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

Genotoxic and stress inductive potential of cadmium in Xenopus laevis larvae

The present investigation evaluates the toxic potential of Cd in larvae of the frog Xenopus laevis after 12 days of exposure to environmentally relevant contamination levels, close to those measured in the river Lot (France). Several genotoxic and detoxification mechanisms were analyzed in the larvae: clastogenic and/or aneugenic effects in the circulating blood by micronucleus (MN) induction, metallothionein (MT) production in whole larvae, gene analyses and Cd content in the liver and also in the whole larvae. The results show: (i) micronucleus induction at environmental levels of Cd contamination (2, 10, 30 μg L−1); (ii) an increased and concentration-dependent quantity of MT in the whole organism after contamination with 10 and 30 μg Cd L−1 (a three- and six-fold increase, respectively) although no significant difference was observed after contamination with 2 μg Cd L−1; (iii) Cd uptake by the whole organism and by the liver as a response to Cd exposure conditions; (4) up-regulation of the genes involved in detoxification processes and response to oxidative stress, while genes involved in DNA repair and apoptosis were repressed. The results confirm the relevance of the amphibian model and highlight the complementarity between a marker of genotoxicity, MT production, bioaccumulation and genetic analysis in the evaluation of the ecotoxicological impact

The micronucleus assay as a biological dosimeter of in vivo ionising radiation exposure

Biological dosimetry, based on the analysis of micronuclei (MN) in the cytokinesis-block micronucleus (CBMN) assay can be used as an alternative method for scoring dicentric chromosomes in the field of radiation protection. Biological dosimetry or Biodosimetry, is mainly performed, in addition to physical dosimetry, with the aim of individual dose assessment. Many studies have shown that the number of radiation-induced MN is strongly correlated with dose and quality of radiation. The CBMN assay has become, in the last years, a thoroughly validated and standardised technique to evaluate in vivo radiation exposure of occupational, medical and accidentally exposed individuals. Compared to the gold standard, the dicentric assay, the CBMN assay has the important advantage of allowing economical, easy and quick analysis. The main disadvantage of the CBMN assay is related to the variable micronucleus ( MN) background frequency, by which only in vivo exposures in excess of 0.2-0.3 Gy X-rays can be detected. In the last years, several improvements have been achieved, with the ultimate goals (i) of further increasing the sensitivity of the CBMN assay for low-dose detection by combining the assay with a fluorescence in situ hybridisation centromere staining technique, (ii) of increasing the specificity of the test for radiation by scoring nucleoplasmic bridges in binucleated cells and (iii) of making the assay optimally suitable for rapid automated analysis of a large number of samples, viz. in case of a large-scale radiation accident. The development of a combined automated MN-centromere scoring procedure remains a challenge for the future, as it will allow systematic biomonitoring of radiation workers exposed to low-dose radiation

Direct MN Test on Peripheral Blood to Detect Chromosomal Breakage: Application in Smokers

The purpose was to assess chromosomal damage in blood mononuclear cells of smokers. Smoker’s peripheral blood samples were screened for micronuclei. Samples from smokers who had an illness were excluded. From each sample, 500 swelled mononuclear leucocytes were screened using a light microscope, with 400x magnification. Frequency distribution of subjects having 0, 1, 2, 3, 4, and 5 micronuclei (MN) according to age and condition were tabulated. From the 102 samples, 5 were excluded, and only 97 were analyzed. There was an increase in MN count in 12.8%, 12.9%, 33.3%, and 25% of normal smokers living in unpolluted area, hypertensive smokers living in unpolluted area, normal smokers living in polluted area, and hypertensive smokers living in polluted area, respectively. Therefore, there was a tendency of increasing MN count in smokers in the productive age group, hypertensive people, and people living in polluted area.&nbsp

A Small Family of Elements with Long Inverted Repeats is Located Near Sites of Developmentally Regulated DNA Rearrangement in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions

The micronucleus assay in radiation accidents

The cytokinesis-block micronucleus assay in peripheral blood lymphocytes is a standardised and validated technique for biodosimetry. Automated scoring of micronuclei allows large scale applications as in population triage in case of radiation accidents or malevolent use of radioactive sources. The dose detection limit (95% confidence) of the micronucleus assay for individual dose assessment is restricted to 0.2 Gy but can be decreased to 0.1 Gy by scoring centromeres in micronuclei using fluorescence in situ hybridization (FISH). In the past the micronucleus assay was applied for a number of large scale biomonitoring studies of nuclear power plant workers and hospital workers. Baseline micronucleus frequencies depend strongly on age and gender. The assay was also already used for biodosimetry of radiation accidents. In a multiple endpoint biodosimetry study for dose assessment of a worker exposed accidentally in 2003 to X-rays, a good agreement was obtained between dose estimates resulting from the micronucleus assay, the scoring of dicentrics and translocations. Automated scoring of micronuclei in combination with centromere signals, allowing systematic biodosimetry of exposed populations, remains a challenge for the future

Increased chromosomal radiosensitivity in asymptomatic carriers of a heterozygous BRCA1 mutation

Background: Breast cancer risk increases drastically in individuals carrying a germline BRCA1 mutation. The exposure to ionizing radiation for diagnostic or therapeutic purposes of BRCA1 mutation carriers is counterintuitive, since BRCA1 is active in the DNA damage response pathway. The aim of this study was to investigate whether healthy BRCA1 mutations carriers demonstrate an increased radiosensitivity compared with healthy individuals. Methods: We defined a novel radiosensitivity indicator (RIND) based on two endpoints measured by the G2 micronucleus assay, reflecting defects in DNA repair and G2 arrest capacity after exposure to doses of 2 or 4 Gy. We investigated if a correlation between the RIND score and nonsense-mediated decay (NMD) could be established. Results: We found significantly increased radiosensitivity in the cohort of healthy BRCA1 mutation carriers compared with healthy controls. In addition, our analysis showed a significantly different distribution over the RIND scores (p = 0.034, Fisher’s exact test) for healthy BRCA1 mutation carriers compared with non-carriers: 72 % of mutation carriers showed a radiosensitive phenotype (RIND score 1–4), whereas 72 % of the healthy volunteers showed no radiosensitivity (RIND score 0). Furthermore, 28 % of BRCA1 mutation carriers had a RIND score of 3 or 4 (not observed in control subjects). The radiosensitive phenotype was similar for relatives within several families, but not for unrelated individuals carrying the same mutation. The median RIND score was higher in patients with a mutation leading to a premature termination codon (PTC) located in the central part of the gene than in patients with a germline mutation in the 5′ end of the gene. Conclusions: We show that BRCA1 mutations are associated with a radiosensitive phenotype related to a compromised DNA repair and G2 arrest capacity after exposure to either 2 or 4 Gy. Our study confirms that haploinsufficiency is the mechanism involved in radiosensitivity in patients with a PTC allele, but it suggests that further research is needed to evaluate alternative mechanisms for mutations not subjected to NMD

Evaluation of the genotoxic and teratogenic potential of a municipal sludge and sludge-amended soil using the amphibian Xenopus laevis and the tobacco: Nicotiana tabacum L. var. xanthi Dulieu

The toxic, genotoxic and teratogenicpotential of amunicipal sewage sludge was assessed using the micronucleus assay on the larvae of the amphibianXenopuslaevis and with the tobacco somatic mutation test using the yellow–green xanthiDulieu mutant a1+/a1 a2+/a2. The teratogenicpotential was assessed by means of the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). Various doses of the pasty sludge added to a crop soil were tested using the three bioassays. The test systems were performed either directly with sludge or sludge-amendedsoil samples (plant model) or with aqueous extracts (aquatic animal model). Using the tobacco model, we found no mutagenic impact of the soilamended with the sludge, perhaps because the clay-like nature of the soil, with its high adsorption capacity, may have prevented the contaminants from reaching the target. All leachates of amendedsoils produced a significant size reduction in Xenopus embryos. Depending on the soil/sludge ratio, some leachates were found to be genotoxic but were never teratogenic. This battery of in vivo test systems enabled us to estimate the global long-term effects under agricultural conditions with various genetic endpoints on ecologically relevant organisms characteristic of the aquatic and terrestrial compartments

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

Chronic ethanol consumption in mice does not induce DNA damage in somatic or germ cells, evaluated by the bone marrow micronucleous assay and the dominant lethal mutation assay

Indexación: Web of Science; ScieloAlthough alcohol is known to be a carcinogen for humans, ethanol-genotoxicity studies are incomplete. Ethanol seems not to be a bacterial mutagen, but the results are conflicting in rodent assays. We investigate the genotoxicity in the bone marrow micronucleus (MN) test and in the dominant lethal mutation (DLM) assay using two long-term ethanol exposure protocols. In the MN test, mice consumed three doses (5, 10 and 15% v/v) for 32 weeks. MN induction was compared to two control groups of 5- and 38-week-old mice (the ages of the treated mice when the treatment was initiated and when they were killed, respectively). For the three groups treated with ethanol there was no significant increase in MN induction as compared to the first control group, but observed MN frequencies were significantly lower than in the 38-week-old control group. This suggests a protective effect against genotoxic damage caused by aging, probably due to ethanol action as a hydroxyl radical scavenger. In the DLM assay, male mice drank ethanol at 15% or 30% (v/v) for 20 weeks. In both groups the number of dead implants was similar to the control, but there was a significant reduction in total implants, indicating a pre-implantation loss.http://ref.scielo.org/7b9mt