No description available.

No description available.

No description available.

The topoisomerase I (TopoI) poisons (e.g. camptothecin, CPT) cause stabilisation of the TopoI-DNA cleavable complex resulting in DNA single strand breaks (SSBs). DNA breakage and cytotoxicity are related to TopoI activity and TopoI poisons are thought to be primarily cytotoxic in S phase due to conversion of SSB to double strand breaks (DSB) through replication fork collision. DNA SSB are repaired by the base excision repair/SSB repair (BER/SSBR) pathway and DSB are predominantly repaired by homologous recombination repair (HRR) or non-homologous end joining (NHEJ). Poly (ADP-ribose) polymerase-1 (PARP-1) recognises SSB and DSB and promotes their repair. Previous studies in this laboratory demonstrate that PARP inhibitors enhance TopoI poison-induced cytotoxicity by a mechanism that involves elevation of DNA breaks level or retarding the repair of TopoI poison-induced DNA strand breaks. The role of BER/SSBR in this process was verified previously however, there is no data linking sensitisation of TopoI poison-induced cytotoxicity by PARP inhibitors to a specific cell cycle phase. The aim of this project was to test the hypothesis that a) PARP inhibition inhibits the repair of CPT-induced SSB, resulting in more replication-associated DSB in S phase cells, b) PARP inhibition further retards DSB repair and c) consequently the greatest sensitisation is seen in S phase cells. To elucidate the mechanisms DNA breakage, and cytotoxicity were determined in asynchronous cells and those separated into G1, S and G2/M phases by centrifugal elutriation, in cells exposed to the clinically active PARP-1 inhibitor, AG014699, and CPT. Cell cycle-specific variations in TopoI and PARP expression and activity were also investigated as possible contributors to ultimate cytotoxicity. Survival of asynchronous Lovo colorectal cancer cells exposed to CPT was substantially reduced by co-incubation with AG014699. CPT cytotoxicity was greater in a 60% pure S phase population than in 90% pure G1 population. AG014699 increased the cytotoxicity of CPT in the S phase cells but not cells in G1. K562 leukaemia cells were used to confirm findings obtained with Lovo cells. Highly purified (>90% pure) cell cycle specific fraction of K562 cells were used to demonstrate that CPT was most cytotoxic to S phase cells and that potentiation of CPT-induced cytotoxicity by AG014699 is predominantly related to S phase cells whose survival was reduced by 2-fold. TopoI activity was greatest in S phase cells, corresponding to the greater cytotoxicity of CPT in this phase. However, the magnitude of the effect of AG014699 on survival did not correspond with the level of PARP-1 activity, which was highest in G2 cells. The potentiation of CPT-induced cytotoxicity by AG014699 in S phase was shown to correlate with the level of SSB and DSB, which was highest in S phase. Repair of CPT-induced SSB and DSB was most rapid during S phase and AG014699 hindered the repair of both SSB and DSB to the greatest extent in S phase compared with other phases of the cell cycle. This suggests that collision with the replication fork is a major mechanism for the induction of DSB and resultant cytotoxicity. and that PARP is involved in the repair of CPT-induced DSB as well as SSB. BER/SSBR requires XRCC1 as well as PARP, furthermore, PARP-1 is proposed to act in a backup NHEJ pathway that also involves XRCC1. To test these hypotheses the effect of AG014699 on the DNA repair and cytotoxicity of CPT, temozolomide (a DNA methylating agent that induces SSB) and neocarzinostatin (an inducer of DSB) was investigated in XRCC1 wild type (AA8) and mutant (EM9) cells. AG014699 enhanced the cytotoxicity of these agents in parallel with increasing the number of DSB and inhibition of their repair in EM9 as well as AA8 cells. Therefore PARP has an additional role in the protection of cells from DNA SSB and DSB independent of XRCC1. Currently there are 9 PARP inhibitors undergoing clinical evaluation, including combinations with TopoI poisons. The data presented here demonstrate that PARP inhibitors reduce the repair of TopoI poison-induced DNA DSB as well as SSB and consequent cell survival primarily in S phase. Furthermore, we demonstrate that the activity of PARP inhibition is independent of XRCC1 indicating that tumours associated with XRCC1 polymorphisms will be similarly sensitive. These findings may have implications for the rational design of clinical trials involving PARP inhibitor – TopoI poison combinations.

The work presented here adopted an in vitro approach with cell types from different species (fish: Epithelioma Papillosum Cyprini (EPCA1), Rainbow Trout Gonad (RTG-2); mammals: Chinese Hamster Ovary (CHO-K01), primary human fibroblast cells (84BR)) to elucidate the potential genotoxic effects of the interaction of the polycyclic aromatic hydrocarbon (PAH), benzo(a)pyrene (B(a)P) (0.0, 0.1, 1.0 and 3.2 µg mlˉ¹) with ultraviolet radiation (UVA/UVB) (typically 25, 50, 100, 200, 500, 1000, 2000, 4000, 6000, 8000 J mˉ²). Initially the experimental techniques and conditions were optimised and validated in the CHO-K1, EPCA1 and RTG-2 cell lines. It was shown that mammalian (CHO-Kl) and fish cells (EPCA1 and RTG-2) exhibited similar sensitivities to chemicals with different modes of action i.e. clastogenic ethyl methansulphonate (EMS) (0.0, 0.8, 1.6 and 3.2 mM) and aneugenic colchicine (COL) (0.0, 0.1, 1.0 and 1.8 µg mlˉ¹) following cytotoxicity experiments with neutral red retention (NRR). Similarly, using the micronucleus assay (Mn) all the cell lines tested showed a similar response to EMS and COL and the use of the anti-kinetochore stain provided a useful approach with which to distinguish between clastogenic and aneugenic effects in the cell. Following comet assay experiments the importance of optimising and validating variables was demonstrated. The optimal variables chosen for the comet assay were 20 minutes unwinding for fish cells (EPCA1 and RTG-2) and 40 minutes unwinding time for mammalian cells (CHO-K1 and 84BR) with 20 minutes electrophoresis for all cell types. Following these validation studies, the cytotoxic and genotoxic effects produced in cells of aquatic (EPCA1, RTG-2) and mammalian (CHO-K1, 84BR) origin following treatment with B(a)P and UVR was investigated. The incubation of all cells (EPCA1, RTG-2, CHO-K1) with B(a)P alone caused limited cytotoxicity (NRR), increased DNA damage (comet assay) and altered cellular functions that were from aneugenic and clastogenic mechanisms (Mn assay). EPCA1, RTG-2 and CHO-K1 cells irradiated with UVB displayed a significant increase in cytotoxicity (NRR) and DNA damage (comet assay). Cells irradiated with UVA (RTG-2, CHO-K1, 84BR) showed no significant increases in cytotoxicity and only CHO-K1 showed increased DNA damage (comet assay). There were significant increases in cellular alterations (Mn assay) following UVA irradiation. All cells (RTG-2, CHO-K1, 84BR) incubated with B(a)P followed by irradiation with UVA showed a synergistically increased cytotoxicity (NRR) and DNA damage (comet assay) from a 1.2-fold increase up to a 4-fold increase in DNA damage. There were also altered cellular mechanisms that may be due to both aneugenic and clastogenic mechanisms (Mn assay). Oxidative stress as a product of the formation of the hydroxyl radical was shown to be a key element in these processes (Electron Spin Resonance (ESR)). It is therefore concluded that the genotoxic effects of the PAH B(a)P and UVA irradiation are synergistically increased when both insults are experienced in combination. This worrying result was observed within both fish and mammalian cell types and appeared to be mediated via an oxidative stress mechanism which included the formation of the hydroxyl radical.

No description available.

The potential reproductive, developmental, immunological, growth and carcinogenetic effects of endocrine disruptors in humans and wildlife is of global concern. Scarce prior risk analyses of these multiple stressors in river watersheds existed. Therefore, this thesis developed an integrated risk assessment of endocrine disruptors at a section of the Lower Uruguay River with industrial (a bleached Kraft pulp mill), domestic (cities) and agricultural (soy crops) sources. A preliminary risk assessment prioritised oestrogens and further compounds of concern in the watershed, notably nonylphenol, glyphosate, endosulfan, chlorophenols, dioxins and furans, polyaromatic hydrocarbons, polychlorinated byphenyls, rosin acids and phytosterols. Models predicted their multimedia distribution, and food web interactions, and then tested. A three-tiered exposure assessment first rated the river status with eutrophication risks using artificial neural networks, while growth effects evidenced in Hyalella curvispina. Then, river sampling sites were determined by hydrodynamic modelling, tracking pollutant transport by clustering and observing reproductive effects in Ceriodapnia dubia. Finally, target compounds were analysed and endocrine disruption studied from gene to population levels. Biomonitoring with Astyanax fasciatus wildfish found no intersex, but smaller testes downstream the pulp mill and lower condition factor near municipal discharges. Spinal malformations were observed exposing Pimephales promelas to sediment elutriates. When exposed to pulp mill effluent, egg production decreased by half. Anti-oestrogenic or androgenic effects were suggested by the toxicogenomic biomarkers ESR1, ESR2, IGF-I and GHR. The oestrogenicity of a stream receiving municipal wastewater was demonstrated by effects like estradiol in ZP3, ESR1 and IGF-I expression, in agreement with the luciferase receptor-binding screen, and the occurrence of oestrogens and nonylphenol. Overall risks of endocrine disruptors were estimated with radar diagrams, pondering nonylphenol and endosulfan as of concern in the watershed. The risks of endocrine disruption to humans through fish and water ingestion were characterised as low, and from low to moderate to freshwater biota.

No description available.

No description available.

Les amphibiens constituent le groupe le plus menacé d’extinction parmi les vertébrés. Néanmoins, peu de travaux en toxicologie des amphibiens tiennent compte des stades précoces de leur cycle de vie. Pourtant, un individu est exposé directement aux substances présentes dans le milieu aquatique dès l’émission des gamètes. Cette thèse de doctorat a pour objectifs d’apporter de nouvelles données sur les effets d’expositions à des xénobiotiques d’ovocytes de Xenopus laevis, de rechercher des cibles au sein de ce gamète et de participer au développement d’un nouveau modèle en écotoxicologie pour évaluer la qualité de milieux aquatiques.Les avantages, que présentent ces ovocytes, nous ont permis de développer des protocoles efficaces pour appréhender la toxicité de substances. Des endpoints ont pu être définis autour de la maturation ovocytaire, de la fécondation, du développement embryonnaire et de la formation de jeunes têtards. Les effets d’expositions au cadmium, au plomb, au cuivre, à la bouillie bordelaise, au glyphosate, au RoundUp® GT Max et à la deltaméthrine ont été déterminés. Des essais ont été également conduits pour des échantillons de milieux soumis à différentes pressions anthropiques.Il est apparu que l’ovocyte de xénope est sensible aux expositions, notamment au cadmium et au glyphosate et différentes signatures d’expositions sont apparues, comme la formation de doubles structures cytologiques induites par le glyphosate.Les réponses mises en évidence prouvent que l’ovocyte de X. laevis est un modèle pertinent et permettent de recommander l’étude des premières étapes du cycle de vie de l’amphibien en toxicologie aquatique. / Amphibians are one of the most imperiled group of extinction. Nevertheless, few toxicological studies are interested in the earliest steps of their life cycle, even if gamete emission, fertilization and embryogenesis are directly exposed to water pollution. In this context, this PhD thesis aims to bring new data about xenobiotic exposure effects on Xenopus laevis oocytes, to highlight targets inside this germ cells and to contribute to the elaboration of a new model in ecotoxicology to assess aquatic environment quality.As a well-known gamete, the xenopus oocyte makes possible to establish suitable experimental designs to assess toxicity. Many endpoints were defined regarding the oocyte maturation, the fertilization and also the development. The experiments were conducted in metal (cadmium, lead, copper) and in phytopharmaceutical (Bordeaux mixture, glyphosate, RoundUp® GT Max, deltamethrin) contaminated conditions, but also in environmental samples from various aquatic habitats.The xenopus oocyte appeared to be sensitive to contaminant exposures and specially to cadmium and both formulations of glyphosate. Never observed effects were reported. Pollutant signatures were also pointed up, like the double cytological structures induced by glyphosate exposures.The observed responses and results from environmental water experimentations show that X. laevis oocyte is a pertinent model in ecotoxicology and allow to recommend the first steps of the amphibian life cycle in aquatic toxicology.