Chronic inflammation is intimately linked with cancer development and progression and therefore reducing or eliminating inflammation represents a logical treatment and prevention strategy. Studies have shown that anti-inflammatory agents have anti-tumour effects in cancers, with reduced metastases and mortality. Current use of anti-inflammatory agents in the treatment and prevention of cancer is limited by their toxicity and side effects. The emerging field of nanotechnology allows the fundamental properties of a drug to be altered, creating a product with improved reactivity and bioavailability, leading to more targeted treatments and reduced dosage. In the present study, the genotoxic effects of three commonly used anti-inflammatory drugs; aspirin, ibuprofen and hydrocortisone, in their bulk and nano forms were evaluated on peripheral blood lymphocytes of healthy donors using the comet assay and the micronucleus assay. In order to determine any anti-cancer effects, these agents were also tested in peripheral blood lymphocytes in patients with haematological cancers. The glucocorticoid hydrocortisone was also evaluated for anti-oxidant capacity. Our results demonstrate that the nano versions of each drug produced a different response than the bulk counterpart, indicating that a reduction in particle size had an impact on the reactivity of the drug. Our results also indicate that the nano versions of each drug were less genotoxic than the bulk formulation, further emphasising the potential of nanoparticles as an improvement to current treatment options. We also found an anti-oxidant effect with hydrocortisone, with a more profound effect seen with the nano formulation.

The effect of coffee and cocoa on oxidative damage to macromolecules has been investigated in several studies, often with controversial results. This study aimed to investigate the effect of one-month consumption of different doses of coffee or cocoa-based products containing coffee on markers of DNA damage and lipid peroxidation in young healthy volunteers. Twenty-one volunteers were randomly assigned into a three-arm, crossover, randomized trial. Subjects were assigned to consume one of the three following treatments: one cup of espresso coffee/day (1C), three cups of espresso coffee/day (3C), and one cup of espresso coffee plus two cocoa-based products containing coffee (PC) twice per day for 1 month. At the end of each treatment, blood samples were collected for the analysis of endogenous and H2O2-induced DNA damage and DNA oxidation catabolites, while urines were used for the analysis of oxylipins. On the whole, four DNA catabolites (cyclic guanosine monophosphate (cGMP), 8-OH-2′-deoxy-guanosine, 8-OH-guanine, and 8-NO2-cGMP) were detected in plasma samples following the one-month intervention. No significant modulation of DNA and lipid damage markers was documented among groups, apart from an effect of time for DNA strand breaks and some markers of lipid peroxidation. In conclusion, the consumption of coffee and cocoa-based confectionery containing coffee was apparently not able to affect oxidative stress markers. More studies are encouraged to better explain the findings obtained and to understand the impact of different dosages of these products on specific target groups.

No / Genotoxic agents can interact with DNA in germ cells possibly resulting in a heritable trait (germline mutation). Thus, in vitro male germ cell tests, which can detect phase specificity of such agents, could be used by regulatory agencies to help evaluate the potential risk of mutation. The male germ cell system now has a well-established model for studying phase specificity using the STA-PUT velocity sedimentation. On treatment with genotoxic agents, differences in chemical structure and metabolic differences in types of male germ cell lead to differing susceptibilities to genotoxicity, so careful investigation is required for phase specificity. This can yield valuable information about the potential mechanisms involved in the genotoxicity responses and thus increase the significance of the findings. This is especially important because mutations induced in the germline could also affect future generations. In this chapter, we briefly review the field of the male germ cell DNA damage response.

In the present investigation peripheral blood lymphocytes from patients with inflammatory bowel disease (IBD) and different cancer states were treated with various agents and compared with lymphocytes from healthy control individuals (HCI) treated in the same way and measured in the Comet assay. For inflammatory bowel disease, patient's responses in IBD patients treated with H2O2 were higher than in HCI and Crohn's patients (CD) were found to have higher responses than Ulcerative colitis (UC) patients. The responses for all IBD and HCI were all reduced in the presence of chaga mushroom extract which behaved in an antioxidant manner. A second group of IBD patients were treated with the heterocyclic amine (food mutagen), IQ and H2O2 and responses were reduced in the presence of the flavonoids, quercetin and epicatechin and compared with HCI similarity treated. In all cells responses were reduced with flavonoids and again CD had higher responses than the UC patients and IBD patients higher than HCI. The responses with CD and UC were that confirmed in two independent studies with IBD, one with chaga mushroom extract and the other with flavonoids. Peripheral lymphocytes from malignant melanoma and suspected melanoma patients and colon cancer and polyposis patients were compared to the lymphocytes from HCI and treated with UVA. There were differential sensitivities when measured in the micronucleus and Comet assays. The cancer patients had higher responses than those in the precancerous states and they in turn were higher than responses in HCI. In all the studies, untreated baseline DNA damage values were also higher in IBD and cancer patients and pre-cancerous patients than HCIs. This would suggest that baseline frequencies of different diseases compared to controls could be an important biomarker in the diagnosis of pre-cancers and early stage cancers. Also peripheral lymphocytes are a useful surrogate for cancers and pre-cancerous disease states since, blood is present in all organs and tissues and DNA is basically the same in all cells.

Ever increasing applications of nanomaterials (materials with one or more dimension less than 100 nm) has raised awareness of their potential genotoxicity. They have unique physico-chemical properties and so could have unpredictable effects. Zinc oxide (ZnO) and titanium dioxide (TiO2) are widely used in a number of commercial products. There are published studies indicating that some forms of these compounds may be photo-clastogenic in mammalian cells. What has not been investigated before is the effect of nanoparticles from these compounds in human germ cells. Thus the present study has examined their effects in the presence and absence of UV light in human sperm and compared responses to those obtained with human lymphocytes using the Comet assay to measure DNA damage. The effect of nanoparticles (40-70nm range) was studied in human sperm and lymphocytes in the dark, after pre-irradiation with UV and simultaneous irradiation with UV. The studies do provide some evidence that there are photo-genotoxic events in sperm and lymphocytes in the absence of overt toxicity. The cytotoxic and genotoxic potentials of ZnO and TiO2 as well as their effect on phosphotyrosine expression, were examined in the human epithelial cervical carcinoma cells (Hela cells). This was done to try and determine the underlying molecular events resulting from their exposure to ZnO and TiO2 nanoparticles occurring at the same time as DNA is damaged. Concentration- and time-dependent cytotoxicity, and an increase in DNA and cytogenetic damage with increasing nanoparticle concentrations were reported in this study. Mainly for zinc oxide, genotoxicity was clearly associated with an increase in tyrosine phosphorylation. Nanotechnology has raced ahead of nanotoxicology and little is known of the effects of nanoparticles in human systems, let alone in diseased individuals. Therefore, the effects of TiO2 nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases (lung cancer, chronic obstructive pulmonary disease (COPD) and asthma) were compared with those in healthy individuals using genotoxic endpoints to determine whether there are any differences in sensitivity to nano-chemical insult between the patient and control groups. The results have shown concentration dependent genotoxic effects of TiO2 in both respiratory patient and control groups in the Comet assay and an increasing pattern of cytogenetic damage measured in the micronucleus assay without being statistically significant except when compared with the untreated controls of healthy individuals. Furthermore, modulation of ras p21 expression was investigated. Regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels that showed modulation as the result of nanoparticle treatment. Results have suggested that both ZnO and TiO2 nanoparticles can be genotoxic over a range of concentrations without either photoa-ctivation or being cytotoxic.

Le sélénium est reconnu comme un micronutriment important pour l'homme et les animaux. Plusieurs études ont montré qu'une supplementation en sélénium dans le régime alimentaire pourrait être bénéfique contre les cancers du foie, du colon, du pancréas et de la prostate. Le mécanisme anti-carcinogène du sélénium se produit au niveau systémique, cellulaire et nucléaire. Ces processus peuvent également impliquer le système immunitaire et ne doivent pas être interprétés par un seul mécanisme. Jusqu'à présent son mécanisme d'action est encore inconnu. L'objectif de cette étude était d'étudier l'effet des composés du sélénium, à faibles concentrations, sur la capacité de réparation de l'ADN dans les cellules du cancer de la prostate LNCaP (p53 compétentes). Ce travail est divisé en trois parties. La première partie du travail a été consacrée à étudier l'effet des deux composés du sélénium (SS et SM) sur les propriétés cytotoxiques et génotoxiques de différents stress oxydatifs et non oxydatifs. Les résultats ont montré qu'un prétraitement avec une faible dose en Se stimulait la synthèse des sélénoprotéines, et protègait contre la toxicité et les dommages oxydatifs à l'ADN induites par les UVA ou H2O2, mais pas par MMS ou UVC. La deuxième partie a été consacrée à l'influence de la supplementation en sélénium sur la capacité de réparation de l'ADN. Notre travail a clairement montré l'augmentation de l'efficacité d'excision de certaines glycosylases que n'est pas nécessairement corrélée à une augmentation de l'expression génique et /ou protéiques. Enfin, la troisième partie de notre travail a été dédiée à l'optimisation de la technique Host Cell Reactivation (HCR) qui nous a permis d'étudier la capacité de réparation de l'ADN in cellulo, afin de cibler les partenaires impliqués dans la voie de signalisation affectées par la supplémentation en sélénium. En conclusion, nous pourront penser que le mécanisme d'action du sélénium est représenté par un délicat équilibre entre l'activation et la répression de l'activité de certaines protéines qui induit des changements conformationnels plus ou moins directement impliqués dans la réparation de l'ADN et la progression de la croissance cellulaire.

Oxidative stress is caused by overproduction and overaccumulation of ROS (reactive oxygen species). This state is responsible for cellular damage during unfavorable environmental conditions such as drought, low temperatures, salinity. In order to directly study oxidative stress at tobacco plants (Nicotiana tabacum cv. Xanthi) I used specific herbicides, MV (methyl viologen) and 3-AT (3- aminotriazole). There were several markers used for monitoring oxidative damage to cellular components: DNA damage detected by a comet assay, lipid peroxidation, carbonylated proteins and modification of activities of antioxidant enzymes CAT (catalase) and APX (ascorbate peroxidase). Fluorescent microscopy documented changes in a redox state of tobacco cells and a specific signal for peroxisomes was observed after treatment with higher concentrations of MV and 3-AT. Application of both herbicides caused significant DNA damage, while they worked in a different concentrations, MV in µM and 3-AT in mM. Another convincing oxidative stress marker for MV was protein carbonylation. The inhibition of antioxidant enzymes CAT and APX was less significant when compared to the effects of 3-AT. Decreasing membrane stability proved to be an universal oxidative stress marker for both herbicides. On the other hand, lipid...

Les zones côtières sont soumises à des pressions anthropiques multiples notamment de nature chimique qui peuvent faire peser un risque réel pour la pérennité des espèces aquatiques. Le bassin d’Arcachon, lagune macrotidale située sur la façade atlantique, est aussi le siège d’une activité ostréicole importante. Cependant depuis plusieurs années, les exploitations ostréicoles sont confrontées à une baisse de recrutement et une forte mortalité des naissains d’huître. La contamination chimique du milieu comme facteur pouvant contribuer aux effets observés sur les huîtres n’a pour l’instant pas été vérifiée. L’étude présentée ici porte sur l’évaluation, à travers différentes approches, de la toxicité potentielle de métaux et pesticides sur les stades précoces de développement de l’huître creuse C. gigas. Les réponses embryotoxiques, génotoxiques et l’expression de gènes d’intérêt ont été étudiés. Les différents pesticides (S-métolachlore, irgarol et diuron) et métaux (cuivre et cadmium) ont tout d’abord été testés séparément pour déterminer leur spectre d’effets et leur mode d’action. Il a été montré qu’une exposition des gamètes ou des embryons d’huître aux pesticides étudiés et au cuivre conduit à une augmentation des malformations larvaires et des dommages à l’ADN, une diminution du succès de fécondation et un impact sur la qualité de la descendance à des teneurs environnementales. Le cadmium, quant à lui, ne présente pas d’effet embryotoxique et génotoxique aux concentrations présentes dans le milieu aquatique. Les métabolites du métolachlore, ESA métolachlore et OA métolachlore sont retrouvés dans le bassin d’Arcachon à de plus fortes concentrations que le composé parent, cependant rien n’est actuellement connu sur les effets toxiques de ces métabolites. Il a été montré que ces métabolites sont moins embryotoxiques et génotoxiques sur les embryons et sur les spermatozoïdes d’huître que le métolachlore. Des variations dans l’expression des gènes impliqués dans les défenses antioxydantes sont observées pour les larves d’huître exposées au métolachlore et au métolachlore ESA. La toxicité d’un mélange de pesticides représentatifs de la contamination du bassin d’Arcachon en présence ou non de cuivre a ensuite été évaluée. L’exposition des embryons d’huître à ces mélanges conduit à des défauts de développement, des dommages à l’ADN et des modifications de l’expression des gènes impliqués majoritairement dans le stress oxydant. Finalement, une cartographie de la toxicité des sédiments du bassin d’Arcachon a été réalisée au cours des 4 saisons de l’année 2011 à l’aide du test embryolarvaire huître. Les sédiments d’Arguin présentent une faible toxicité quelle que soit la saison considérée. En revanche, les sédiments du Tès montrent un effet embryotoxique plus important au printemps et en été par rapport à la saison hivernale. L’ensemble de ce travail permet d’émettre l’hypothèse d’un risque chimique accru pour le développement des premiers stades de vie de huître creuse dans le bassin d’Arcachon. / The coastal areas are subject to multiple anthropogenic pressures including chemical pollution that can pose a real risk to the sustainability of aquatic species. The Arcachon Bay, macrotidal lagoon located on the French Atlantic coast, is the important ecosystem for oyster farming. But for several years, the oyster farms face lower recruitment and high mortality of oyster spat. Chemical contamination of the environment as a factor that may contribute to the observed effects on oysters has so far not been investigated.The present thesis aimed at evaluating through different approaches, of the potential toxicity of heavy metals and pesticides representative of the Arcachon Bay contamination on the early life stages of the Pacific oyster, Crassostrea gigas. Embryotoxicity, genotoxicity and expression levels of eleven targeted genes were studied. Firstly, different pesticides (S-metolachlor, irgarol, and diuron) and metals (copper and cadmium) were separately tested to determine their spectrum of effects. It were shown that exposure of gametes and embryos of oyster to environmental concentrations of pesticides and copper increased developmental abnormalities and DNA damage, and reduced fertilization success and affected offpring quality. Cadmium, meanwhile, showed no embryotoxic and genotoxic effects at the concentrations found in the Arcachon Bay. Metabolites of metolachlor, metolachlor ESA and metolachlor OA, are found in the Arcachon Bay at higher concentrations than their parent compound. The results showed that these metabolites were less embryotoxic and genotoxic on oyster embryos and spermatozoa than metolachor. Significant changes in expression of genes involved in antioxidant defense were observed for oyster larvae exposed to metolachlor and metolachlor ESA. Toxicity of mixtures of pesticides representative of the Arcachon Bay contamination with and without copper was then evaluated. Exposures of oyster embryos to these mixtures lead to development defects, DNA damage and changes in the expression of genes involved mainly in oxidative stress responses. Finally, mapping of toxicity of sediments from the Arcachon Bay was conducted for four seasons of 2011 with the oyster embryo-larvae assay. Sediments collected from Arguin exhibited low toxicity, regardless any season. In contrast, sediments from Le Tès showed higher toxicity in spring and summer seasons compared to winter season.From this work, it can be hypothesized that chemical contamination of the Arcachon Bay represents a threat for oyster reproduction and development.

La toxicologie génétique est une discipline qui vise à détecter des facteurs chimiques ou physiques interagissant avec l'ADN des cellules et qui, en l'absence de réparation fidèle, sont susceptibles de provoquer des mutations géniques et/ou chromosomiques. Le test des comètes est un test court de génotoxicité simple, reproductible et rapide pour étudier la survenue de lésions primaires de l'ADN. Il s'agit d'une technique micro-électrophorétique très sensible permettant la mise en évidence des lésions de l'ADN de cellules eucaryotes individuelles exposées à des agents génotoxiques. En présence de cassures de l'ADN, les fragments d'ADN ainsi formés migrent plus rapidement que l'ADN intact lors de l'électrophorèse, donnant aux noyaux cellulaires l'aspect de comètes. La cryoconservation des ovocytes matures par vitrification a de nombreuses applications: alternative à la congélation d'embryons en FIV, préservation de la fertilité avant traitement gonadotoxique, développement du don d'ovocyte. La vitrification consiste à transformer un liquide en un état vitreux et utilise des agents cryoprotecteurs (CP) à haute concentration. Plusieurs centaines de naissances d'enfants en bonne santé ont été décrites . Néanmoins peu d'études ce sont intéressées aux effets à long terme de cette technique en particulier au plan génétique. L'objectif de ce travail a été dans un premier temps de développer et de valider une technique de test des comètes sur ovocyte de souris, puis d'utiliser ce test pour évaluer la génotoxicité du PrOH sur les ovocytes de souris qu'il soit employé seul ou inclus dans les solutions de vitrifications commercialisées pour la cryoconservation d'ovocytes humains. / Genetic toxicology is a discipline that aims to detect chemical or physical factors interact with the DNA of somatic and / or germ cells and in the absence of accurate repair, are likely to cause gene and / or chromosomal mutations. The comet assay is a simple, reproductive and rapid test to study primary DNA damage. This microelectrophoretic technique, is used to visualize denatured DNA fragments migrating out of the cell nucleus during electrophoresis. The image obtained is a ''comet'' with a distinct head consisting of intact DNA and a tail containing relaxed DNA loops or broken pieces of DNA. Oocyte vitrification techniques is booming in the world, with the key to many applications: alternative to IVF embryo freezing, fertility preservation before gonadotoxic treatment, development of oocyte donation. Oocyte Vitrification traps all the aqueous solutions in a vitreous solid phase, preventing any ice crystal formation, because of very high cooling rates and high cryoprotectants concentrations. vitrification-cryopreservation has led to several hundred live births with reassuring obstetrical and perinatal outcomes. However, little is known about the possible long-term consequences on human live births after oocyte vitrification. The objective of this work was initially to develop and validate a technique comet assay on mouse oocyte, then to evaluate on mature oocytes the genotoxic effects of PrOH solution and finally the genotoxic effects of three oocyte vitrification protocols used in human ART the genotoxic of three oocyte vitrification protocols used in human.

The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...