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Thierry Douki

Publications and source records attributed to Thierry Douki.

At least 19 recordsLinked to original sources
PubMed2025

p53 Regulates Nuclear Architecture to Reduce Carcinogen Sensitivity and Mutagenic Potential.

The p53 tumor suppressor is an indispensable regulator of DNA damage responses that accelerates carcinogenesis when mutated. In this report, we uncover a new mechanism by which p53 maintains genomic integrity in the absence of canonical DNA damage response activation. Specifically, loss of p53 dramatically alters chromatin structure at the nuclear periphery, allowing increased transmission of an environmental carcinogen, ultraviolet (UV) radiation, into the nucleus. Genome-wide mapping of UV-induced DNA lesions in p53-deficient primary cells reveals elevated lesion abundance in regions corresponding to locations of high mutation burden in malignant melanomas. These findings uncover a novel role of p53 in the suppression of mutations that contribute to cancer and highlight the critical influence of nuclear architecture in regulating sensitivity to carcinogens.

Journal Article↗
PubMed2006

Influence of static magnetic field on cadmium toxicity: study of oxidative stress and DNA damage in rat tissues.

In the present study, we investigated the effect of co-exposure to static magnetic field (SMF) and cadmium (Cd) on the biochemical parameters, antioxidant enzymes activity and DNA damage in rat tissues. Animals were treated with cadmium (CdCl(2), 40mg/L, per os) in drinking water during 4 weeks. Cd treatment induced an increase of plasma lactate dehydrogenase (LDH) and transaminases levels. Moreover, Cd treatment increased malondialdehyde (MDA) and 8-oxodGuo levels in rat tissues. However, the antioxidant enzymes activity such as the glutathione peroxidase (GPx), catalase (CAT) and the superoxide dismutase (SOD) were decreased in liver and kidney, while we noted a huge increase of hepatic and renal cadmium content. Interestingly, the combined effect of SMF (128mT, 1h/day during 30 consecutive days) and Cd (40mg/L, per os) decreased the GPx and CAT activities in liver compared to cadmium treated group. However, the association between SMF and Cd failed to alter transaminases, MDA and 8-oxodGuo concentration. Cd treatment altered antioxidant enzymes and DNA in liver and kidney of rats. Moreover, SMF associated to Cd disrupt this antioxidant response in liver compared to Cd-treated rats.

Animals↗
PubMed2006

Soil fungi reduce the iron content and the DNA damaging effects of asbestos fibers.

Some soil fungi growing on asbestos fibers release chelators and antioxidants. The bioweathering potential of fungi has thus been envisaged as a possible route for bioremediation of asbestos rich soils, where no inactivation procedures have been established so far. The present study reports fungal-mediated modification of the surface reactivity of the fibers and of their potential to damage DNA in vitro. Verticillium sp. and Paecilomyces sp. were selected among the fungi isolated from fragments of chrysotile bearing rocks, as the most potent in iron extraction, and studied in parallel with F. oxysporum, previously reported to modify the surface reactivity of asbestos fibers. One sample of chrysotile from the Western Alps and a sample of UICC (Union Internationale Contre le Cancer) crocidolite were incubated with or without fungi. All fungi extracted iron from both fibers (7.3% from crocidolite and 33.6% from chrysotile by Verticillium sp.), releasing it into the medium. F. oxysporum and Paecilomyces sp. suppressed the potential of the fibers to release hydroxyl radical, while Verticillium sp. suppressed it on crocidolite but enhanced it on chrysotile, a hallmark of ongoing mobilization of reactive iron. Fibers incubated in the growth medium, but in the absence of fungi, exhibited a remarkable potential to damage DNA in vitro, measured by the generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, while all the fungi reduced such effect. Fungi may thus be regarded as appropriate candidates for bioremediation of asbestos rich soils whereby the reactive iron ions responsible for DNA damage are progressively removed from the fibers.

Asbestos, Crocidolite↗
PubMed2006

Cyclobutane pyrimidine dimers are predominant DNA lesions in whole human skin exposed to UVA radiation.

Solar UV radiation is the most important environmental factor involved in the pathogenesis of skin cancers. The well known genotoxic properties of UVB radiation (290-320 nm) mostly involve bipyrimidine DNA photoproducts. In contrast, the contribution of more-abundant UVA radiation (320-400 nm) that are not directly absorbed by DNA remains poorly understood in skin. Using a highly accurate and quantitative assay based on HPLC coupled with tandem mass spectrometry, we determined the type and the yield of formation of DNA damage in whole human skin exposed to UVB or UVA. Cyclobutane pyrimidine dimers, a typical UVB-induced DNA damage, were found to be produced in significant yield also in whole human skin exposed to UVA through a mechanism different from that triggered by UVB. Moreover, the latter class of photoproducts is produced in a larger amount than 8-oxo-7,8-dihydro-2'-deoxyguanosine, the most common oxidatively generated lesion, in human skin. Strikingly, the rate of removal of UVA-generated cyclobutane pyrimidine dimers was lower than those produced by UVB irradiation of skin. Finally, we compared the formation yields of DNA damage in whole skin with those determined in primary cultures of keratinocytes isolated from the same donors. We thus showed that human skin efficiently protects against UVB-induced DNA lesions, whereas very weak protection is afforded against UVA. These observations emphasize the likely role played by the UVA-induced DNA damage in skin carcinogenesis and should have consequences for photoprotection strategies.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2006

Low ionic strength reduces cytosine photoreactivity in UVC-irradiated isolated DNA.

Exposure of DNA to far-UV radiation leads to the formation of several types of dimeric lesions, including cyclobutane dimers and (6-4) photoproducts. In order to gain insights into the main parameters driving DNA photochemistry, the effect of ionic strength on the yield of formation of these photoproducts was investigated in UVC-irradiated samples of isolated genomic DNA. The main consequence of lowering the ionic strength was a decrease in the UVC-induced formation of thymine-cytosine and cytosine-cytosine photoproducts. The reactivity of thymine-thymine and cytosine-thymine doublets was hardly affected. Evidence was obtained against a major role played by duplex denaturation in these observations. A more likely explanation is a change in the DNA structure as the result of a larger extent of protonation at low counter-ions concentration.

Animals↗
PubMed2006

Ionic liquid droplet as e-microreactor.

A powerful approach combining a droplet-based, open digital microfluidic lab-on-a-chip using task-specific ionic liquids as soluble supports to perform solution-phase synthesis is reported as a new tool for chemical applications. The negligible volatility of ionic liquids enables their use as stable droplet reactors on a chip surface under air. The concept was validated with different ionic liquids and with a multicomponent reaction. Indeed, we showed that different ionic liquids can be moved by electrowetting on dielectric (EWOD), and their displacement was compared with aqueous solutions. Furthermore, we showed that mixing ionic liquids droplets, each containing a different reagent, in "open" systems is an efficient way of carrying supported organic synthesis. This was applied to Grieco's tetrahydroquinolines synthesis with different reagents. Analysis of the final product was performed off-line and on-line, and the results were compared with those obtained in a conventional reaction flask. This technology opens the way to easy synthesis of minute amounts of compounds ad libitum without the use of complex, expensive, and bulky robots and allows complete automation of the process for embedded chemistry in a portable device. It offers several advantages, including simplicity of use, flexibility, and scalability, and appears to be complementary to conventional microfluidic lab-on-a-chip devices usually based on continuous-flow in microchannels.

Ions↗
PubMed2006

Dinucleotide spore photoproduct, a minimal substrate of the DNA repair spore photoproduct lyase enzyme from Bacillus subtilis.

The overwhelming majority of DNA photoproducts in UV-irradiated spores is a unique thymine dimer called spore photoproduct (SP, 5-thymine-5,6-dihydrothymine). This lesion is repaired by the spore photoproduct lyase (SP lyase) enzyme that directly reverts SP to two unmodified thymines. The SP lyase is an S-adenosylmethionine-dependent iron-sulfur protein that belongs to the radical S-adenosylmethionine superfamily. In this study, by using a well characterized preparation of the SP lyase enzyme from Bacillus subtilis, we show that SP in the form of a dinucleoside monophosphate (spore photoproduct of thymidilyl-(3'-5')-thymidine) is efficiently repaired, allowing a kinetic characterization of the enzyme. The preparation of this new substrate is described, and its identity is confirmed by mass spectrometry and comparison with authentic spore photoproduct. The fact that the spore photoproduct of thymidilyl-(3'-5')-thymidine dimer is repaired by SP lyase may indicate that the SP lesion does not absolutely need to be contained within a single- or double-stranded DNA for recognition and repaired by the SP lyase enzyme.

Bacillus subtilis↗
PubMed2006

Adducts of oxylipin electrophiles to glutathione reflect a 13 specificity of the downstream lipoxygenase pathway in the tobacco hypersensitive response.

The response to reactive electrophile species (RES) is now considered as part of the plant response to pathogen and insect attacks. Thanks to a previously established high-performance liquid chromatography tandem mass spectrometry methodology, we have investigated the production of oxylipin RES adducts to glutathione (GSH) during the hypersensitive response (HR) of plants. We have observed that RES conjugation to GSH in tobacco (Nicotiana tabacum) leaves is facile and nonspecific. In cryptogein-elicited tobacco leaves, we show that the oxylipin RES adducts to GSH are produced in correlation with GSH consumption, increase in glutathione S-transferase activity, and the appearance of the cell death symptoms. In this model, the adducts arise mainly from the downstream 13 lipoxygenase (LOX) metabolism, although the induced 9 LOX pathway leads massively to the accumulation of upstream metabolites. The main adducts were obtained from 2-hexenal and 12-oxo-phytodienoic acid. They accumulate transiently as 1-hexanol-3-GSH, a reduced adduct, and 12-oxo-phytodienoic acid-GSH, respectively. RES conjugation does not initiate cell death but explains part of the GSH depletion that accompanies HR cell death. The nature of these GSH conjugates shows the key role played by the 13 LOX pathway in RES signaling in the tobacco HR.

Acrolein↗
PubMed2006

Age-dependent DNA repair and cell cycle distribution of human skin fibroblasts in response to UVA irradiation.

Ageing process in cells is associated with oxidative stress. Ultraviolet A produces reactive oxygen species responsible for accumulation of DNA and cellular damage. After the evaluation of antioxidant enzyme activities and oxidative stress markers at the basal state, we have studied the responses to UVA stress of coetaneous fibroblasts, isolated from different male donors (2-88 years, n=23) in terms of cytotoxicity, genotoxicity and DNA repair capacities. For this purpose, we have determined level of DNA damage using the comet assay (single strand breaks and alkali-labile sites) and the cell cycle distribution after a 5 J/cm2 irradiation. No differences with age were observed for antioxidant enzyme activities and oxidative stress markers. DNA strand breaks after UVA irradiation (5-20 J/cm2), was found to be age-dependent. DNA repair was slow and also significantly affected by ageing. The cell cycle distribution analysis showed that high repair correlated with high proliferative capacities at basal level. Twenty-four hours after the stress, fraction of young fibroblasts blocked in G1 phase was significantly increased whereas significant modifications concerned the G2-M phase for adult and older fibroblasts. These results indicate an age-dependent decline in the DNA repair capacities correlated with modifications of the cell cycle parameters.

Adolescent↗
PubMed2006

Exposure to static magnetic field of pregnant rats induces hepatic GSH elevation but not oxidative DNA damage in liver and kidney.

BACKGROUND: The present investigation was designed to evaluate the effects of subacute exposure to static magnetic field (SMF) on some parameters indicative of oxidative stress and on oxidative DNA damage in pregnant rat. METHODS: Females rats (n = 6) were exposed to a SMF (128 mT; 1 h/day) from day 6 to day 19 of pregnancy and were allowed to deliver normally. The control group (n = 6) was not exposed to SMF. Dams were sacrificed 3 days after delivery. The effects of subacute exposure to SMF on oxidative states were assessed on the measurements of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) and catalase (CAT). The level of 8-oxo-dG was measured using high-performance liquid chromatography coupled to electrochemical detection. RESULTS: Subacute exposure to SMF failed to alter plasma GPx, MDA, CAT and SOD respectively in liver and kidney. By contrast, SMF increased total GSH (+56%, p <0.05) and reduced GSH (+108%, p <0.05) in liver. Our results showed that the exposure to SMF did not induce oxidative DNA lesions in liver and kidney. CONCLUSIONS: The data do not provide evidence that subacute SMF exposure causes DNA damage in liver and kidney in pregnant rats. The present results suggest that hepatic GSH plays an important role in protection against SMF during pregnancy. These changes in antioxidant status (GSH) lead to some adaptive responses due to activation of systems controlling the body oxidative mechanism balance.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2006

Effects of subchronic exposure to static magnetic field on testicular function in rats.

BACKGROUND: The aim of this study was to investigate the effect of static magnetic field (SMF) exposure on testicular function, antioxidant status and DNA oxidation in rats. METHODS: Male adult rats were exposed to SMF (128 mT; 1 h/day for 30 days). After sacrifice, the epididymal sperm number was counted. Testosterone concentration in plasma and testis was measured by radioimmunoassay. MDA level and GPx, CAT and SOD activities were used as markers of oxidative stress in testis. The 8-oxo-dG level is measured by the HPLC-EC system. RESULTS: Subchronic exposure to SMF has no effect on epididymal sperm count, spermatozoa motility and genital organ weight. In contrast, SMF induces a decrease of testicular and plasmatic testosterone levels, respectively (1.48 +/- 0.56 vs. 4.66 +/- 0.51 ng/g, p<0.05; 0.97 +/- 0.16 vs. 1.64 +/- 0.18 ng/mL, p<0.05). Exposed rats displayed an increase of malondialdehyde (2.01 +/- 0.03 vs. 1.47 +/- 0.06 micromol/g protein, p<0.05), metallothioneins (1.04 +/- 0.22 vs. 0.37 +/- 0.06 microg/g, p<0.05) and 8-oxo-dG concentrations (3.38 +/- 0.30 vs. 2.36 +/- 0.28 8-oxo-dG/10(6) bases, p<0.05) in the testis. In the gonad, SMF decreases the CAT (14.33 +/- 1.16 vs. 21.67 +/- 2.05 U/mg protein, p<0.05), GPx (177.40 +/- 5.97 vs. 237.20 +/- 15.65 U/mg protein, p<0.05) and mitochondrial Mn-SOD (2.95 +/- 0.10 vs. 3.53 +/- 0.29 U/mg protein, p<0.05) activities. However, cytosolic CuZn-SOD activity is unaffected. CONCLUSIONS: Subchronic exposure to SMF failed to alter spermatogenesis in rat testis. In contrast, the same treatment decreased testosterone levels and induced DNA oxidation.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2006

Oxidative DNA damage and antioxidant defenses in the European common lizard (Lacerta vivipara) in supercooled and frozen states.

The European common lizard (Lacerta vivipara) tolerates long periods at sub-zero temperatures, either in the supercooled or the frozen state. Both physiological conditions limit oxygen availability to tissues, compelling lizards to cope with potential oxidative stress during the transition from ischemic/anoxic conditions to reperfusion with aerated blood during recovery. To determine whether antioxidant defenses are implicated in the survival of lizards when facing sub-zero temperatures, we monitored the activities of antioxidant enzymes and oxidative stress either during supercooling or during freezing exposures (20 h at -2.5 degrees C) and 24 h after thawing in two organs of lizards--muscle and liver. Supercooling induced a significant increase in the total SOD and GPx activity in muscle (by 67 and 157%, respectively), but freezing had almost no effect on enzyme activity, either in muscle or in liver. By contrast, thawed lizards exhibited higher GPx activity in both organs (a 133% increase in muscle and 59% increase in liver) and a significant decrease in liver catalase activity (a 47% difference between control and thawed lizards). These data show that supercooling (but not freezing) triggers activation of the antioxidant system and this may be in anticipation of the overgeneration of oxyradicals when the temperature increases (while thawing or at the end of supercooling). Oxidative stress was assessed from the content of 8-oxodGuo and the different DNA adducts resulting from lipid peroxidation, but it was unaltered whatever the physiological state of the lizards, thus demonstrating the efficiency of the antioxidant system that has been developed by this species. Overall, antioxidant defenses appear to be part of the adaptive machinery for reptilian tolerance to sub-zero temperatures.

Adaptation, Physiological↗
PubMed2006

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) gamma-rays. MATERIAL AND METHODS: DNA samples and human monocytes were exposed either to gamma-rays emitted by a (60)Co source or to (12)C(6+) or (36)Ar(18+) 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 gamma-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 degrees 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.

DNA↗
PubMed2005

Conjugation of keto fatty acids to glutathione in plant tissues. Characterization and quantification by HPLC-tandem mass spectrometry.

Both biotic and abiotic stress activate the oxylipin pathway in plants. As reactive electrophile species (RES), some oxylipins are expected to bind cellular nucleophiles in a Michaël-type addition reaction. Using the HPLC-tandem mass spectrometry techniques, we have established the analytical basis for the investigation of oxylipin conjugation to glutathione (GSH) in plant extracts. The GSH adducts to the four keto fatty acid isomers issued from both linoleic and linolenic acids were first produced and their mass spectrometric features analyzed in the positive electrospray ionization mode. In all cases, the main fragmentation (MS2 mode) of the pseudomolecular ion leads to the neutral loss of a glutamyl moiety (-129 Da), affording an ion that gives structural information upon an additional fragmentation (MS3 mode). The glutamyl loss was confirmed by the analysis of other GSH adducts to oxylipin RES and appeared as being characteristic of GSH adducts. It is thus proposed to search GSH adducts in plant extracts by HPLC-MS/MS, using initially the neutral loss mode and then the MS2 mode to further characterize the identified compounds. This methodology was successfully applied to the analysis of GSH adducts upon infiltration into leaves of the four previous keto fatty acids at 5 mM, a concentration inducing cell death. The production of GSH adducts to oxylipin RES was observed for the first time in plant tissues. Furthermore, the levels of adduct production explain in part the observed GSH depletion. These results support the role of RES in altering protein activities and cellular redox balance of plant cells, via addition reactions to cellular nucleophiles.

Chromatography, High Pressure Liquid↗
PubMed2005

An aromatic hydroxylation reaction catalyzed by a two-component FMN-dependent Monooxygenase. The ActVA-ActVB system from Streptomyces coelicolor.

The ActVA-ActVB system from Streptomyces coelicolor isatwo-component flavin-dependent monooxygenase that belongs to an emerging class of enzymes involved in various oxidation reactions in microorganisms. The ActVB component is a NADH:flavin oxidoreductase that provides a reduced FMN to the second component, ActVA the proper monooxygenase. In this work, we demonstrate that the ActVA-ActVB system catalyzes the aromatic monohydroxylation of dihydrokalafungin by molecular oxygen. In the presence of reduced FMN and molecular oxygen, the ActVA active site accommodates and stabilizes an electrophilic flavin FMN-OOH hydroperoxide intermediate species as the oxidant. Surprisingly, we demonstrate that the quinone form of dihydrokalafungin is not oxidized by the ActVA-ActVB system, whereas the corresponding hydroquinone is an excellent substrate. The enantiomer of dihydrokalafungin, nanaomycin A, as well as the enantiomer of kalafungin, nanaomycin D, are also substrates in their hydroquinone forms. The previously postulated product of the ActVA-ActVB system, the antibiotic actinorhodin, was not found to be formed during the oxidation reaction.

Anthraquinones↗
PubMed2005

Effect of denaturation on the photochemistry of pyrimidine bases in isolated DNA.

The influence of denaturation on DNA photochemistry was studied by quantifying the yield of formation of all possible bipyrimidine photolesions within isolated genomic DNA samples exposed to UVC radiation. Effects of DNA melting was studied either by carrying out irradiation over a wide range of temperature (0-90 degrees C) or by decreasing the ionic strength of the solution at 30 degrees C. A first observation was a much larger decrease in the photoreactivity upon increasing the temperature in single-stranded than in double-stranded DNA. Secondly, formation of trans,syn cyclobutane dimers and, to a lesser extent, modification in the ratio between the yields of cyclobutane dimers and (6-4) photoproducts, were found to be other main features associated with denaturation. These results emphasize the modulating role of structure in the yield and nature of UV-induced DNA damage.

Cyclobutanes↗
PubMed2005

Repair of the three main types of bipyrimidine DNA photoproducts in human keratinocytes exposed to UVB and UVA radiations.

Induction of DNA damage by solar UV radiation is a key event in the development of skin cancers. Bipyrimidine photoproducts, including cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (64 PPs) and their Dewar valence isomers, have been identified as major UV-induced DNA lesions. In order to identify the predominant and most persistent lesions, we studied the repair of the three types of photolesions in primary cultures of human keratinocytes. Specific and quantitative data were obtained using HPLC associated with tandem mass spectrometry. As shown in other cell types, 64 PPs are removed from UVB-irradiated keratinocytes much more efficiently than CPDs. In contrast, CPDs are still present in high amounts when cells recover their proliferation capacities after cell cycle arrest and elimination of a part of the population by apoptosis. The predominance of CPDs is still maintained when keratinocytes are exposed to a combination of UVB and UVA. Under these conditions, 64 PPs are converted into their Dewar valence isomers that are as efficiently repaired as their (6-4) precursors. Exposure of cells to pure UVA radiation generates thymine cyclobutane dimers that are slightly less efficiently repaired than CPDs produced upon UVB irradiation. Altogether, our results show that CPDs are the most frequent and the less efficiently repaired bipyrimidine photoproducts irrespectively of the applied UV treatment.

Apoptosis↗