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International audience; Some amount of furanose in a southern conformation, possibly in both, but certainly in one of the two adjacent nucleotides of a dipyrimidine site, is necessary for (6-4) photoproduct formation in oligonucleotides. To explore the necessity, role, and most favorable location of each South sugar conformer on the formation of the (6-4) adduct in the thymine dinucleotide TpT, the photochemical behavior of two synthetic analogues, in which the South sugar conformation is prohibited for one of their two sugars, has been examined. Herein, we experimentally demonstrate that the presence of one sugar presenting some amount of South puckering, at any of the extremities, is sufficient to trigger (6-4) adduct formation. Nonetheless, the photochemical behavior of the dinucleotide with a South-puckered conformation at the 5'end, mimics more closely that of TpT. In addition, using the 5' North 3' South-dilocked dinucleotide, we demonstrate that the flexibility of the South pucker at the 3'-end has little influence on the (6-4) adduct formation.

Sunlight induces actinic keratosis, skin cancers and photoaging. Photoprotection is thus a major issue in public health to prevent the harmful effects of solar ultraviolet (UV) radiations. Recent data have shown that the visible (VIS) and infrared (IR) radiations can lead to skin damage by oxidative stress, suggesting that a balanced protection across the entire spectrum of sunlight is necessary to prevent cutaneous alterations. In this context, we developed a new generation of sunfilter called Phenylene Bis-Diphenyltriazine or TriAsorB (CAS N°55514-22-2). The aim of the present study was to assess the photoprotective efficacy of TriAsorB from UV to IR light. Spectrophotometric assays were performed to measure absorption and reflectance of TriAsorB in the different spectral ranges of sunlight: UV, VIS including blue light or high energy visible (HEV) and IR. DNA damage was evaluated using reconstructed human epidermis (RHE): 8-hydroxy-2'-deoxyguanosine (8OHdG) in response to HEV exposure, pyrimidine dimers (CPDs) and (6-4) photoproducts following solar-simulated radiation (SSR). TriAsorB is a broad spectrum UVB + UVA filter including long UVA. Interestingly, it also absorbs VIS radiations, especially in the HEV region. These radiations are also reflected. Protection in the IR spectral range is weak. Furthermore, the sunfilter specifically protects the skin against the oxidative lesions 8OHdG induced by HEV and prevents SSR-induced DNA damage. Thus, TriAsorB is an innovative sunfilter that might be used in sun care products for skin photoprotection from UV to VIS radiations. Finally, it prevents sunlight genotoxicity and protected the skin against solar radiations, especially blue light.

International audience; Introduction: Unlike "light" cylobutane pyrimidine dimers (CPD) formed during ultraviolet radiation (UVR) exposure, dark CPD (dCPD) are formed afterwards. Studies have attributed this to delayed melanin sensitization. There are no data on the role of melanin in dCPD formation in human skin.Methods and results: Volunteers of Fitzpatrick skin types (FST I/II vs. VI) were exposed to erythemally equivalent doses of solar simulated radiation. CPD were assessed by semi-quantitative immunostaining in whole epidermis and in three epidermal zones, and quantitative HPLC-MS/MS (whole epidermis) at different times post-exposure up to 24 hr. A CPD peak that appeared at 1-2 hr post-exposure in whole epidermis measurements, in all skin types, demonstrated dCPD. However, both dCPD and light CPD were absent in the basal layer of FST VI with the greatest melanin concentration. Modelling the whole epidermis data showed no differences between the repair kinetics of FST I/II and VI.Discussion: Melanin may be a sensitizer or "sunscreen" for dCPD depending on its location and concentration. Previous CPD repair studies in human skin have assumed peak CPD immediately after UVR exposure and so have overestimated total repair.

Sulfur mustard (SM), a chemical warfare agent, is a strong alkylating compound that readily reacts with numerous biomolecules. The goal of the present work was to define and validate new biomarkers of exposure to SM that could be easily accessible in urine or plasma. Because investigations using SM are prohibited by the Organization for the Prohibition of Chemical Weapons, we worked with 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of SM. We developed an ultra-high-pressure liquid chromatography - tandem mass spectrometry approach (UHPLC-MS/MS) to the conjugate of CEES to glutathione and two of its metabolites, the cysteine and the N-acetyl-cysteine conjugates. The N7-guanine adduct of CEES (N7Gua-CEES) was also targeted. After synthesizing the specific biomarkers, a solid phase extraction protocol and a UHPLC-MS/MS method with isotopic dilution were optimized. We were able to quantify N7Gua-CEES in the DNA of HaCaT keratinocytes and of explants of human skin exposed to CEES. N7Gua-CEES was also detected in the culture medium of these two models, together with the glutathione and the cysteine conjugates. In contrast, the N-acetyl-cysteine conjugate was not detected. The method was then applied to plasma from mice cutaneously exposed to CEES. All four markers could be detected. Our present results thus validate both the analytical technique and the biological relevance of new, easily quantifiable biomarkers of exposure to CEES. Because CEES behaves very similarly to SM, the results are promising for application to this toxic of interest., Analytical and Bioanalytical Chemistry, Springer Verlag, 2020

The UVA and UVB components of sunlight can produce three classes of bipyrimidine DNA photolesions [cyclobutane pyrimidine dimers (CPDs), pyrimidine (6‐4) pyrimidone photoproducts (6‐4PPs) and related Dewar valence isomers (DewarPPs)]. The UVA/UVB ratio of sunlight is high in winter and low in summer in the Northern Hemisphere. Since UVB radiation produces 6‐4PPs and UVA radiation converts them into DewarPPs through photoisomerization, it is expected that there may be differences in the photoisomerization of 6‐4PPs between summer and winter, although that has never been documented. To determine that, isolated DNA was exposed to natural sunlight for 8 h in late summer and in winter, and absolute levels of the three classes of photolesions were quantified using calibrated ELISAs. It was found that sunlight produces CPDs and 6‐4PPs in DNA at a ratio of about 9:1 and converts approximately 80% of 6‐4PPs into DewarPPs within 3 h. Moreover, photoisomerization is more efficient in winter than in late summer after sunlight irradiation for the same duration, at similar solar UV doses and with the same induction level of CPDs. These results demonstrate that seasonal differences in the UVA/UVB ratio influence the efficiency of the photoisomerization of 6‐4PPs into DewarPPs., The UVA and UVB components of sunlight produce three main classes of bipyrimidine DNA photolesions. The UVA/UVB ratio of sunlight is high in winter and low in summer in the Northern Hemisphere. Since UVB produces (6‐4) photoproducts (6‐4PPs) and UVA converts them into Dewar valence isomers (DewarPPs) through photoisomerization, it is expected that there may be seasonal differences in the photoisomerization of 6‐4PPs. Indeed, photoisomerization is more efficient in winter than in late summer after sunlight irradiation, demonstrating that seasonal differences in the UVA/UVB ratio influence the efficiency of the photoisomerization of 6‐4PPs into DewarPPs.

UV-induced formation of photoproducts in DNA is a major initiating event of skin cancer. Consequently, many analytical tools have been developed for their quantification in DNA. In the present work, we extended our previous liquid chromatography-mass spectrometry method to the quantification of the short DNA fragments containing photoproducts that are released from cells by the repair machinery. We designed a robust protocol including a solid-phase extraction step (SPE), an enzymatic treatment aimed at releasing individual photoproducts, and a liquid chromatography method combining on-line SPE and ultra-high-performance liquid chromatography for optimal specificity and sensitivity. We also added relevant internal standards for a better accuracy. The method was validated for linearity, repeatability, and reproducibility. The limits of detection and quantification were found to be in the fmol range. The proof of concept of the use of excreted DNA repair products as biomarkers of the genotoxicity of UV was obtained first in in vitro studies using cultured HaCat cells and ex vivo on human skin explants. Further evidence was obtained from the detection of pyrimidine dimers in the urine of human volunteers collected after recreational exposure in summer. An assay was designed to quantify the DNA photoproducts released from cells within short fragments by the DNA repair machinery. These oligonucleotides were isolated by solid-phase extraction and enzymatically hydrolyzed. The photoproducts were then quantified by on-line SPE combined with UHPLC-MS/MS with isotopic dilution.

International audience; Photobiological effects are known to greatly depend on the wavelength of the incident photons that define the nature of the activated chromophores. A growing number of experimental data show that considering the effect of complex light sources as a sum of the effects of monochromatic exposures can be misleading. Indeed, the combined exposure to several wavelength ranges may modulate photobiological responses or even induce novel processes. These observations are similar to a well known topic in chemical toxicology: the non additivity of effects in mixtures where either antagonism or synergy are often observed. In the present work, we investigated if a data analysis tool first developed for studying non-additivity in mixtures of drugs, the combination index, could be applied to photobiological processes. We chose to work on the formation of UV induced DNA photoproducts where additive, antagonist and synergistic effects take place simultaneously. In addition to this application, we worked on the mathematical bases of the concept in order to broaden its applicability to phenomena exhibiting various dose response patterns. We also addressed the question of the evaluation of the error on the determination of the combination index.

In the Puna region, the total solar irradiation and the UV incidence is the highest on Earth, thus, restraining the physiology of individual microorganisms and the composition of microbial communities. UV-resistance of microbial strains thriving in High-Altitude Andean Lakes was demonstrated and their mechanisms were partially characterized by genomic analysis, biochemical and physiological assays. In this work, we present the molecular events involved in the adaptive response of the model HAAL extremophilic actinobacterium Nesterenkonia sp. Act20 under artificial UV-B radiation, herein called as UV-resistome. Proteomic profiles of cultures exposed to different UV-experimental conditions showed that the leading systems for adaptation to the UV-challenge in-vitro are DNA repair and antioxidant mechanisms.

International audience; The cyclobutane pyrimidine dimer (CPD) is a main mutagenic photolesion in DNA produced by UVR. We previously studied the wavelength-dependent kinetics of mutation induction efficiency using monochromatic UVR sources and transgenic mice developed for mutation assay and established the action spectra of UVR mutagenicity in the mouse epidermis and dermis. Here, we further established the action spectra of CPD and pyrimidine(6-4)pyrimidone photoproduct formation in the same tissues and in naked DNA using the same sources and mouse strain. Quantitative ELISA helped us estimate the photolesion formation efficiencies on a molecule-per-nucleotide basis. Using these action spectra, we confirmed that the UVR mutation mostly depends on CPD formation. Moreover, the mutagenicity of a CPD molecule (CPD mutagenicity) was found to vary by wavelength, peaking at approximately 313 nm in both the epidermis and dermis with similar wavelength-dependent patterns. Thus, the CPD formation efficiency is a main determinant of UVR mutagenicity in mouse skin, whereas a wavelength-dependent variation in the qualitative characteristics of CPD molecules also affects the mutagenic consequences of UVR insults. In addition, the CPD mutagenicity was always higher in the epidermis than in the dermis, suggesting different cellular responses to UVR between the two tissues irrespective of the wavelength.

All photobiological events depend on the wavelength of the incident radiation. In real-life situations and in the vast majority of laboratory experiments, exposure always involves sources with various emission spectra spreading over a wide wavelength range. Action spectra are often used to describe the efficiency of a process at different wavelengths and to predict the effects of a given light source by summation of the individual effects at each wavelength. However, a full understanding of the biological effects of complex sources requires more than considering these concomitant events at each specific wavelength. Indeed, photons of different energies may not have additive but synergistic or inhibitory effects on photochemical processes and cellular responses. The evolution of a photobiological response with post-irradiation time must also be considered. These two aspects may represent some limitations to the use of action spectra. The present review, focused on mammalian cells, illustrates the concept of action spectrum and discusses its drawbacks using theoretical considerations and examples taken from the literature. Emphasis is placed on genotoxicity for which wavelength effects have been extensively studied. Other effects of UV exposure are also mentioned.

International audience; This chapter is intended to provide a concise overview of some basic and general concepts of photophysics/photochemistry useful for the understanding of the following chapters. After a general and easy introduction to the theoretical basis of the interaction between light and molecules, we shall briefly describe the principle of the most used steady-state and time-resolved experimental spectroscopy techniques. Then we shall introduce the most used analytical techniques to quantify and characterize the photodamage and describe the main biological phenomena associated with DNA damage and repair.

International audience; Some bacterial species enter a dormant state in the form of spores to resist to unfavorable external conditions. Spores are resistant to a wide series of stress agents, including UV radiation, and can last for tens to hundreds of years. Due to the suspension of biological functions, such as DNA repair, they accumulate DNA damage upon exposure to UV radiation. Differently from active organisms, the most common DNA photoproducts in spores are not cyclobutane pyrimidine dimers, but rather the so‐called spore photoproducts. This noncanonical photochemistry results from the dry state of DNA and its binding to small, acid‐soluble proteins that drastically modify the structure and photoreactivity of the nucleic acid. Herein, multiscale molecular dynamics simulations, including extended classical molecular dynamics and quantum mechanics/molecular mechanics based dynamics, are used to elucidate the coupling of electronic and structural factors that lead to this photochemical outcome. In particular, the well‐described impact of the peculiar DNA environment found in spores on the favored formation of the spore photoproduct, given the small free energy barrier found for this path, is rationalized. Meanwhile, the specific organization of spore DNA precludes the photochemical path that leads to cyclobutane pyrimidine dimer formation.

Synthetic amorphous silica (SAS) is used in a plethora of applications and included in many daily products to which humans are exposed via inhalation, ingestion, or skin contact. This poses the question of their potential toxicity, particularly towards macrophages, which show specific sensitivity to this material. SAS represents an ideal candidate for the adsorption of environmental contaminants due to its large surface area and could consequently modulate their toxicity. In this study, we assessed the toxicity towards macrophages and intestinal epithelial cells of three SAS particles, either isolated SiO2 nanoparticles (LS30) or SiO2 particles composed of agglomerated-aggregates of fused primary particles, either food-grade (E551) or non-food-grade (Fumed silica). These particles were applied to cells either alone or in combination with genotoxic co-contaminants, i.e., benzo[a]pyrene (B[a]P) and methane methylsulfonate (MMS). We show that macrophages are much more sensitive to these toxic agents than a non-differenciated co-culture of Caco-2 and HT29-MTX cells, used here as a model of intestinal epithelium. Co-exposure to SiO2 and MMS causes DNA damage in a synergistic way, which is not explained by the modulation of DNA repair protein mRNA expression. Together, this suggests that SiO2 particles could adsorb genotoxic agents on their surface and, consequently, increase their DNA damaging potential.

TiO2 particles are broadly used in daily products, including cosmetics for their UV-absorbing property, food for their white colouring property, water and air purification systems, self-cleaning surfaces and photoconversion electrical devices for their photocatalytic properties. The toxicity of TiO2 nano- and microparticles has been studied for decades, and part of this investigation has been dedicated to the identification of their potential impact on DNA, i.e., their genotoxicity. This review summarizes data retrieved from their genotoxicity testing during the past 6 years, encompassing both in vitro and in vivo studies, mostly performed on lung and intestinal models. It shows that TiO2 particles, both nano- and micro-sized, produce genotoxic damage to a variety of cell types, even at low, realistic doses.

Some bacterial species enter a dormant state in the form of spores to resist to unfavorable external conditions. Spores are resistant to a wide series of stress agents, including UV radiation, and can last for tens to hundreds of years. Due to the suspension of biological functions such as DNA repair, they accumulate DNA damage upon exposure to UV radiation. Differently from active organisms, the most common DNA photoproduct in spores are not cyclobutane pyrimidine dimers, but rather the so-called spore photoproduct. This non-canonical photochemistry results from the dry state of DNA and the binding to small acid soluble proteins that drastically modify the structure and photoreactivity of the nucleic acid. In this contribution, we use multiscale molecular dynamics simulations including extended classical molecular dynamics and QM/MM biased dynamics to elucidate the coupling of electronic and structural factors leading to this photochemical outcome. In particular, we rationalize the well-described impact of the peculiar DNA environment found in spores on the favored formation of the spore photoproduct, given the small free energy barrier found for this path. Meanwhile, the specific organization of spore DNA precludes the photochemical path leading to cyclobutane pyrimidine dimers formation.TOC GRAPHICS

This study examined the microbicidal activity of 222-nm UV radiation (UV(222)), which is potentially a safer alternative to the 254-nm UV radiation (UV(254)) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, and Clostridioides difficile and a herpesvirus were all killed or inactivated by UV(222) and at lower fluences than with UV(254). B. subtilis spores and cells lacking the major DNA repair protein RecA were more sensitive to UV(222), as were spores lacking their DNA-protective proteins, the α/β-type small, acid-soluble spore proteins. The spore cores’ large amount of Ca(2+)-dipicolinic acid (∼25% of the core dry weight) also protected B. subtilis and C. difficile spores against UV(222), while spores’ proteinaceous coat may have given some slight protection against UV(222). Survivors among B. subtilis spores treated with UV(222) acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV(222) resistance. UV(222)-treated B. subtilis spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV(222) does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV(222). IMPORTANCE Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of Bacillus anthracis. Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV(254)) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV(222)) may be less harmful to people than UV(254) yet may still kill bacteria and at lower fluences than UV(254). The present work has identified the damage by UV(222) that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV(222) also inactivates a herpesvirus.

International audience; Skin has the potential to be exposed to both solar UV radiation and polycyclic aromatic hydrocarbons, especiallyin occupational environments. In the present work, we investigated how benzo[a]pyrene (B[a]P) modulatescellular phototoxicity and impacts formation and repair of pyrimidine dimers induced by simulated sunlight(SSL) in normal human keratinocytes (NHK). We were especially interested in determining whether the arylhydrocarbon receptor (AhR) was involved since it was recently shown to negatively impact repair. Addition of1 μM B[a]P after exposure to 2 minimal erythemal doses of SSL had little impact on NHK. The inverse protocolinvolving incubation with B[a]P followed by irradiation led to a strong increase in phototoxicity. Repair of DNAphotoproducts was drastically impaired. Using agonists and antagonists of AhR allowed us to conclude that thisfactor was not involved in these results. Observation of a strong increase in the level of the oxidative marker 8-oxo-7,8-dihydroguanine in the protocol involving B[a]P treatment followed by exposure to SSL strongly suggestedthat a photosensitized oxidative stress was responsible for cell death and inhibition of DNA repair.Accordingly, both adverse effects were diminished with a lower concentration of B[a]P and a lower SSL dose,leading to less oxidative stress.

International audience; Keratinocytes and melanocytes, two cutaneous cell types located within the epidermis, are the origin of most skin cancers, namely carcinomas and melanomas. These two types of tumors differ in many ways. First, carcinomas are almost 10 times more frequent than melanomas. In addition, the affected cellular pathways, the mutated genes and the metastatic properties of the tumors are not the same. This review addresses another specificity of melanomas: the role of photo-oxidative stress. UVA efficiently produces reactive oxygen species in melanocytes, which results in more frequent oxidatively generated DNA lesions than in other cell types. The question of the respective contribution of UVB-induced pyrimidine dimers and UVA-mediated oxidatively generated lesions to mutagenesis in melanoma remains open. Recent results based on next generation sequencing techniques strongly suggest that the mutational signature associated with pyrimidine dimers is overwhelming in melanomas like in skin carcinomas. UVA-induced oxidative stress may yet be indirectly linked to the genotoxic pathways involved in melanoma through its ability to hamper DNA repair activities.

International audience; Formation of pyrimidine dimers in DNA is a major initiating event in the induction of skin cancer. Model experiments suggest that, upon absorption of UVA, one type of dimers induced by UVB, the pyrimidine (6‐4) pyrimidone photoproducts, photosensitizes the formation of mutagenic cyclobutane pyrimidine dimers by triplet ‐triplet energy transfer (TTET). We investigated whether this photoreaction actually took place when 64PP were located within a DNA duplex rather than added as external sensitizers like in available data. Our results show that this process is not detectable in DNA and double‐stranded oligonucleotides exposed to a combination of UVB and UVA. TTET could only be observed, as a very minor photoreaction, in a short single‐stranded oligonucleotide bearing a 64PP. It may be concluded that 64PP‐mediated TTET does not significantly contribute to UV‐induced DNA damage. In contrast, the photoisomerization of 64PP into their Dewar valence isomers is very efficient.

MiaE (2-methylthio-N6-isopentenyl-adenosine37-tRNA monooxygenase) is a unique non-heme diiron enzyme that catalyzes the O2-dependent post-transcriptional allylic hydroxylation of a hypermodified nucleotide 2-methylthio-N6-isopentenyl-adenosine (ms2i6A37) at position 37 of selected tRNA molecules to produce 2-methylthio-N6–4-hydroxyisopentenyl-adenosine (ms2io6A37). Here, we report the in vivo activity, biochemical, spectroscopic characterization and X-ray crystal structure of MiaE from Pseudomonas putida. The investigation demonstrates that the putative pp-2188 gene encodes a MiaE enzyme. The structure shows that Pp-MiaE consists of a catalytic diiron(III) domain with a four alpha-helix bundle fold. A docking model of Pp-MiaE in complex with tRNA, combined with site directed mutagenesis and in vivo activity shed light on the importance of an additional linker region for substrate tRNA recognition. Finally, krypton-pressurized Pp-MiaE experiments, revealed the presence of defined O2 site along a conserved hydrophobic tunnel leading to the diiron active center.

International audience; CONSPECTUS. Guanine (G) radicals are precursors to DNA oxidative damage, correlated with carcinogenesis and aging. During the past few years, we demonstrated clearly an intriguing effect: G radicals can be generated upon direct absorption of UV radiation with energy significantly lower than the G ionization potential. Using nanosecond transient absorption spectroscopy, we studied the primary species, ejected electrons and guanine radicals, which result from photoionization of various DNA systems in aqueous solution. The DNA propensity to undergo electron detachment at low photon energies greatly depends on its secondary structure. Non-detected for monomers or unstacked oligomers, it may be one order of magnitude higher for G-quadruplexes than for duplexes. The experimental results point toward non-vertical processes, associated with the relaxation of electronic excited states. Theoretical studies are required to validate the mechanism and determine the factors that enter in play. In any case, such a mechanism, which may be operative over a broad excitation wavelength range, explains the occurrence of oxidative damage observed upon UVB and UVA irradiation. Quantification of G radical populations and their time evolution questioned some widespread views. It appeared that G radicals may be generated with the same probability as pyrimidine dimers, which are considered to be the major lesions induced upon absorption of low-energy UV radiation by DNA. As the most important part of the initially created radical cations undergoes deprotonation, the vast majority of the final reaction products is expected to stem from long-lived deprotonated radicals. Consequently, the widely used oxidation marker 8-oxodG is not representative of the extent of oxidative damage when this is triggered by generation of radical cations. Beyond the biological consequences, photo-generation of electron holes in G-quadruplexes may inspire applications in nano-electronics; although four-stranded structures are currently studied as molecular wires, their behavior as photoconductors has not been explored so far.In the present Account, after highlighting some key experimental issues, we first describe the photo-ionization process and, then, we focus on radicals. We use as show-cases new results obtained for genomic DNA and Oxytricha G-quadruplexes. Generation and reaction dynamics of G radicals in these systems provide a representative picture of the phenomena reported previously for duplexes and G-quadruplexes, respectively.

International audience; Biodiesel is considered as a valuable and less toxic alternative to diesel. However, cellular and molecular effects of repeated exposure to biodiesel emissions from a recent engine equipped with a diesel particle filter (DPF) remain to be characterized. To gain insights about this point, the lung transcriptional signatures were analyzed for rats (n = 6 per group) exposed to filtered air, 30% rapeseed biodiesel (B30) blend or reference diesel (RF0), upstream and downstream a DPF, for 3 weeks (3 h/day, 5 days/week). Genomic analysis revealed a modest regulation of gene expression level (lower than a 2-fold) by both fuels and a higher number of genes regulated downstream the DPF than upstream, in response to either RF0 or to B30 exhaust emissions. The presence of DPF was found to notably impact the lung gene signature of rats exposed to B30. The number of genes regulated in common by both fuels was low, which is likely due to differences in concentrations of regulated pollutants in exhausts, notably for compound organic volatiles, polycyclic aromatic hydrocarbons, NO or NOx. Nevertheless, we have identified some pathways that were activated for both exhaust emissions, such as integrin-, IGF-1- and Rac-signaling pathways, likely reflecting the effects of gas phase products. By contrast, some canonical pathways relative to "oxidative phosphorylation" and "mitochondrial dysfunction" appear as specific to B30 exhaust emission; the repression of transcripts of mitochondrial respiratory chain in lung of rats exposed to B30 downstream of DPF supports the perturbation of mitochondria function. This study done with a recent diesel engine (compliant with the European IV emission standard) and commercially-available fuels reveals that the diesel blend composition and the presence of an after treatment system may modify lung gene signature of rats repeatedly exposed to exhaust emissions, however in a rather modest manner.

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Ultraviolet B (UVB) radiation induces mutagenic DNA photoproducts, in particular cyclobutane pyrimidine dimers (CPDs), in epidermal keratinocytes (KC). To prevent skin carcinogenesis, these DNA photoproducts must be removed by nucleotide excision repair (NER) or apoptosis. Here we report that the UVB-sensitive transcription factor aryl hydrocarbon receptor (AHR) attenuates the clearance of UVB-induced CPDs in human HaCaT KC and skin from SKH-1 hairless mice. Subsequent RNA interference and inhibitor studies in KC revealed that AHR specifically suppresses global genome but not transcription-coupled NER. In further experiments, we found that the accelerated repair of CPDs in AHR-compromised KC depended on a modulation of the p27 tumor suppressor protein. Accordingly, p27 protein levels were increased in AHR-silenced KC and skin biopsies from AHR−/− mice, and critical for the improvement of NER. Besides increasing NER activity, AHR inhibition was accompanied by an enhanced occurrence of DNA double-strand breaks triggering KC apoptosis at later time points after irradiation. The UVB-activated AHR thus acts as a negative regulator of both early defense systems against carcinogenesis, NER and apoptosis, implying that it exhibits tumorigenic functions in UVB-exposed skin. In fact, AHR−/− mice developed 50% less UVB-induced cutaneous squamous cell carcinomas in a chronic photocarcinogenesis study than their AHR+/+ littermates. Taken together, our data reveal that AHR influences DNA damage-dependent responses in UVB-irradiated KC and critically contributes to skin photocarcinogenesis in mice.

DNA is considered a potential biomarker for life-detection experiments destined for Mars. Experiments were conducted to examine the photochemistry of bacterial DNA, either unprotected or within Bacillus subtilis spores, in response to exposure to simulated martian surface conditions consisting of the following: temperature (−10°C), pressure (0.7 kPa), atmospheric composition [CO2 (95.54%), N2 (2.7%), Ar (1.6%), O2 (0.13%), and H2O (0.03%)], and UV–visible–near IR solar radiation spectrum (200–1100 nm) calibrated to 4 W/m2 of UVC (200–280 nm). While the majority (99.9%) of viable spores deposited in multiple layers on spacecraft-qualified aluminum coupons were inactivated within 5 min, a detectable fraction survived for up to the equivalent of ∼115 martian sols. Spore photoproduct (SP) was the major lesion detected in spore DNA, with minor amounts of cyclobutane pyrimidine dimers (CPD), in the order TT CPD > TC CPD >> CT CPD. In addition, the (6-4)TC, but not the (6-4)TT, photoproduct was detected...

International audience; Sulfur mustard, a chemical warfare agent known to be a vesicant of skin, readily diffuses in the blood stream and reaches internal organs. In the present study, we used the analog (2-chloroethyl)-ethyl-sulfide (CEES) to provide novel data on the systemic diffusion of vesicants and on their ability to induce brain damage, which result in neurological disorders. SKH-1 hairless mice were topically exposed to CEES and sacrificed at different time until 14 days after exposure. A plasma metabolomics study showed a strong systemic impact following a selfprotection mechanism to alleviate the injury of CEES exposure. This result was confirmed by the quantification of specific biomarkers in plasma. Those were the conjugates of CEES with glutathione (GSH-CEES), cysteine (Cys-CEES) and N-acetyl-cysteine (NAC-CEES), as well as the guanine adduct (N7Gua-CEES). In brain, N7Gua-CEES could be detected both in DNA and in organ extracts. Similarly, GSH-CEES, Cys-CEES and NAC-CEES were present in the extracts until day14. Altogether, these results, based on novel exposure markers, confirm the ability of vesicants to induce internal damage following dermal exposure. The observation of alkylation damage to glutathione and DNA in brain provides an additional mechanism to the neurological insult of SM.

Objectifs L’yperite est un toxique de guerre appartenant a la famille des vesicants causant de graves brulures cutanees, possedant une toxicite oculaire, pulmonaire et systemique. En plus des effets aigues, cet agent entraine des consequences a long terme, physiques, psychiques, possedant un pouvoir carcinogene et mutagene. Aujourd’hui, il n’existe ni antidote ni traitement specifique ni marqueur d’exposition a l’yperite qui soit dose en routine comme preuve d’intoxication ou aidant a la prise en charge des intoxiques. Afin d’avoir une methode d’analyse fiable, les biomarqueurs d’expositions persistants et specifiques doivent etre valides. Methode Une premiere classe de marqueurs potentiels sont les dommages a l’ADN induits par addition de l’yperite sur les bases. Le 2-chloroethyl ethyl sulfure (CEES), l’analogue monofonctionnel de l’yperite, est egalement souvent utilise dans ces etudes car ils possedent une reactivite chimique tres proche. Les molecules electrophiles telles que l’yperite et le CEES se lient aussi a un tripeptide present en forte quantite dans les cellules, le glutathion. Ce dernier est metabolise successivement jusqu’a obtenir un derive de l’acide mercapturique (N-acetylcysteine), facilement eliminable par les urines. Pour evaluer la pertinence de ces metabolites comme biomarqueurs, des cellules, des explants de peau humaine et des souris ont ete exposes au CEES. Leur analyse est basee sur un couplage entre chromatographie liquide et spectrometrie de masse en mode tandem (UHPLC-MS/MS). La preparation de l’echantillon, basee sur l’extraction sur phase solide (solid phase extraction), se fait en ligne i.e. integree au systeme chromatographique. Resultats Grâce a la methode analytique developpee, nous avons pu quantifier l’adduit N7Guanine-CEES a la fois dans l’ADN et dans le milieu de culture de cellules HaCaT (lignee de keratinocyte) exposees au CEES. Les conjugues du glutathion (GSH-CEES) et de la cysteine (Cys-CEES) au CEES sont bien detectes dans le milieu de culture. Des experiences similaires faites sur des explants de peau humaine « fraiche » ont conduit aux memes resultats. Ces experiences in vitro montrent que les biomarqueurs choisis sont biologiquement pertinents puisque produits dans les cellules puis excretes dans le milieu extracellulaire. Des souris ont ete exposees par voie cutanee au CEES et sacrifiees jusqu’a 14 jours apres le traitement. Cette experience permet d’evaluer la persistance des metabolites dans un organisme. Dans le plasma, l’adduit N7Guanine-CEES et les conjugues Cys-CEES et N-Acetyl-Cysteine-CEES (NAC-CEES) ont ete detectes jusqu’a 14 jours apres l’exposition. A ce stade, en moyenne, la quantite de NAC-CEES est encore 12 fois superieure a la limite de detection. Conclusion A travers ces diverses experimentations biologiques, a la fois in vitro et in vivo, nous avons obtenus des resultats coherents permettant de conclure sur la pertinence des biomarqueurs etudies. L’adduit de l’ADN, N7Guanine-CEES, a ete detecte pour tous les modeles utilises. Dans le plasma, il est persistant jusqu’a 14 jours. GSH-CEES est ephemere et proche des limites de detection puisqu’il est rapidement transforme en CYS-CEES des qu’il est excrete par les cellules. La quantite de Cys-CEES est elevee par rapport aux autres biomarqueurs quel que soit le modele biologique mais son analyse est complexe de par son caractere polaire et la presence d’interferents. Ce conjugue est transforme en acide mercapturique essentiellement au niveau des reins et du foie. Ainsi, NAC-CEES n’a ete quantifie que lors de l’exposition animale. Il est cependant persistant pendant deux semaines avec une limite de detection de l’ordre de 17 fmol ce qui en fait un biomarqueur de choix. L’adaptation de cette methode analytique a l’yperite est en cours, avec de premiers resultats encourageants. A terme, l’approche pourrait permettre le diagnostic rapide de contaminations a l’yperite par un prelevement sanguin dried blood spot (DBS) sur le terrain suivi de la desorption integree au systeme SPE-UHPLC-MS/MS.

Objectif L’yperite (ou gaz moutarde) est un toxique de guerre appartenant a la famille des vesicants causant de graves brulures cutanees, et possedant egalement une toxicite oculaire, pulmonaire et systemique. De plus, cet agent entraine des effets a long terme, physiques, psychiques, et possede un pouvoir carcinogene et mutagene. Cet alkylant bifonctionnel reagit rapidement avec des biomolecules nucleophiles telles que l’ADN, les proteines et le glutathion. La mesure non-invasive de biomarqueurs dans l’urine ou le sang est necessaire pour diverses raisons : l’etablissement de la preuve de contamination, le diagnostic et le pronostic medical en cas de blessure avec d’anciennes munitions ou d’attaques terroristes. Methode Ainsi, nous avons mis en place une approche analytique basee sur un couplage entre chromatographie liquide et spectrometrie de masse en mode tandem (UHPLC-MS/MS) pour quantifier des derives du 2-chloroethyl ethyl sulfure (CEES), l’analogue monofonctionnel de l’yperite. Pour mettre au point une methode specifique et quantitative, les standards des biomarqueurs d’interet ont ete synthetises. Une premiere classe de marqueurs potentiels sont les dommages a l’ADN. L’adduit Guanine-CEES a donc ete synthetise. Pour notre second type de cibles, nous avons tire parti du fait que les molecules electrophiles telles que l’yperite et le CEES se lient, par voie chimique ou enzymatique, a un tripeptide present en forte quantite dans les cellules, le glutathion. Le conjugue Glutathion-CEES ainsi genere est ensuite metabolise pour donner successivement les conjugues Cysteine-CEES et N-acetylcysteine-CEES. Nous avons synthetise ces molecules standards ainsi que leurs derives marques par des isotopes stables. Ces etalons internes permettent de travailler en dilution isotopique par detection en spectrometrie de masse. La preparation de l’echantillon biologique, pour purifier et concentrer les biomarqueurs, est basee sur l’extraction sur phase solide (Solid Phase Extraction, SPE). Une phase polymerique hydrophobe a capacite de charge elevee, de 100 mg est utilisee. Le protocole optimise permet d’avoir des rendements de recuperation entre 60 % et 100 %. Resultats Grâce a la methode analytique developpee, nous avons pu quantifier l’adduit Guanine-CEES a la fois dans l’ADN et dans le milieu de culture de cellules HaCaT (lignee de keratinocyte) exposees au CEES. Les conjugues du glutathion et de la cysteine au CEES sont bien detectes dans le milieu de culture. Des experiences similaires ont ete faites sur des explants de peau humaine « fraiche ». Les LOQ dans les milieux de culture se situent entre 20 et 120 fmol en fonction du biomarqueur. Ces experiences in vitro montrent que les biomarqueurs choisis sont biologiquement pertinents puisque produits dans les cellules puis excretes dans le milieu extracellulaire. Ce sont donc de bons candidats pour un diagnostic non-invasif. En partant de cette methode validee, une comparaison entre la SPE « manuelle » et la SPE on-line a ete faite. Ce traitement d’echantillon « en ligne » est tres interessant car le volume d’echantillon biologique est faible, il permet egalement un gain de temps important car la preparation prend environ 20 min au lieu de 7 h. La SPE on-line permet d’obtenir de meilleures limites de quantification (entre 9 et 33 fmol) et moins de variabilite. La purification de l’echantillon est amelioree par une separation orthogonale i.e. l’echantillon est lave sur une phase PentaFluoroPhenyle (PFP) avant d’etre separe sur une colonne C18. Ainsi, il a ete possible de detecter et quantifier des biomarqueurs dans le plasma de souris intoxiquees au CEES jusqu’a 14 jours apres exposition. Conclusion Cette preparation en ligne d’echantillons biologiques, sang et urine, permet de diagnostiquer rapidement des contaminations au CEES. L’extension a l’yperite se fera par la synthese des composes standards correspondant et simple adaptation de la methode analytique.

International audience; Both UV and ionizing radiations damage DNA. Two main mechanisms, so-called direct and indirect pathways, are involved in the degradation of DNA induced by ionizing radiations. The direct effect of radiation corresponds to direct ionization of DNA (one electron ejection) whereas indirect effects are produced by reactive oxygen species generated through water radiolysis, including the highly reactive hydroxyl radicals, which damage DNA. UV (and visible) light damages DNA by again two distinct mechanisms. UVC and to a lesser extend UVB photons are directly absorbed by DNA bases, generating their excited states that are at the origin of the formation of pyrimidine dimers. UVA (and visible) light by interaction with endogenous or exogenous photosensitizers induce the formation of DNA damage through photosensitization reactions. The excited photosensitizer is able to induce either a one-electron oxidation of DNA (type I) or to produce singlet oxygen (type II) that reacts with DNA. In addition, through an energy transfer from the excited photosensitizer to DNA bases (sometime called type III mechanism) formation of pyrimidine dimers could be produced. Interestingly it has been shown recently that pyrimidine dimers are also produced by direct absorption of UVA light by DNA, even if absorption of DNA bases at these wavelengths is very low. It should be stressed that some excited photosensitizers (such as psoralens) could add directly to DNA bases to generate adducts. The review will described the differences and similarities in terms of damage formation (structure and mechanisms) between these two physical genotoxic agents.

Combined exposure to complex mixtures of polycyclic aromatic hydrocarbons (PAHs) and ultraviolet radiation (UVR) is suspected to enhance PAH skin permeability and skin cancer risk depending on PAH bioactivation. The impact of PAH mixtures (exposure dose, composition, and complexity) and UVR was assessed for PAH cutaneous absorption and metabolism using realistic exposure conditions and human skin explants. PAH complex mixtures were extracted from the industrial products coal tar pitch (CTP-I) and petroleum coke (PC-I). The synthetic mixture (CTP-S) was identically reconstituted using PAH standards. The applied dose was adjusted to 1 (PC-I, CTP-I) or 10 nmol (CTP-I, CTP-S) of benzo[a]pyrene (B[a]P). Unmetabolized PAHs were recovered from the skin surface, skin and medium, and then quantified by HPLC-fluorescence detection. PAH metabolites were collected from the medium and analyzed by GC–MS/MS. B[a]P and PAH penetration was lower for the highest B[a]P dose, industrial mixtures, and CTP-I compared to PC-I. Skin irradiation increased PAH penetration only for CTP-I. PAH uptake was poorly influenced by the different experimental conditions. PAH metabolism markedly decreased in the application of mixtures, leading to unmetabolized PAH accumulation in human skin. PAH metabolism was similar between CTP-I and PC-I, but was lower for the highest dose and the industrial mixtures, suggesting a saturation of xenobiotic metabolizing enzymes, as confirmed in a time-course study. UVR strongly inhibited all PAH metabolism. Altogether, these results underline the necessity to consider the reality of human exposure (PAH complex mixtures and UVR) during in vitro experiments to properly estimate skin absorption and metabolism.

International audience; G-quadruplexes are four-stranded DNA structures playing a key role in many biological functions and are promising for G-quadruplexes are four-stranded DNA structures playing a key role in many biological functions and are promising for applications in the field of nanoelectronics. Characterizing the generation and fate of radical cations (electron holes) within these systems is important in relation to the DNA oxidative damage and/or conductivity issues. This study focuses on guanine radicals in G-quadruplexes formed by association of four TGGGGT strands in the presence of Na+ cations, (TG4T)4/Na+. Using nanosecondtransient spectroscopy with 266 nm excitation, we quantitatively characterize hydrated ejected electrons and three types of guanine radicals. We show that, at an energy lower by 2.7 eV than the guanine ionization potential, one-photon ionization occurs with quantum yield of (3.5 ± 0.5) × 10−3. Deprotonation of the radical cations is completed within 20 μs, leading to the formation of (G-H2)• radicals, following a strongly nonexponential decay pattern. Within 10 ms, the latter undergoes tautomerization to deprotonated (G-H1)• radicals. The dynamics of the various radicals determined for (TG4T)4/Na+, in connection to those reported previously for telomeric G-quadruplexes TEL21/Na+, is correlated with energetic factors computed by quantum chemical methods. The faster deprotonation of radical cations in (TG4T)4/Na+ compared to TEL21/Na+ explainsthat irradiation of the former does not generate 8-oxodGuo, which is readily detected by high-performance liquid chromatography/mass spectrometry in the case of TEL21/Na+..