and food starvation were studied. For both factors, a hypermethylation was observed after 1 month of exposure. In a third step, gammarids were exposed to a range of environmentally relevant cadmium concentrations (0.05-5 μg/L) in order to assess the effect of a chemical stress. Whatever the cadmium concentration used, a significant hypomethylation was observed after 14 days followed by a trend for hypermethylation after 1 month of exposure. These results are the first ones dealing with the 5C-methylation status in gammarids. The results constitute potential markers of environmental stresses in relevant sentinel species widely used in ecotoxicological studies.