Germ Line-Specific DNA Sequences are Present on All Five Micronuclear Chromosomes in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The development of the macronucleus from the zygotic micronucleus in the ciliated protozoan Tetrahymena spp. involves the elimination of specific DNA sequences (M. C. Yao and M. Gorovsky, Chromosoma 48:1-18 1974). The present study demonstrates that micronucleus-specific DNA is present on all five of the micronuclear chromosomes. Fragments of micronuclear DNA from Tetrahymena thermophila were cloned in the plasmid vector pBR322. A procedure was developed to examine the organization of the cloned sequences in micro- and macronuclear DNA without nick translating each individual probe. Twenty-three percent of randomly selected DNA sequences examined by this method were micronucleus (germ line) specific. They were all members of families of repeated sequences. Hybridization of six micronucleus-specific DNA sequences to micronuclear DNA from nullisomic strains of T. thermophila, which are lacking one or more pairs of chromosomes in the micronucleus, suggested that these sequences are present on several chromosomes. One micronucleus-specific sequence was shown by in situ hybridization to be present on all five of the micronuclear chromosomes

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

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

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

Biomonitoring of the genotoxic potential (micronucleus assay) and detoxifying activity (EROD induction) in the River Dadou (France), using the amphibian Xenopus laevis

Within the framework of a general survey of the water quality of the river Dadou (Tarn, France), different physicochemical parameters were measured and an inventory of the fish population was made along the water course, around the Rassisse dam.With the aim of monitoring the potential genotoxic effects and the detoxifying activities induced in organisms exposed to the river water, two in vivo bioassays were performed in laboratory experiments, using larvae of the amphibian Xenopus laevis.The first was the micronucleus test, using red blood cells, and the second the assay of ethoxyresorufin-O-deethylase (EROD) induction in the liver of exposed animals.Eight water samples were taken from the river and at outlet points from the two major industrial activities of the studied section of the water course: a spar-fluor mine and a water treatment plant.Genotoxic impact and EROD induction were measured in the larvae.The effluent of the filter-washing process from the water treatment plant was found to be particularly genotoxic, even after dilution in pure reconstituted water, but no particular genotoxicity was found, either in Dadou river water, or in the effluents from the mine.On the other hand, most of the water samples tested produced a clear induction of EROD activity compared to the level of enzymatic activity found in the liver of larvae reared in the river water sampled upstream of the industrial activities.These results were interpreted taking into account (i) the high concentrations of pollutants (fluorine and manganese) measured in the river water, (ii) the very low population levels inventoried in the downstream section of the river and (iii) the possible interactions between the substances present in the river water, particularly the classical EROD inducers PAHs and PCBs

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

Biomonitoring of the genotoxic potential of aqueous extracts of soils and bottom ash resulting from municipal solid waste incineration, using the comet and micronucleus tests on amphibian (Xenopus laevis) larvae and bacterial assays (MutatoxR and Ames tests)

The management of contaminated soils and wastes is a matter of considerable human concern. The present study evaluates the genotoxic potential of aqueous extracts of two soils (leachates) and of bottom ash resulting from municipal solid waste incineration (MSWIBA percolate), using amphibian larvae (Xenopus laevis). Soil A was contaminated by residues of solvents and metals and Soil B by polycyclic aromatic hydrocarbons and metals. MSWIBA was predominantly contaminated by metals. Two genotoxic endpoints were analysed in circulating erythrocytes taken from larvae: clastogenic and/or aneugenic effects (micronucleus induction) after 12 days of exposure and DNA-strand-breaking potency (comet assay) after 1 and 12 days of exposure. In addition, in vitro bacterial assays (MutatoxR and Ames tests) were carried out and the results were compared with those of the amphibian test. Physicochemical analyses were also taken into account. Results obtained with the amphibians established the genotoxicity of the aqueous extracts and the comet assay revealed that they were genotoxic from the first day of exposure. The latter test could thus be considered as a genotoxicity-screening tool. Although genotoxicity persisted after 12 days’ exposure, DNA damage decreased overall between days 1 and 12 in the MSWIBA percolate, in contrast to the soil leachates. Bacterial tests detected genotoxicity only for the leachate of soil A (Mutatox). The results confirm the ecotoxicological relevance of the amphibian model and underscore the importance of bioassays, as a complement to physicochemical data, for risk evaluation

Assessment of the genotoxicity of olive mill waste water (OMWW) with the Vicia faba micronucleus test

The present study concerns the genotoxicity of olive mill waste water (OMWW) generated in mills producing olive oil in Morocco. The Vicia faba micronucleus test was used to evaluate the genotoxicity of OMWW and the six major phenolic compounds identified by HPLC in this effluent. Five dilutions of OMWW were tested: 0.1, 1, 5, 10 and 20%. Maleic hydrazide was used as a positive control. The results showed that OMWW was genotoxic at 10% dilution. In order to investigate the components involved in this genotoxicity, the six major phenols present in this effluent, oleuropein, gallic acid, 4-hydroxyphenyl acetic acid, caffeic acid, paracoumaric acid and veratric acid, were studied at concentrations corresponding to the genotoxic concentration of the OMWW itself. Two phenols, gallic acid and oleuropein induced a significant increase in micronucleus frequency in Vicia faba; the four other phenols had no significant genotoxic effect. These results suggest that under the experimental conditions of our assay, OMWW genotoxicity was associated with gallic acid and oleuropein

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

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