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PubMed2005

Biomonitoring of the genotoxic potential of draining water from dredged sediments, using the comet and micronucleus tests on amphibian (Xenopus laevis) larvae and bacterial assays (Mutatox and Ames tests).

Management of contaminated dredged sediments is a matter of great human concern. The present investigation evaluates the genotoxic potential of aqueous extracts of five sediments from French channels (draining water from dredged sediments), using larvae of the frog Xenopus laevis. Two genotoxic endpoints were analyzed in larvae: clastogenic and/or aneugenic effects (micronucleus induction after 12 d of exposure) and DNA-strand breaking potency (comet assay after 1 and 12 d of exposure) in the circulating blood. Additionally, in vitro bacterial assays (Microtox and Ames tests) were carried out and the results were compared with those obtained with larvae. Physicochemical analyses were also taken into account. Analytical analyses highlighted in the five draining waters a heavy load of contaminants such as metals and hydrocarbons. The results obtained with the micronucleus test established the genotoxicity of three draining waters. The comet assay showed that all 5 draining waters were genotoxic after 1 d of exposure. Although 3 of them were still genotoxic after 12 d of exposure, DNA damage globally decreased between d 1 and 12. The comet assay can be considered as a genotoxicity-screening tool. Data indicate that both tests should be used in conjunction in Xenopus. Bacterial tests (Ames) revealed genotoxicity for only one draining water. The results confirm the relevance of the amphibian model and the need to resort to bioassays in vivo such as the Xenopus micronucleus and comet assays for evaluation of the ecotoxicological impact, an essential complement to the physicochemical data.

Animals↗
PubMed2001

Micronucleus test in fish from a pampasic pond (Argentina): an estimation of the presence of genotoxic compounds.

The Los Padres pond is one of the commonly shallow, polymictic lakes from the so-called depressed Pampa (Argentina). Its watershed includes one affluent, named Los Padres creek, which flows through horticultural lands wherein great amounts of pesticides are applied. Opposite to this stream, the pond drains into La Tapera creek that is the effluent running toward the sea. Many studies have confirmed the capacity of various pesticides to induce genetic damage. The use of micronucleus (MN) tests in fish has enabled us to detect the presence of contaminants in the lakewater and to evaluate theirgenotoxic effects. For this purpose, water samples were collected during April, August, and December 1999 from both creeks characterized by different environmental conditions. In the laboratory, specimens oftetras Cheirodon interruptus (Pisces, Characidae) were reared in water samples from the two creeks. Control fish were kept in drinking water. Fifteen individuals from each experimental group were sacrificed after 24-, 48-, and 72-hour exposure intervals. Micronucleus frequency in fish erythrocytes was determined, and the Kruskal-Wallis test for statistic analysis was used. We made the following observations: (1) Highly significant differences occurred in MN frequency between the control group and the samples from both creeks. (2) An increase in MN frequency was evident in specimens sampled from the affluent input during the month of December. These results allowed us to conclude that the increase in MN frequency observed in fish belonging to both sampling sites would indicate the existence of genotoxic compounds in the Los Padres pond. The high MN frequency in fish collected near Los Padres creek inlet might be related to the polluted load transported by the affluent and discharged into the lake's surface waters. Future work would allow us to develop efficient methods for predicting the presence of genotoxic contaminants. It would be possible then to propose strategies for regulating and decreasing the sources of pollution that affect human health.

Animals↗
PubMed1992

A collaborative study on the improvement of the micronucleus test in cultured human lymphocytes.

A collaborative study between two laboratories has been carried out in order to investigate the reproducibility of the cytokinesis-block micronucleus assay in human lymphocytes. A modified protocol to obtain good quality slides from whole blood cultures has been developed. The spontaneous frequencies of micronuclei in binucleated lymphocytes from five male subjects were evaluated in each laboratory at two cytochalasin-B concentrations (3 and 6 micrograms/ml). Our data show that 6 micrograms/ml cytochalasin-B was more effective than 3 micrograms/ml to arrest cytokinesis in all the donors analysed. The mean frequency of micronuclei in binucleated cells was significantly lower at 6 micrograms/ml compared to 3 micrograms/ml cytochalasin-B in both laboratories. The results obtained indicate a very high concordance between both sets of data supporting the validity of the proposed protocol.

Adult↗
PubMed2002

The single laser flow cytometric micronucleus test: a time course study using colchicine and urethane in rat and mouse peripheral blood and acetaldehyde in rat peripheral blood.

A single laser flow cytometric procedure to quantify micronucleus frequency in rat and mouse peripheral blood was evaluated. Reticulocytes express the transferrin receptor (also known as the CD71-defined antigen). When combined with a DNA stain, antibodies against this antigen can be used to differentially label and quantify micronucleated reticulocytes. The object of this study was to evaluate the method for rat and mouse peripheral blood using flow cytometry and compare the results obtained between two laboratories (GlaxoWellcome and Litron Laboratories). The compounds selected were the rodent carcinogens colchicine, urethane and acetaldehyde. Colchicine gives a positive response in the rat bone marrow micronucleus assay and an inconclusive result in the rat peripheral blood micronucleus assay. The latter two are both established rat carcinogens readily detected in both the bone marrow and peripheral blood micronucleus assays. In these experiments both rat and mice were treated with either colchicine or urethane and rats alone treated with acetaldehyde. After a single treatment, repeat sampling of peripheral blood was made at 0, 24, 48 and 72 h. Replicate blood samples were obtained and fixed for flow cytometric analysis at both facilities. The micronucleated reticulocyte frequency of each blood sample was determined by analysing 20 000 total reticulocytes per blood sample. The data suggest that the single laser flow cytometric procedure resulted in consistent reticulocyte and micronucleated reticulocyte frequencies between laboratories. Furthermore, these flow cytometric data compare favourably with previously published data.

Acetaldehyde↗
PubMed1994

Micronucleus test and metaphase analyses in mice exposed to known and suspected spindle poisons.

Micronucleus (Mn) and metaphase chromosome analyses were performed in mouse bone marrow cells with two known and eight suspected mitotic spindle poisons. Polychromatic (PCEs) and normochromatic (NCEs) erythrocytes were scored for presence of Mn, while structural (CAs) and numerical chromosome aberrations (NCAs), i.e. hyperploid cells, were evaluated by metaphase analysis. CAs were scored in first, and NCAs in the second metaphases, identified by BrdUrd differential staining. Hydroquinone induced Mn, NCAs and CAs; colchicine, vinblastine and, to a lesser extent, chloral hydrate, diazepam and econazole induced both Mn and NCAs; cadmium chloride and thimerosal induced Mn and CAs, while pyrimethamine and thiabendazole induced Mn only. The proposed stepwise protocol allowed satisfactory statistical evaluation of the effects induced with a reduction in the number of animals killed. An acceptable agreement was found between induction of Mn and NCAs, suggesting a possible use of the Mn test for revealing compounds with aneugenic properties.

Animals↗
PubMed2003

DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the comet assay and micronucleus tests.

A population study is reported in which the DNA damage induced by g-radiation (2 Gy) and the kinetics of the subsequent repair were estimated by the comet and micronucleus assays in isolated lymphocytes of 82 healthy donors and patients with head and neck cancer before radiotherapy. The parameters of background and radiation-induced DNA damage, rate of repair, and residual non-repaired damage were measured by comet assay, and the repair kinetics for every donor were computer-fitted to an exponential curve. The level of background DNA damage before irradiation measured by comet assay as well as the level of micronuclei were significantly higher in the head and neck cancer patient group than in the healthy donors, while the parameters of repair were widely scattered in both groups. Cancer patient group contained significantly more individuals, whose irradiated lymphocytes showed high DNA damage, low repair rate and high non-repaired DNA damage level. Lymphocytes of donors belonging to this subgroup showed significantly lower inhibition of cell cycle after irradiation.

Carcinoma, Squamous Cell↗
PubMedDate not supplied

Cytokinesis-block micronucleus test in patients undergoing radioiodine therapy for differentiated thyroid carcinoma.

Cytokinesis-blocked micronucleus assay (MNT) was applied in the peripheral blood lymphocytes of patients undergoing radioiodine-131 ((131)I) therapy for differentiated thyroid carcinoma (DTC) after thyroidectomy to assess the genotoxic risk of this therapy. The amount of administered (131)I activity varied from 3,330 to 4,030 MBq. Blood samples were taken immediately before (131)I administration and one month later, along with routine blood tests. Twenty-six patients underwent radioiodine ablation (RIA) or radioiodine therapy (RIT) after radical thyroidectomy. The aim of this therapy was to ablate residual thyroid tissue, to treat lymph node metastases and/or distant lung metastases. The amount of orally administered activity of (131)I ranged between 3,330 and 4,030 MBq according to the kind of therapy (RIA or RIT). In five patients the cytogenetic analysis was performed immediately before and one month after the second therapy which was given to them 6 months to 1 year after the first. Three patients were male and 23 female. The age of the patients ranged between 23 and 76 years (mean age: 48.6 years). Results show that after radioiodine therapy there is a significant increase in the frequency of micronuclei. Comparing the average frequency of micronuclei in the patients studied before and after (131)I, a more than doubling increase was found. Mean values +/- SD of the patients before and after (131)I therapy were 10.72 per thousand +/- 5.84 per thousandand 25.28 per thousand +/- 12.6 per thousand respectively. These findings indicate a genotoxic activity of (131)I therapy estimated after a period of one month.

Adult↗
PubMedDate not supplied

[Application of micronucleus test for the demonstration of antimutagenic properties of natural substances].

Authors have used a reliable and reproductible original model of controlled production of free radicals for the cytokinesis-block micronucleus assay. This mutagenicity test is of easy realization and quick interpretation. An experimental model is proposed to test mutagenic activity of free radical generators and antioxydant properties of many substances, such as ascorbic acid. Its addition to the culture medium showed a significant decrease in the rate of micronucleated cells exposed to the free radical generator. The study of other antioxydant compounds (beta-caroten and 5-hydroxyquinolin) have confirmed results previously obtained with ascorbic acid. Thus, the free radical generator coupled to the cytokinesis-block micronucleus assay represents a reliable test to study and evaluate the antioxydant power of natural substances.

Analysis of Variance↗
PubMed1996

In vitro mutagenicity and genotoxicity study of 1,2-dichloroethylene, 1,1,2-trichloroethane, 1,3-dichloropropane, 1,2,3-trichloropropane and 1,1,3-trichloropropene, using the micronucleus test and the alkaline single cell gel electrophoresis technique (comet assay) in human lymphocytes.

The main objective of this study was to compare the cytotoxic genotoxic and mutagenic activity of a number of chlorinated aliphatic hydrocarbons, which are widely used as chemical intermediates, solvents, degreasing agents etc. in industry, and to establish the structure-toxicity relationship of the chemicals by using the most adequate determinants in estimating their toxicity. The mutagenicity and cytotoxicity of some of the candidate chemicals, namely 1,2-dichloroethylene, 1,1,2-trichloroethane, 1,3-dichloropropane, 1,2,3-trichloropropane and 1,1,3-trichloropropene were evaluated in an in vitro micronucleus assay. The cytokinesis-block methodology was applied on human lymphocytes in the presence or absence of an external metabolic activation system (S9-mix). In the micronucleus assay, all test substances, except 1,2,3-trichloropropane with and without S9-mix and 1,1,2-trichloroethane without S9-mix in the repeated experiment, exhibited a low but statistically significant mutagenic activity, compared to the concurrent control. However, none of the five chemicals was able to induce a clear and reproducible linear dose-dependent increase in micronucleus frequencies in this assay. Generally, mutagenic activity of the chemicals was found in the absence of severe cytotoxicity and/or cell cycle delay. The DNA breakage capacity and the cytotoxicity of these chemicals were also assessed in the alkaline single cell gel (SCG) electrophoresis test (comet assay) with and without S9-mix in isolated human lymphocytes. All chemical compounds induced DNA breakage, in the presence or absence of the metabolic activation system, at the doses tested. The data showed that the DNA reactivity of the chemicals increased with increasing degree of halogenation. The results of the present work suggested that the comet assay might be a more suitable and sensitive screening method than the micronucleus test for this particular class of compound. However, both assays do detect different endpoints.

Adult↗
PubMed2006

Evaluation of antimutagenic activity and mechanisms of action of beta-glucan from barley, in CHO-k1 and HTC cell lines using the micronucleus test.

Due to the need to identify new antimutagenic agents and to determine their mechanism of action, the present study examined the mechanism of action of the beta-glucan with regard to antimutagenicity using the micronucleus assay in CHO-k1 and HTC cell lines. The mutagenicity experiments were performed with three different concentrations of beta-glucan (5, 10, and 20 microg/mL), in wich only the highest dose showed mutagenic activity. In the antimutagenicity experiments, the same concentrations of beta-glucan were combined with a mutagenic agent, methylmethane sulfonate, or 2-aminoanthracene, using four different treatment protocols: pre-treatment, simultaneous treatment (simple and with pre-incubation), and post-treatment. The results indicate that the CHO-k1 cell line treated with MMS presented a chemopreventive activity for all the doses of beta-glucan in the different treatment protocols, except for the lowest dose in post-treatment. When HTC cell line treated with MMS is analysed, a chemopreventive activity can be verified for the highest dose in both pre- and post-treatment. For the simple simultaneous treatment, the three doses demonstrated efficacy, while for the simultaneous treatment with pre-incubation only the intermediate concentration was effective. In HTC treated with 2AA both the lowest dose in the pre-treatment protocol and the post-treatment protocol did not show efficacy in preventing DNA damage. The evaluation of the different protocols and the damage decrease percentages observed suggest that beta-glucan has both desmutagenic and bioantimutagenic activity. It is necessary, however, to note that efficacy and mechanism of action are subject to variation when compared the two cell lines, since in HTC, representing a drug-metabolizing system, this substance can show a diminished chemopreventive capacity.

Animals↗