Photogenotoxicity of mammalian cells: a review of the different assays for in vitro testing

During the past several years, phototoxicity has been studied at the molecular level, and these studies have provided new insights in the field of DNA lesion characterization, DNA repair and cell response to ultraviolet (UV)-induced stress. The development of new antibiotics and antiinflammatory drugs has highlighted the necessity to develop the assessment of phototoxicity in the safety evaluation of new chemical compounds. This paper aims at reviewing the known molecular mechanisms of the cellular response to UV-induced stress, the in vitro methods that can be proposed and used to screen for toxicity of sunlight and the photosensitization process resulting from the activation of drugs by light. UV sources, biological systems and endpoints of interest in that particular objective are listed. Phototoxic effects span from the cytotoxic-apoptotic effect to the induction of primary DNA damage, DNA repair and a variety of stress genes acting on the cell cycle and the fate of the cell. Ultimately, it can lead to the induction of hereditary DNA modification. A variety of assays are proposed to specifically address all these particular consequences of UV-induced toxicity.     

Widely tunable THz synthesizer

The generation of cw-THz radiation by photomixing is particularly suited to the high resolution spectroscopy of gases; nevertheless, until recently, it has suffered from a lack of frequency metrology. Frequency combs are a powerful tool that can transfer microwave frequency standards to optical frequencies and a single comb has permitted accurate (10⁻⁸) THz frequency synthesis with a limited tuning range. A THz synthesizer composed of three extended cavity laser diodes phase locked to a frequency comb has been constructed and its utility for high resolution gas phase spectroscopy demonstrated. The third laser diode allows a larger tuning range of up to 300 MHz to be achieved without the need for large frequency excursions, while the frequency comb provides a versatile link to be established from any traceable microwave frequency standard. The use of a single frequency comb as a reference for all of the cw-lasers eliminates the dependency of synthesized frequency on the carrier envelope offset frequency. This greatly simplifies the frequency comb stabilization requirements and leads to a reduced instrument complexity.     

Assessment of the photoprotection properties of sunscreens by chromatographic measurement of DNA damage in skin explants

Evaluation of the photoprotection provided by sunscreens is performed either through the induction of erythema and expressed as the sun protection factor (SPF), or by the UVA-mediated persistent pigment darkening (PPD). None of these two endpoints has a link with skin cancer, the most deleterious consequence of excess exposure to solar UV radiation. We thus set up a complementary approach to evaluate the protection provided by sunscreens to the genome of human skin. This is based on the quantification of the thymine cyclobutane dimer (TT-CPD), the main DNA lesion induced by both UVB and UVA radiations. Irradiations were performed ex vivo on human skin explants and the level of TT-CPD in DNA was determined by HPLC associated with tandem mass spectrometry. The technique was first optimized and validated with three standard sunscreens. The study was then extended to the evaluation of a commercial high SPF sunscreen exhibiting efficient UVA photoprotection. The DNA protecting factor was found to reflect the ratio between UVB and UVA photoprotection, although the absolute values of the genomic protection were, as a general trend, lower than either SPF or PPD. These data show the usefulness of the proposed approach for the evaluation of the genoprotection afforded by sunscreens.     

Theoretical and experimental studies of CH3X-Y2 rotational line shapes for atmospheric spectra modelling: application to room-temperature CH3Cl-O2

The case of symmetric tops CH(3)X (X = Br, Cl, F, …) perturbed by non-polar diatoms Y(2) (Y = N(2), O(2), …) is analysed from the viewpoint of theoretical collisional broadening of their rotational lines observed in atmospheric spectra. A semi-classical approach involving an exponential representation of the scattering operator and exact trajectories governed by the isotropic potential is presented. For the first time the active molecule is strictly treated as a symmetric top and the atom-atom interactions are included in the intermolecular potential model. It is shown for the CH(3)Cl-O(2) system that these interactions contribute significantly to the line width for all values of the rotational quantum numbers J and K. Additional testing of modifications required in the semi-classical formalism for a correct application of the cumulant expansion is performed and it is shown that the use of the cumulant average on the rotational states of the perturbing molecule leads to entirely negligible effects for the not very strongly interacting CH(3)Cl-O(2) system. In order to check the theoretical predictions and to extend the scarce experimental data available in the literature to higher values of the rotational quantum numbers, new measurements of room-temperature O(2)-broadened CH(3)Cl rotational lines are carried out by a photomixing continuous-wave terahertz spectrometer. The experimental line widths extracted with a Voigt profile model demonstrate an excellent agreement with theoretical results up to very high J-values (J = 31, 37, 40, 45, 50).     

Gas-phase vibrational spectroscopy and ab initio study of organophosphorus compounds: discrimination between species and conformers

Gas phase vibrational spectra of dimethyl methylphosphonate (DMMP), trimethyl phosphate (TMP), and triethyl phosphate (TEP) have been measured using FTIR spectroscopy. For DMMP, TMP, and TEP, most of the infrared active vibrational modes have been observed in the 50-5000 cm (-1) spectral range, allowing an unambiguous discrimination between the three molecules. The vibrational analysis of the spectra was performed by comparing with MP2 and B3LYP harmonic and anharmonic force field ab initio calculations. The extension to anharmonic calculations provides the best agreement for the mid-infrared and the near-infrared spectra, but they do not improve the harmonic frequency predictions in the far-infrared domain. This part of the vibrational spectra associated with collective and nonlocalized vibrational modes presents the largest frequency differences between the two lowest energy conformers of DMMP and TMP. These two conformers were taken into account in the vibrational assignment of the spectra. Their experimental evidence was obtained by deconvoluting vibrational bands in the mid-infrared and in the far-infrared regions, respectively. For TEP, the conformational landscape appears very complicated at ambient temperature, and a further analysis at low temperature is required to explain the vibrational features of each conformer.     

Pickering Emulsion Stabilized by Catalytic Polyoxometalate Nanoparticles: A New Effective Medium for Oxidation Reactions

Decyl‐, dodecyl‐, and tetradecyltrimethylammonium cations were combined with the catalytic polyoxometalate [PW₁₂O₄₀]^3? anion to give spherical and monodisperse nanoparticles that are able to stabilize emulsions in the presence of water and an aromatic solvent. This triphasic liquid/solid/liquid system, based on a catalytic surfactant, is particularly efficient as a reaction medium for epoxidation reactions that involve hydrogen peroxide. The reactions proceed at competitive rates with straightforward separation of the phases by centrifugation. Such catalytic “Pickering” emulsions combine the advantages of heterogeneous catalysis and biphasic catalysis without the drawbacks (e.g., catalyst leaching or separation time).     

Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions(†)

The survey focuses on recent aspects of photochemical reactions to cellular DNA that are implicated through the predominant formation of mostly bipyrimidine photoproducts in deleterious effects of human exposure to sunlight. Recent developments in analytical methods have allowed accurate and quantitative measurements of the main DNA photoproducts in cells and human skin. Highly mutagenic CC and CT bipyrimidine photoproducts, including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are generated in low yields with respect to TT and TC photoproducts. Another striking finding deals with the formation of Dewar valence isomers, the third class of bipyrimidine photoproducts that is accounted for by UVA-mediated isomerization of initially UVB generated 6-4PPs. Cyclobutadithymine (T<>T) has been unambiguously shown to be involved in the genotoxicity of UVA radiation. Thus, T<>T is formed in UVA-irradiated cellular DNA according to a direct excitation mechanism with a higher efficiency than oxidatively generated DNA damage that arises mostly through the Type II photosensitization mechanism. C<>C and C<>T are repaired at rates intermediate between those of T<>T and 6-4TT. Evidence has been also provided for the occurrence of photosensitized reactions mediated by exogenous agents that act either in an independent way or through photodynamic effects.     

THz media characterization by means of coherent homodyne detection, results and potential applications

This paper describes the performance of a coherent homodyne spectrometer to preserve phase information of the THz electric field. Thereby, optical constants of a high resistive silicon sample are presented between 200 GHz and 1.2 THz. The phase shift introduced by such a thick sample is much greater than 2π, and the manner of solving the ambiguity of the modulo 2π is discussed and can be used to surround the refractive index mainly at high frequency where the signal to noise ratio decreases. The analysis of the coherent homodyne signal as a function as the frequency at a fixed delay line permits deduction of sample thickness or measurement of distance without any mechanical displacement, and so facilitates potential ranging and tomography studies. PACS 73.40.sx; 33.20.Bx; 39.30.+w     

Gas filter correlation instrument for air monitoring at submillimeter wavelengths

A gas filter correlation (GFC) instrument for air monitoring at submillimeter wavelengths has been developed. We used a high-power terahertz radiation source in combination with a specific gas filter cell to obtain a highly selective instrument to differentiate species present in an unknown mixture. This approach provides a new method for survey measurements in the spectrum from 100 to 1000 GHz, in which many molecular rotational lines appear. Basic operational considerations and preliminary experiments with hydrogen sulfide are described.     

The Human Melanocyte as a Particular Target for UVA Radiation and an Endpoint for Photoprotection Assessment

The induction of DNA breaks by UVA (320-400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin-related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.