Giemsa versus acridine orange staining in the fish micronucleus assay and validation for use in water quality monitoring

This study concerns a comparative analysis of the acridineorange and Giemsastaining procedures for the fish erythrocyte micronucleusassay. The goal was to optimize the assay in the context of field watermonitoring. Fish (Carassius carassius) were exposed to a reference genotoxic agent, cyclophosphamide monohydrate 5 mg l−1 for 2, 4, and 6 days before testing. Slides from each individual were scored using the two procedures. The results show that the assay was more sensitive when acridineorange was used. When slides were Giemsa stained, the presence of ambiguous artefacts, leading to false positives and increasing random variance, reduced the contrast between exposed and control samples. AcridineOrangestaining was then applied in the context of waterqualitymonitoring. Fish were exposed for 4 days to water sampled in two hydrological contexts: basal flow and spring flood. The results show that exposure to spring flood water in an agricultural stream can induce mutagenicity

Mutagenic impact on fish of runoff events in agricultural areas in south-west France

When heavy rainfall follows herbicide application, the intense surface runoff causes stream water contamination. Aquatic organisms are then briefly exposed to a complex mixture of contaminants. The aim of the present study is to investigate the genotoxic impact of such events on fish. A model fish, the Crucian carp (Carassius carassius) was exposed in controlled conditions, for 4 days, to water sampled daily in the Save River (France). The watershed of this stream is representative of agricultural areas in southwest France. Three hydrological conditions were compared: basal flow, winter flood, and spring flood. Chemical analysis of the water samples confirmed the higher contamination of the spring flood water,mainly explained by a peak of metolachlor. Genotoxicity was evaluated by micronucleus (MN) test and comet assay in peripheral erythrocytes. A significant increase in DNA breakdowns compared to controls was detected by the comet assay for all conditions. Exposure to spring flood water resulted in the highest damage induction. Moreover, induced chromosomal damage was only detected in this condition. In addition, fish were exposed, for 4 days, to an experimental mixture of 5 herbicides representative of the spring flood water contamination. Fish exhibited moderate DNA damage induction and no significant chromosomal damage. The mutagenicity induced by field-collected water is then suspected to be the result of numerous interactions between contaminants themselves and environmental factors, stressing the use of realistic exposure conditions. The results revealed a mutagenic impact of water contamination during the spring flood, emphasizing the need to consider these transient events in water quality monitoring programs

Oc031--Generic Substitution Of Antiepileptic Drug (Aed) And Loss Of Seizure Control: A Population-Based Case-Crossover Study

Open access CC-BYInternational audienceThere are still controversies over pill substitution among AEDs: some studies claimed that switching between brand and generic AED (generic substitution) can lead to breakthrough seizures; other studies have refuted these concerns. France and some US states recommend limiting substitution of generic AED. We aimed at further estimating the association between generic substitution and loss of seizure control

Caractérisation des effets génotoxiques sur poissons de produits phytosanitaires en période de crue

Les recherches effectuées dans le cadre de cette thèse visent à évaluer l'impact biologique des flashs de contaminations d'origine agricole associés aux crues. Dans un premier temps, nous avons cherché à évaluer les effets génotoxiques de tels événements. Ces mesures renseignent sur des effets précoces et pertinents écologiquement. Après avoir optimisé et validé le test micronoyaux dans nos conditions expérimentales, nous l'avons utilisé conjointement avec l'essai comète pour tester le potentiel génotoxique de 3 conditions hydrologiques contrastées. Il a ainsi été mis en évidence une plus grande génotoxicité de l'eau de la Save prélevée lors d'une crue de printemps par rapport à une crue d'hiver ou un débit de base. Ces résultats ont ensuite été confrontés avec ceux observés lors d'expositions à des mélanges expérimentaux reconstituant les contaminations observées dans le milieu. Les effets génotoxiques des mélanges d'herbicides ont été confirmés, et la complexité des mécanismes induisant la toxicité lors des épisodes de crues a été mise en évidence. Dans un second temps, afin de caractériser la contamination des organismes lors des épisodes de crue, un protocole d'extraction et d'analyse des herbicides accumulés dans les tissus de poissons a été évalué.This study aims at evaluating the biological impact of transient agricultural contamination events associated with floods. First, we investigated the genotoxic impact of such contamination events. These measures provide early and ecologically relevant data. Then, after the optimization and validation of the micronucleus assay in our experimental conditions, this test has been used together with the comet assay in order to test the genotoxic potential of 3 contrasted hydrological conditions. Spring flood water has been found to be more genotoxic than winter flood water or water sampled during the basal flow. These results have been compared with those of exposure to experimental mixtures, mimicking field contamination. The genotoxic potential of herbicides mixture has been confirmed, and the complexity of the processes inducing the toxicity during flood events has been highlighted. Second, in order to investigate the contamination pattern during flood events, a protocol allowing the extraction and quantification of the herbicides accumulated in fish tissues has been evaluated

Exposure to phosphine in maritime transport: a real and important occupational risk: a report of three cases

In maritime transport, to assess the risks of insect pests spreading, fumigation is recommended by the Food and Agriculture Organisation. Fumigant mostly used for foodstuffs is the phosphine gas generated by the reaction of aluminium phosphide and moisture in the atmosphere. In this article, we first discuss phosphine toxicity to humans and then we describe three cases of occupational exposure in maritime transport of cereals. We found phosphine level higher than 20 ppm in tank atmosphere of bulk carriers and levels from 2 to 3.5 ppm in port silos and port warehouses where cereals were unloaded. Two weeks later, atmospheric measurements in a silo were still at 0.8 ppm. In this case, 3 workers described symptoms which could be linked with phosphine. Exposures to phosphine and cases in maritime transport are surely underestimated. Exposure could occur at sea, in harbour but also in port warehouses, trucks and silos or warehouses along logistic chain. All workers in the chain could be exposed. We can recommend research aiming at the development of alternative techniques using a less harmful gas for humans. At individual level, we propose that, along with the training for employees, workers potentially exposed should wear a test strip (phosphine detector strips) or a personal gas badge with appropriate maintenance

The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks

We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSBCter) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSBCter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSBCter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSBCter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome

Ordered association of helicase loader proteins with the Bacillus subtilis origin of replication in vivo

January 1, 2011The essential proteins DnaB, DnaD and DnaI of Bacillus subtilis are required for initiation, but not elongation, of DNA replication, and for replication restart at stalled forks. The interactions and functions of these proteins have largely been determined in vitro based on their roles in replication restart. During replication initiation in vivo, it is not known if these proteins, and the replication initiator DnaA, associate with oriC independently of each other by virtue of their DNA binding activities, as a (sub)complex like other loader proteins, or in a particular dependent order. We used temperature-sensitive mutants or a conditional degradation system to inactivate each protein and test for association of the other proteins with oriC in vivo. We found that there was a clear order of stable association with oriC; DnaA, DnaD, DnaB, and finally DnaI-mediated loading of helicase. The loading of helicase via stable intermediates resembles that of eukaryotes and the established hierarchy provides several potential regulatory points. The general approach described here can be used to analyse assembly of other complexes.Netherlands Organization for Scientific Research (Rubicon fellowship)National Institutes of Health (U.S.) (Public Health Service Grant GM41934

Co-directional replication-transcription conflicts lead to replication restart

August 24, 2011Head-on encounters between the replication and transcription machineries on the lagging DNA strand can lead to replication fork arrest and genomic instability1, 2. To avoid head-on encounters, most genes, especially essential and highly transcribed genes, are encoded on the leading strand such that transcription and replication are co-directional. Virtually all bacteria have the highly expressed ribosomal RNA genes co-directional with replication3. In bacteria, co-directional encounters seem inevitable because the rate of replication is about 10–20-fold greater than the rate of transcription. However, these encounters are generally thought to be benign2, 4, 5, 6, 7, 8, 9. Biochemical analyses indicate that head-on encounters10 are more deleterious than co-directional encounters8 and that in both situations, replication resumes without the need for any auxiliary restart proteins, at least in vitro. Here we show that in vivo, co-directional transcription can disrupt replication, leading to the involvement of replication restart proteins. We found that highly transcribed rRNA genes are hotspots for co-directional conflicts between replication and transcription in rapidly growing Bacillus subtilis cells. We observed a transcription-dependent increase in association of the replicative helicase and replication restart proteins where head-on and co-directional conflicts occur. Our results indicate that there are co-directional conflicts between replication and transcription in vivo. Furthermore, in contrast to the findings in vitro, the replication restart machinery is involved in vivo in resolving potentially deleterious encounters due to head-on and co-directional conflicts. These conflicts probably occur in many organisms and at many chromosomal locations and help to explain the presence of important auxiliary proteins involved in replication restart and in helping to clear a path along the DNA for the replisome.Biotechnology and Biological Sciences Research Council (Great Britain) (Grant BB/E006450/1)Wellcome Trust (London, England) (Grant 091968/Z/10/Z)National Institutes of Health (U.S.) (Grant GM41934)National Institutes of Health (U.S.) (Postdoctoral Fellowship GM093408)Biotechnology and Biological Sciences Research Council (Great Britain) (Sabbatical Visit

IS911 transpososome assembly as analysed by tethered particle motion

Initiation of transposition requires formation of a synaptic complex between both transposon ends and the transposase (Tpase), the enzyme which catalyses DNA cleavage and strand transfer and which ensures transposon mobility. We have used a single-molecule approach, tethered particle motion (TPM), to observe binding of a Tpase derivative, OrfAB[149], amputated for its C-terminal catalytic domain, to DNA molecules carrying one or two IS911 ends. Binding of OrfAB[149] to a single IS911 end provoked a small shortening of the DNA. This is consistent with a DNA bend introduced by protein binding to a single end. This was confirmed using a classic gel retardation assay with circularly permuted DNA substrates. When two ends were present on the tethered DNA in their natural, inverted, configuration, Tpase not only provoked the short reduction in length but also generated species with greatly reduce effective length consistent with DNA looping between the ends. Once formed, this ‘looped’ species was very stable. Kinetic analysis in real-time suggested that passage from the bound unlooped to the looped state could involve another species of intermediate length in which both transposon ends are bound. DNA carrying directly repeated ends also gave rise to the looped species but the level of the intermediate species was significantly enhanced. Its accumulation could reflect a less favourable synapse formation from this configuration than for the inverted ends. This is compatible with a model in which Tpase binds separately to and bends each end (the intermediate species) and protein–protein interactions then lead to synapsis (the looped species)