Phenylene Bis-Diphenyltriazine (TriAsorB), a new sunfilter protecting the skin against both UVB + UVA and blue light radiations.

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.

Comparison of the DNA damage response in BEAS-2B and A549 cells exposed to titanium dioxide nanoparticles.

For some decades production of titanium dioxide nanoparticle (TiO2-NP) has been increasing at a considerable rate; concerns as to the toxicity of these particles upon inhalation have been raised. Indeed, TiO2-NPs have been shown to induce significant genotoxicity and to adversely affect both major DNA repair mechanisms: base excision repair (BER) and nucleotide excision repair (NER). The aims of the present study were to (i) compare the genotoxicity of TiO2-NPs and their impact on DNA repair processes on A549 alveolar carcinoma and BEAS-2B normal bronchial lung cell lines and (ii) delve deeper into the mechanisms leading to these effects. To achieve these goals, TiO2-NPs effects on cytotoxicity, genotoxicity, DNA repair activity and DNA repair gene expression were investigated in both cell lines upon exposure to 1-100 µg/mL of anatase/rutile, 21 nm TiO2-NPs. Our results show that TiO2-NPs induce comparable cytotoxic and genotoxic responses in BEAS-2B and A549 cells. Functional response to DNA damage is observed in both cell lines and consists of an overall downregulation in DNA repair processes. When evaluating the relative importance of the two DNA repair pathways, we observed a lower impact on BER compared with NER activities, suggesting that repair of oxidatively generated DNA damage is still triggered in these cells. This response becomes measureable at 4 h of exposure in BEAS-2B but only after 48 h of exposure in A549 cells. The delayed response in A549 cells is due to an initial overall and intense downregulation of the genes encoding DNA repair proteins. This overall downregulation correlates with increased methylation of DNA repair gene promoters and downregulation of NRF2 and BRCA1, which may thus be considered as upstream regulators. These results strengthen the evidence that TiO2-NP induces indirect genotoxicity in lung cells, via modulation of DNA repair processes, and shed some light on the mechanisms behind this effect.

Dewar valence isomers, the third type of environmentally relevant DNA photoproducts induced by solar radiation.

UV-induced DNA damage is the main initiating event in solar carcinogenesis. UV radiation is known to induce pyrimidine dimers in DNA, including cyclobutane dimers and (6-4) photoproducts which have been extensively studied. In contrast, much less attention has been paid to Dewar valence isomers, the photoisomerisation product of (6-4) photoproducts. Yet, the available data show that Dewar isomers can be produced by exposure to sunlight and may lead to mutations. Dewars are thus environmentally and biologically relevant. The present review summarizes currently available information on the formation, mutagenic properties and repair of this class of UV-induced DNA damage.

A new hair follicle-derived human epidermal model for the evaluation of sunscreen genoprotection.

Induction of skin cancer is the most deleterious effect of excessive exposure to sunlight. Accurate evaluation of sunscreens to protect the genome is thus of major importance. In particular, the ability of suncare products to prevent the formation of DNA damage should be evaluated more directly since the Sun Protection Factor is only related to erythema induction. For this purpose, we developed an in vitro approach using a recently characterized reconstituted human epidermis (RHE) model engineered from hair follicle. The relevance of this skin substitute in terms of UV-induced genotoxicity was compared to ex vivo explants exposed to solar-simulated radiation (SSR). The yield of bipyrimidine photoproducts, their rate of repair, and the induction of apoptosis were very similar in both types of skin samples. In order to evaluate the protection afforded by sunscreen against DNA damage, bipyrimidine photoproducts were quantified in tissue models following SSR exposure in the presence or absence of a SPF50+ formula. A rather high DNA protection factor of approximately 20 was found in RHE, very similar to that determined for explants. Thus, RHE is a good surrogate to human skin, and also a convenient and useful tool for investigation of the genoprotection of sunscreens.

Radiation-mediated formation of complex damage to DNA: a chemical aspect overview.

During the last three decades, a considerable amount of work has been undertaken to determine the nature, the mechanism of formation and the biological consequences of radiation-induced DNA lesions. Most of the information was obtained via the development of chemical approaches, including theoretical, analytical and organic synthesis methods. Since it is not possible to present all the results obtained in this review article, we will focus on recent data dealing with the formation of complex DNA lesions produced by a single oxidation event, as these lesions may play a significant role in cellular responses to ionizing radiation and also to other sources of oxidative stress. Through the description of specific results, the contribution of different chemical disciplines in the assessment of the structure, the identification of the mechanism of formation and the biological impacts in terms of repair and mutagenicity of these complex radiation-induced DNA lesions will be highlighted.

Cytotoxic and genotoxic impact of TiO2 nanoparticles on A549 cells.

Titania nanoparticles are produced by tons, and included in commercial products, raising concerns about their potential impact on human health. This study relates their cytotoxic and genotoxic impact on a cell line representative of human lung, namely A549 alveolar epithelial cells.

Changes in antioxidant status and biochemical parameters after orally cadmium administration in females rats.

The research was conducted to investigate the toxic effects of cadmium chloride (CdCl2), administered during gestation period on female Wistar rats. Pregnant rats received CdCl2 (20 mg/l, orally) from Day 6 to Day 19 of pregnancy. Results showed that Cd treatment induced a decrease in body weight gain. The relative liver weight increased significantly, with a marked decrease of glycogen and total lipids content. The administration of Cd induced hepatotoxicity as indicated by elevations in plasma alanine aminotransferase (ALT), aspartate aminotransferase and lactate dehydrogenase (LDH) activities (p < 0.05). Treatment with CdCl2 caused a significant (p < 0.05) increase in glucose. A significant increase was observed in the level of MDA and 8-oxodGuo tissues in the cadmium-exposed group compared to the control group (p < 0.05). Results showed that cadmium given to dams led to an oxidative stress and DNA damage in tissues of pregnant rats.

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.

Determination of new types of DNA lesions in human sperm.

Careful attention has been focused recently on DNA quality in human IVF. Therefore a variety of methods has been developed to evaluate DNA integrity, especially concerning fragmentation. Using liquid chromatography and mass spectrometry (LC/MS/MS) for our best sperm samples, we have established reference values for several oxidative lesions, in order to gain insights into the cause of DNA lesions. Besides 8-oxodeoxyguanosine, we found rather high levels of two ethenonucleosides: 1,N6-ethenoadenosine and 1,N2-ethenoguanosine. These compounds probably arise from a reaction with 4-hydroxy-2-nonenal, the main aldehyde compound released during lipid peroxidation, or after occupational exposure to vinyl chloride. The quantity of chlorinated bases detected is low. All of this decay has to be repaired by the oocytes at the time of fertilization or immediately after. This aspect should not be overlooked in assisted reproductive technology, in order to understand risks and limitations.

Sensitivity to polychromatic UV-radiation of strains of Deinococcus radiodurans differing in their DNA repair capacity.

PURPOSE: To characterize the ultraviolet (UV) sensitivity and establish the UV-induced DNA damage profile of cells of four Deinococcus radiodurans strains. The investigated strains differ in their radiation susceptibility, leading to a classification into a UV-sensitive (UVS78 and 1R1A) and a UV-resistant class (wild type strain R1 and 262). MATERIALS AND METHODS: Deinococcus radiodurans cells were exposed in suspension to monochromatic 254 nm (UV-C) and polychromatic UV radiations; the surviving fraction was determined by assessing the ability of the bacteria to form colonies. The UV-induced DNA lesions were measured quantitatively using an accurate and highly specific assay that involves the combination of high performance liquid chromatography (HPLC) with tandem mass spectrometry detection. RESULTS: Analysis of the DNA photoproducts showed that the TC (6-4) photoproduct and the TT and TC cyclobutane dimers were the major lesions induced by UV-C and UV-(>200 nm)-radiation. The UV-sensitive class was approx. 10 times more susceptible to UV-C and UV-(>200 nm)-radiations than the resistant class. Interestingly, the survival curves of all investigated strains become similar with longer UV wavelengths in the UV-(>315 nm)-radiation range. This observation suggests that the repair mechanisms of the UV-resistant class are not specifically effective for damage produced by UV of the >315 nm range. However, the initial amount of DNA photoproducts produced upon irradiation was found to be the same in resistant and sensitive strains for each wavelength range. CONCLUSION: Compared to mammalian cells, the DNA of Deinococcus radiodurans cells is less susceptible to the photo-induced formation of thymine cyclobutane dimers as inferred from comparative analysis. The ongoing investigations may contribute to a better understanding of the mechanism of DNA photoprotection against the direct effects of UV radiation. This may be of interest in the present context of a possible continuous decrease in the ozone layer thickness.

Resveratrol enhances UVA-induced DNA damage in HaCaT human keratinocytes.

Resveratrol, a polyphenolic phytoalexin, is a very effective antioxidant that also exhibits strong antiproliferative and anti-inflammatory properties. Recent studies have provided support for the use of resveratrol in human cancer chemoprevention, in combination with either chemotherapeutic drugs or cytotoxic factors for a most efficient treatment of drug refractory tumor cells. Resveratrol is also widely used in topical preparations, as a chemoprotective compound against development of several cutaneous disorders, including skin cancer. Nevertheless, the combined effect of resveratrol and UVA irradiation on cellular toxicity and DNA damage has never been assessed. The aim of this work was to investigate the effect of resveratrol on cell fate in immortalized human keratinocytes HaCaT cells. The results indicated that resveratrol potentiates the production of significant amounts of 8-oxo-7,8-dihydro-2'-deoxyguanosine in UVA-irradiated genomic DNA. Moreover, the combination of resveratrol with UVA significantly enhances the induction of DNA strand breaks and cell death in HaCaT keratinocytes. The conclusion is a potential hazardous effect of topical application of resveratrol, particularly on regions exposed to sunlight.

Direct and indirect effects of UV radiation on DNA and its components.

In this survey, emphasis was placed on the main photoreactions of nucleic acid components, involving both direct and indirect effects. The main UVB- and UVA-induced DNA photoproducts, together with the mechanisms of their formation, are described. Information on the photoproduct distribution within cellular DNA is also provided, taking into account the limitations of the different analytical methods applied to monitor the formation of the DNA damage. Thus, the formation of the main DNA dimeric pyrimidine lesions produced by direct absorption of UVB photons was assessed using a powerful HPLC-tandem mass spectrometry assay. In addition, it was found that UVA photooxidation damage mostly involves the guanine residues of cellular DNA as the result of singlet oxygen generation by still unknown endogenous photosensitizers.

Individual determination of the yield of the main UV-induced dimeric pyrimidine photoproducts in DNA suggests a high mutagenicity of CC photolesions.

Bipyrimidine photoproducts induced in DNA by UVB radiation include cyclobutane dimers, (6-4) photoproducts, and their related Dewar valence isomers. Even though these lesions have been extensively studied, their rate of formation within DNA is still not known for each possible bipyrimidine site (TT, TC, CT, and CC). Using a method based on the coupling of liquid chromatography to mass spectrometry, we determined the distribution of the 12 possible bipyrimidine photoproducts within isolated and cellular DNA. TT and TC were found to be the most photoreactive sequences, whereas lower amounts of damage were produced at CT and CC sites. In addition to this quantitative aspect, sequence effects were observed on the relative yield of (6-4) adducts with respect to cyclobutane pyrimidine dimers. Another interesting result is the lack of formation of Dewar valence isomers in detectable amounts within the DNA of cells exposed to low doses of UVB radiation. The photoproduct distribution obtained does not fully correlate with the UV mutation spectrum. A major striking observation deals with the low yield of cytosine-cytosine photoproducts which are likely to be associated with the UV-specific CC to TT tandem mutation.

The role of duck hepatitis B virus and aflatoxin B1 in the induction of oxidative stress in the liver.

The aim of our study was to use the Pekin duck model to investigate the interactions between hepadnaviral infection and aflatoxin B1 (AFB1) exposure including the role of both factors in the induction of oxidative stress in the liver. AFB1 exposure of duck hepatitis B virus (DHBV) infected Pekin ducks induced a significant increase in viral replication associated with an intense biliary ductular cells proliferation. Interestingly, extremely high levels of AFB1-DNA adducts (40-120 pmol AFB1-Fapy/mg DNA) and AFB1-albumin adducts (1,500-3,000 pg AFB1-lys Eq/mg albumin) were detected in duck liver and serum respectively, as compared to other animal species exposed to a similar AFB1 dose. DHBV infection was found to induce a non-significant increase in AFB1-albumin adduct levels in duck serum. During the treatment duration there was no effect on formation of oxidative base damage within DNA and no effect on oxidative lipid peroxidation following either viral infection or AFB1 exposure. In terms of hepatic antioxidant enzymes (catalase, superoxide dismutase (SOD), glutathione peroxidase) a significant increase in SOD activity occurred following AFB1 exposure, but not DHBV infection, but this was observed only after the cessation of treatment, when biliary ductular cells proliferation was reduced.

Oxidative damage and induced mutations in m13mp2 phage DNA exposed to N-nitrosopyrrolidine with UVA radiation.

N:-Nitrosopyrrolidine (NPYR) is carcinogenic in rodents and undergoes alpha-hydroxylation upon microsomal CYP450 metabolism, giving rise to mutations. Previously, we reported the direct mutagenicity of NPYR, under ultraviolet A (UVA) irradiation, towards Salmonella typhimurium and phage M13mp2. In the present study, we measured the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in a replicative form of M13mp2 DNA exposed to NPYR plus UVA. Formation of 5-hydroxy-2'-deoxycytidine in calf thymus DNA treated with NPYR plus UVA was also observed. Singlet oxygen is likely to account for the formation of 8-oxodGuo. We analyzed the spectrum of mutations in lacZalpha of M13mp2 phages produced on transfecting Escherichia coli with the replicative form of phage DNA that had been treated with NPYR plus UVA. The role of oxidative DNA damage in mutagenesis was explored using mutM-proficient and -deficient E.coli strains as the hosts. A higher level of mutation was observed with the mutM-deficient host than with the -proficient host. Base substitutions at GC pairs predominated in both mutM-proficient and -deficient hosts. With the mutM-deficient host, we observed an overall increase in the percentage of GC-->TA transversions. In addition we noted that there were fewer GC-->AT transitions than in the mutM-proficient host. With these hosts, different hot spots were observed and a new GC-->TA hot spot was produced. The formation of 8-oxodGuo in DNA, which is known to induce GC-->TA transversion, may contribute to mutagenesis by NPYR plus UVA.

High-performance liquid chromatography--tandem mass spectrometry measurement of radiation-induced base damage to isolated and cellular DNA.

A method involving high-performance liquid chromatography (HPLC) separation associated with tandem mass spectrometry (MS/MS) detection in the multiple-reaction monitoring mode was set up for the assessment of radiation-induced degradation products of DNA bases. This sensitive and specific assay is aimed at assessing six oxidized 2'-deoxyribonucleosides and two modified purine bases within both isolated and cellular DNA. For this purpose, stable isotopically labeled internal standards were prepared and used for isotope dilution mass spectrometry measurements. The latter method was validated through a comparison with two other assays, including HPLC associated with electrochemical detection and gas chromatography coupled to mass spectrometry. Using the specific and sensitive HPLC-MS/MS approach, 5,6-dihydroxy-5,6-dihydrothymidine, 5-hydroxy-2'-deoxyuridine, 5-(hydroxymethyl)-2'-deoxyuridine, 5-formyl-2'-deoxyuridine, 8-oxo-7,8-dihydro-2'-deoxyadenosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, 4, 6-diamino-5-formamidopyrimidine, and 2, 6-diamino-4-hydroxy-5-formamidopyrimidine were quantified within both isolated and cellular DNA upon exposure to gamma-radiation.

DNA damage induced in cells by gamma and UVA radiation as measured by HPLC/GC-MS and HPLC-EC and Comet assay.

The aim of the work was to measure DNA damage induced within tumoral human monocytes by gamma rays, UVA radiation, and exogenous photosensitizers. The accurate HPLC-EC assay was used to determine the level of 8-oxodGuo. The formation of FapyGua and FapyAde was monitored by HPLC/GC-MS analyses after formic acid hydrolysis at room temperature. For this purpose, cells were exposed to relatively high doses of gamma rays and UVA radiation. The extent of formation of FapyGua in the DNA of cells exposed to gamma rays was estimated to be more than 2-fold higher than that of 8-oxodGuo, i.e., about 0. 027 lesion per 10(6) bases per Gy. The yield of FapyAde was estimated to be 1 order of magnitude lower. The latter results were used to calibrate the alkaline comet assay associated with DNA N-glycosylases. The latter approach allowed the determination of the background level (0.11-0.16 Fpg-sensitive site/10(6) bases) and the yields of strand breaks and DNA base damage upon low irradiation doses. Insights into the mechanism of radiation-induced DNA damage were gained from these measurements. A major involvement of (1)O(2) with respect to hydroxyl radicals and type I photosensitization was thus observed within cells exposed to UVA radiation.

Electrospray-mass spectrometry characterization and measurement of far-UV-induced thymine photoproducts.

Far-UV-induced formation of dimeric pyrimidine photoproducts within DNA is a major cause of the carcinogenic effects of solar light. The chemical structure of this class of lesion has been mostly determined by studies on model compounds. The present work is aimed at providing mass spectrometry data on the thymine-thymine photoproducts, including the diastereoisomers of the cyclobutane dimer, the (6-4) adduct, the related Dewar valence isomer and the spore photoproduct. Fragmentation mass spectra of the modified bases, nucleosides, dinucleoside monophosphates and dinucleotides were recorded following electrospray ionization with either triple-quadrupolar or ion-trap detection. The results showed differences in fragmentation pattern between the different types of photoproducts. In addition, a drastic effect of the diastereoisometry was observed for the cyclobutane dimers. A sensitive detection technique has been developed for the analysis of dinucleoside monophosphate photoproducts by high-performance liquid chromatography associated with mass spectrometry in the negative mode with multiple reaction-monitoring detection.