Michel De Méo

CeO2 nanoparticles induce DNA damage towards human dermal fibroblasts in vitro

Cerium dioxide nanoparticles have been proposed for an increasing number of applications in biomedicine, cosmetic, as polishing materials and also as byproducts from automotive fuel additives. The aim of this study was to examine the potential in vitro cyto- and genotoxicity of nano-sized CeO2 (7 nm) on human dermal fibroblasts. By combining a physico-chemical and a (geno)toxicological approach, we defined the causal mechanisms linking the physico-chemical properties of nano-CeO2 with their biological effects. Using X-ray absorption spectroscopy, we observed a reduction of 21±4% of the Ce4+ atoms localized at the surface of CeO2 nanoparticles due to the interactions with organic molecules present in biological media. These particles induced strong DNA lesions and chromosome damage related to an oxidative stress. These genotoxic effects occurred at very low doses, which highlighted the importance of a genotoxicological approach during the assessment of the toxicity of nanoparticles.

The expression of genes induced in melanocytes by exposure to 365-nm UVA: study by cDNA arrays and real-time quantitative RT-PCR.

Ultraviolet A radiation (UVA; 320-400 nm) constitutes more than 90% of the terrestrial UV solar energy. This type of radiation generates reactive oxygen species and consequently induces DNA damage. UVA irradiation is now considered to be an important carcinogen agent especially in the development of melanoma. UVA radiation is known to activate several pathways in mammalian cells. We have used cDNA arrays to analyze differential gene expression in primary cultures of human melanocytes in response to 365-nm UVA. Among 588 genes studied, 11 were overexpressed. These genes included genes involved in cell cycle regulation (GADD45, CIP1/WAF1), in stress response (HSP70, HSP40, HSP86), in apoptosis (GADD153, tristetraproline) and genes encoding transcription factors (EGR-1, ETR-101, c-JUN, ATF4). This coordinate gene regulation was confirmed by real-time quantitative RT-PCR.

Genotoxicity of visible light (400–800 nm) and photoprotection assessment of ectoin, l-ergothioneine and mannitol and four sunscreens

This study was designed to determine the genotoxic effects of visible (400-800nm) and ultraviolet A (UVA)/visible (315-800nm) lights on human keratinocytes and CHO cells. The alkaline comet assay was used to quantify DNA-damage. In addition, photo-dependent cytogenetic lesions were assessed in CHO cells by the micronucleus test. Three protective compounds [ectoin, l-ergothioneine (ERT) and mannitol] were tested with the comet assay for their effectiveness to reduce DNA single-strand breaks (SSB). Finally, the genomic photoprotections of two broad-band sunscreens and their tinted analogues were assessed by the comet assay. The WST-1 cytotoxicity assay revealed a decrease of the keratinocyte viability of 30% and 13% for the highest UVA/visible and visible irradiations (15 and 13.8J/cm(2), respectively). Visible as well as UVA/visible lights induced DNA SSB and micronuclei, in a dose-dependent manner. The level of DNA breakage induced by visible light was 50% of the one generated by UVA/visible irradiation. However, UVA radiations were 10 times more effective than visible radiations to produce SSB. The DNA lesions induced by visible and UVA/visible lights were reduced after a 1-h preincubation period with the three tested compounds. The maximal protective effects were 92.7%, 97.9% and 52.0% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against visible light and 68.9%, 59.8% and 62.7% for ectoin (0.1mM), ERT (0.5mM) and mannitol (1.5mM), respectively, against UVA/visible light. Thus, visible light was genotoxic on human keratinocytes and CHO cells through oxidative stress mechanisms similar to the ones induced by UVA radiations. The four tested sunscreens efficiently prevented DNA lesions that were induced by both visible and UVA/visible irradiations. The tinted sunscreens were slightly more effective that their colorless analogues. There is a need to complement sunscreen formulations with additional molecules to obtain a complete internal and external photoprotection against both UVA and visible lights.

Lowering of 5-nitroimidazole's mutagenicity: Towards optimal antiparasitic pharmacophore

To improve the antiparasitic pharmacophore, 20 5-nitroimidazoles bearing an arylsulfonylmethyl group were prepared from commercial imidazoles. The antiparasitic activity of these molecules was assessed against Trichomonas vaginalis, the in vitro cytotoxicity was evaluated on human monocytes and the mutagenicity was determined by the Salmonella mutagenicity assay. All IC(50) on T. vaginalis were below the one of metronidazole. The determination of the specificity indexes (SIs), defined as the ratios of the cytotoxic activity and the antitrichomonas activity, indicated that 11 derivatives had a SI over the one of metronidazole. Molecules, bearing an additional methyl group on the 2-position, showed a lower mutagenicity than metronidazole. Moreover, three derivatives were characterized by a low mutagenicity and an efficient antitrichomonas activity.

Evaluation of the Genotoxicity of River Sediments From Industrialized and Unaffected Areas Using a Battery of Short-Term Bioassays

The present investigation evaluated the capacity of the Salmonella mutagenicity test, the comet assay, and the micronucleus assay to detect and characterize the genotoxic profile of river sediments. Three stations were selected on an urban river (Bouches du Rhône, France) exposed to various sources of industrial and urban pollution (StA, StB, and StC) and one station on its tributary (StD). One station in a nonurban river was included (REF). The concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) were determined by HPLC, and the genotoxicity of the sediments was monitored by the Salmonella mutagenicity test (TA98 + S9, YG1041 ± S9), the comet assay, and the micronucleus assay on CHO cells. Chemical analysis showed that the total PAH concentrations ranged from 23 μg kg−1 dw (REF) to 1285 μg kg−1 dw (StD). All the sediments were mutagenic in the Salmonella mutagenicity test. The mutagenicity was probably induced by the presence of nitroarenes (StA, StB, StC, and StD) and aromatic amines (REF) as deduced from the mutagenicity profiles of strains YG1041 ± S9 and TA98 + S9. The comet assay revealed direct DNA lesions in REF, StA, and StB sediments and metabolization‐dependent DNA damage in StC and StD. The micronucleus assay showed an absence of clastogenicity for StA ± S9 and StC‐S9, and a significant clastogenicity ± S9 for the three other stations. The genotoxicity ranking determined by the comet assay + S9 matched the ranking of total and carcinogenic PAH concentrations, and this assay was found to be the most sensitive. Environ. Mol. Mutagen., 2008.

Comet assay on mouse oocytes: an improved technique to evaluate genotoxic risk on female germ cells.

OBJECTIVE To develop and validate an efficient comet assay on mouse oocytes without depellucidation. DESIGN In vitro experiments using a murine model. SETTING Biogenotoxicology research laboratory in Aix-Marseille II University, France. ANIMAL(S) CD1 prepubescent female mice. INTERVENTION(S) DNA lesions in oocytes were evaluated by the alkaline comet assay. After oocyte retrieval, we first studied the effect of zona pellucida (ZP) on comet morphology. For this study, we applied the comet assay to mature oocytes with and without ZP after exposure to simulated sunlight irradiation (SSI) compared with negative controls. Next, nondepellucidated mouse oocytes were exposed to three well-known genotoxic agents (SSI, methylmethanesulfonate [MMS], and hydrogen peroxide [H(2)O(2)]) and compared with negative controls. Images of oocytes were analyzed with Komet software. MAIN OUTCOME MEASURE(S) DNA damages were quantified and expressed as olive tail moment (OTM), defined as the product of the tail length and the fraction of total DNA in the tail. OTMχ(2) were calculated from OTM; they corresponded to the degrees of freedom (n) of each OTM distribution obtained from at least 50 oocytes. OTMχ(2) is an indicator of DNA lesions. The test was considered positive and statistically significant when OTMχ(2) increased in oocytes compared with the medium-only control cells. RESULT(S) There was no difference in comet aspect between oocyte groups with and without ZP. The three genotoxic agents significantly increased DNA damages as compared with the control groups. The OTMχ(2) values were (mean ± SD): 2.1 ± 0.07, 7.73 ± 0.35, 3.35 ± 0.15, and 12.4 ± 0.51 in control, SSI, MMS, and H(2)O(2) groups, respectively. CONCLUSION(S) Comet assay on non depellucidated mouse oocytes is a rapid and easy test. This assay would be useful to assess the genotoxicity on female germ cells of chemicals, drugs, or environmental pollutants and the efficiency of antioxidant molecules.

Evaluation of Sunscreen Protection in Human Melanocytes Exposed to UVA or UVB Irradiation Using the Alkaline Comet Assay

The in vivo assessment of sunscreen protection does not include the photogenotoxicity of UVA or UVB solar radiation. Using the comet assay we have developed a simple and rapid technique to quantify sunscreen efficacy against DNA damage induced by UV light. Cutaneous human melanocytes from primary cultures were embedded in low‐melting point (LPM) agarose and exposed to UVA (0.8 J/cm2) or to UVB (0.06 J/cm2) through a quartz slide covered with 10 μL volumes of sunscreens. DNA single‐strand breaks induced directly by UVA at 4°C and indirectly through nucleotide excision repair by UVB following a 35 min incubation period at 37°C were quantified using the comet assay. Tail moments (TM) (tail length ×%tail DNA) of 100 cells/sample were determined by image analysis. DNA damage was evaluated with a nonlinear regression analysis on the normalized distribution frequencies of TM using a χ2 function. The coefficients of genomic protection (CGP) were defined as the percentage of inhibition of DNA lesions caused by the sunscreens. Twenty‐one sunscreens were evaluated, and the calculated CGP were compared with the in vivo sun protective factor (SPF) and with the protection factor UVA (PFA). Nonlinear relationships were found between SPF and CGPUVB and between PFA and CGPUVA.

Identification of disinfection by-products in freshwater and seawater swimming pools and evaluation of genotoxicity.

Exposure to disinfection byproducts (DBPs) in swimming pools has been linked to adverse health effects. Numerous DBPs that occur in swimming pools are genotoxic and carcinogenic. This toxicity is of a greater concern in the case of brominated DBPs that have been shown to have substantially greater toxicities than their chlorinated analogs. In chlorinated seawater swimming pools, brominated DBPs are formed due to the high content of bromide. Nevertheless, very little data is reported about DBP occurrence and mutagenicity of water in these pools. In the present study, three seawater and one freshwater swimming pools located in Southeastern France were investigated to determine qualitatively and quantitatively their DBP contents. An evaluation of the genotoxic properties of water samples of the freshwater pool and a seawater pool was conducted through the Salmonella assay (Ames test). The predominant DBPs identified in the freshwater pool were chlorinated species and included trichloroacetic acid, chloral hydrate, dichloroacetonitrile, 1,1,1-trichloropropanone and chloroform. In the seawater pools, brominated DBPs were the predominant species and included dibromoacetic acid, bromoform and dibromoacetonitile. Bromal hydrate levels were also reported. In both types of pools, haloacetic acids were the most prevalent chemical class among the analyzed DBP classes. The distribution of other DBP classes varied depending on the type of pool. As to genotoxicity, the results of Ames test showed higher mutagenicity in the freshwater pool as a consequence of its considerably higher DBP contents in comparison to the tested seawater pool.

Procedures for destruction of patulin in laboratory wastes

Patulin is immunosuppressive and there is limited evidence of its carcinogenicity in experimental animals. The International Agency for Research on Cancer (IARC) initiated a programme for the development of degradation techniques for the commonly investigated mycotoxins. As a part of this programme, the following techniques were tested for the degradation of patulin: treatment with ammonia, treatment with ascorbic acid, and treatment with potassium permanganate in acidic or in alkaline conditions. Patulin analysis was performed by using HPLC with UV detection. Mutagenic activity of degradation residues was tested by in Salmonella typhimurium strains TA 97a, TA 98, TA 100, and TA 102. Complete disappearance of patulin was not achieved after 92 h of treatment with ascorbic acid. All the other methods tested led to complete removal of the molecule. However, the technique using potassium permanganate in acidic conditions produced residues which were mutagenic without activation to Salmonella typhimurium strains TA 100 and TA 102, which was attributed later to Mn2+. The two other techniques gave satisfactory results and were selected for further validation studies.

Influence of the Length of Imogolite-Like Nanotubes on Their Cytotoxicity and Genotoxicity toward Human Dermal Cells

Physical-chemical parameters such as purity, structure, chemistry, length, and aspect ratio of nanoparticles (NPs) are linked to their toxicity. Here, synthetic imogolite-like nanotubes with a set chemical composition but various sizes and shapes were used as models to investigate the influence of these physical parameters on the cyto- and genotoxicity and cellular uptake of NPs. The NPs were characterized using X-ray diffraction (XRD), small angle X-ray scattering (SAXS), and atomic force microscopy (AFM). Imogolite precursors (PR, ca. 5 nm curved platelets), as well as short tubes (ST, ca. 6 nm) and long tubes (LT, ca. 50 nm), remained stable in the cell culture medium. Internalization into human fibroblasts was observed only for the small particles PR and ST. None of the tested particles induced a significant cytotoxicity up to a concentration of 10(-1) mg·mL(-1). However, small sized NPs (PR and ST) were found to be genotoxic at very low concentration 10(-6) mg·mL(-1), while LT particles exhibited a weak genotoxicity. Our results indicate that small size NPs (PR, ST) were able to induce primary lesions of DNA at very low concentrations and that this DNA damage was exclusively induced by oxidative stress. The higher aspect ratio LT particles exhibited a weaker genotoxicity, where oxidative stress is a minor factor, and the likely involvement of other mechanisms. Moreover, a relationship among cell uptake, particle aspect ratio, and DNA damage of NPs was observed.