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.     

Quantitative and qualitative studies of micronucleus induction in mouse erythrocytes using flow cytometry. I. Measurement of micronucleus induction in peripheral blood polychromatic erythrocytes by chemicals with known and suspected genotoxicity

Quantitative and qualitative aspects of the in vivo micronucleus-inducingpotential of five chemicals were studied using flow cytometricenumeration of micronucleated polychromatic peripheral blooderythrocytes in mice. The chemicals were hydroquinone, vinblastinesulphate, chloral hydrate (tested in two different mouse strains),5-bromo-2-deoxyuridine and 2-chlorobenzylidene malonitrile.Repeat samplings of peripheral blood were made at 0, 24, 40,48 and 72 h and for low doses of 5-bromo-2-deoxyuridine 96 hafter i.p. treatment. The agents hydroquinone (lowest effectivedose 25 mg/kg), vinblastine sulphate (lowest effective dose0.05 mg/kg) and 5-bromo-2-deoxyuridine (lowest effective dose200 mg/kg) gave rise to significant increases in the frequenciesof micronucleated polychromatic erythrocytes. No significantinduction of micronucleated polychromatic erythrocytes by 2-chlorobenzylidenemalonitrile or chloral hydrate was found. The frequencies ofinduced micronucleated polychromatic erythrocytes peaked at40 h after hydroquinone treatment, at 48 h after vinblastinetreatment and at 72 h after 5-bromo-2-deoxyuridine treatmentwith evident dose-dependent differences in the kinetics of theinduction of micronucleated polychromatic erythrocytes. Themean relative Hoechst 33342 fluorescence of the populationsof induced micronucleated polychromatic erythrocytes was usedas an indicator of the DNA content of induced micronuclei. Thesevalues were found to be in agreement with the presumed mechanismsof micronucleus induction for hydroquinone, vinblastine sulphateand 5-bromo-2-deoxyuridine. Flow cytometric enumeration of micronucleatedpolychromatic erythrocytes in peripheral blood is an efficientmethod for the study of in vivo micronucleus induction, combiningrapid analysis and high sensitivity with information on possiblemechanisms of micronucleus induction. The method also allowsa substantial reduction in the number of animals needed. ⁴To whom correspondence should be addressed     

Combination effects of clastogens in the mouse peripheral blood micronucleus assay

In order to study how two chemicals interact to induce micronuclei,simple ethylating agents [ethyl methanesulfonate (EMS), ethylethanesulfonate (EES) and N-ethyl-N-nitrosourea (ENU)], spindlepoisons [vincristine sulfate (VINC) and colchicine (COL)] andan oxidizing agent [potassium bromate (KBrO₃)] were used asmodel chemicals for combination treatments. The frequency ofmicronucleated reticulocytes (MNRETs) was evaluated in micetreated with two of these chemicals at a time. The combinationsof ethylating agents (EMS and EES; EMS and ENU) and of spindlepoisons (VINC and COL) induced more micronuclei than those expectedon an additive basis. The apparent synergism was due to a ‘combineddose’ which could be calculated by the dosimetric conversionof one chemical to the other, when damage induced by each chemicalwas ‘equivalent’ in the induction of MNRETs. Incontrast, no apparent synergism in induction of micronucleiwas observed when two chemicals with different modes of clastogenicaction (EMS and KBrO₃ or EMS and VINC) were combined. ¹Present address: Department of Drug Safety Research, EisaiCo. Ltd, 1-3 Tokodai 5-Chome, Tsukuba-shi, Ibaraki 300-26, Japan ²Present address: Department of Biological Sciences, RiversState University, PMB 5080, Portharcourt, Nigeria ³To whom correspondence should be addressed      

Effects of griseofulvin in medium-term liver carcinogenesis assay and peripheral blood micronucleus test in rat.

Published data have suggested a possible link between the tumor promoting activity and the aneugenic properties of griseofulvin. The present study was conducted to explore this relationship. Griseofulvin was evaluated both for its potential promoting activity in liver carcinogenesis in partially hepatectomized F344 male rats initiated by diethylnitrosamine and for its genotoxic potential in the peripheral blood micronucleus assay. Rats were treated daily with 2,000 mg/kg body weight by oral gavage for 12 weeks in the medium-term carcinogenesis bioassay. GST-P-positive foci (mean number and surface area) and altered cell foci were compared in the liver of rats treated with griseofulvin alone, diethylnitrosamine alone,and griseofulvin in addition to diethylnitrosamine by using immunohistochemical and histopathological evaluation, respectively. This evaluation allowed the conclusion that griseofulvin did not initiate the carcinogenic process but rather had a potential in the liver for tumor promoting activity. Griseofulvin was found to be negative in the rat peripheral blood micronucleus test when given at a daily oral dose of 2,000 mg/kg body weight for at least 3 weeks.     

Micronucleus morphology as a means to distinguish aneugens and clastogens in the mouse bone marrow micronucleus assay

Detailed dose-response data for two reference aneugens, vincristine and nocodazole, have been derived for the mouse bone marrow micronucleus assay. Positive data have also been described for six other micronucleus-inducing agents: cyclophosphamide; aniline; urethane; 1,2-dimethylhydrazine; 7,12-dimethylbenzanthracene and procarbazine. Micronuclei (MN) were classified according to the morphological criteria listed by Yamamoto and Kikuchi, i.e. normal, double, multiple, ring, crescent and large. Aniline and control slides exhibited low incidences of aberrant MN (less than 3%). The remaining seven chemicals gave incidences of aberrant MN that varied from 3 to 25%. Only the aneugens vincristine and nocodazole, and the possible aneugen urethane, gave both crescent shaped and large MN (greater than 0.25 cell diameter). It is therefore concluded that only these last two classes of aberrant MN can be taken as indicative of aneugenic activity. Vincristine and nocodazole gave the highest incidences of aberrant MN and these were shown to be strongly dose-related in the case of vincristine.     

Flow cytometry peripheral blood micronucleus test in vivo: Determination of potential thresholds for aneuploidy induced by spindle poisons

Non‐DNA binding genotoxins (e.g., aneugens), unlike DNA‐binding genotoxins, are theoretically expected to show thresholded concentration‐effect response curves. This is a major issue in genetic toxicology testing because the identification of thresholds in vivo can provide a safety margin for exposure to a particular compound. In the current study we measured micronucleus induction by flow cytometry to determine the dose‐response curves for tubulin interacting agents, a specific class of aneugens. All experiments with aneugens, which include colchicine, vinblastine, vincristine, as well as the clastogen cyclophosphamide (CP) were performed in mice to avoid the splenic elimination of micronucleated reticulocytes, which has been described in rats. Flow cytometry analysis revealed a non‐linear dose‐dependent increase in micronuclei frequencies for all tested aneugens, and a linear dose response curve for the clastogen, CP. To determine whether micronucleus induction at higher doses was due to chromosome loss (aneuploidy) or chromosome breakage (clastogenicity), flow sorting of the micronucleated reticulocytes and fluorescent in situ hybridization (FISH) with a mouse pan centromeric probe were performed for vinblastine, vincristine, and colchicine. Statistical evaluation of the flow cytometry and FISH data was performed to determine the threshold levels for chromosome loss in vivo. The threshold concentrations for vinblastine, vincristine, and colchicine were found at 0.35, 0.017, and 0.49 mg kg^?1, respectively. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Improved detection of rare CALLA-positive cells in peripheral blood using multiparameter flow cytometry

A major limitation to the detection of rare cell types in the peripheral blood using monoclonal antibodies is nonspecific binding of the antibody reagent to normal cells. Detection of rare common acute lymphoblastic leukemia antigen (CALLA)-positive cells in peripheral blood is significantly improved by using multiple flow cytometric parameters to exclude a variety of mature blood cells which may nonspecifically bind the antibody reagent. Monocytes and granulocytes are excluded by gating out cells with high 90 degrees light scatter. By gating on red fluorescence, a variety of mature cell types binding to phycoerythrin (PE)-conjugated Leu 3, Leu 2, and M3 monoclonal antibodies are also excluded. CALLA-positive lymphoblasts from 6 consecutive patients were not excluded on the basis of these parameters. Gating on log 90 degrees light scatter and log red fluorescence in this fashion reduced the incidence of nonspecific binding to peripheral blood mononuclear cells of a fluorescein-conjugated irrelevant monoclonal antibody by 98% from 308 cells per million to 5 cells per million. One CALLA-positive lymphoblast per 100,000 peripheral blood mononuclear cells could be detected in mixture experiments using this method. The normal range of CALLA-positive cells in adults is less than 16 cells per million peripheral blood mononuclear cells. This low background of CALLA-positive peripheral blood cells may permit the detection of early leukemic relapse in acute lymphoblastic leukemia by analysis of the peripheral blood. This methodology can be applied to the detection of any rare cell type by using phycoerythrin-conjugated antibodies to markers that the cell type does not possess.     

Protocol recommended by the CSGMT/JEMS.MMS for the short-term mouse peripheral blood micronucleus test

Although the target cells for the bone marrow (BM) and peripheralblood (PB) micronucleus tests are the same, erythroblasts, thePB method offers important advantages over the BM method. Wepropose a protocol for the shorterm peripheral blood micronucleustest. This assay is intended primarily for the identificationof a wide variety of chemical clastogens and spindle poisons,and secondarily for risk assessment. The recommended experimentsize, seemingly small, has adequate detection power. Experimentalresults obtained from Collaborative Study Group for the MicronucleusTest (CSGMT) studies and data collected from a survey of theliterature provided the basis of the proposed protocol. Ourprotocol, designed for mice, includes the following features.(i) The maximum tolerated dose (MTD) is determined experimentallywith a small number of animals treated i.p. or per os (or byother routes, if called for) in a dose-finding test, which canbe conducted simultaneously with tests for finding both numberof treatments and optimal sampling time. (ii) At least threegroups of five mice (at least four effective animals per group),males or females, are given i.p. or per os doses of, for example,the MTD, 1/2 MTD and 1/4 MTD once, twice or more, 24 h apart.(iii) Peripheral blood samples are taken before treatment (the0 time control) and twice at 48 and 72 h for a single treatment,once between 24 and 36 h after the second treatment for doubletreatments, or once 24 h after the final treatment for multipledosing. Or, if an optimal sample time is established in a preliminarytest, samples are taken at that time. (iv) Samples are stainedwith acridine orange, and 2000 immature erythrocytes per animalsare examined. (v) The combined data of 0 time samples are thenegative control. When statistical analysis shows that the negativecontrol is out of the historical control range or experimentalresults are equivocal, a repeat test is needed. Correspondence should be addressed to: Shizuyo Sutou, Itoham Central Research Institute, 1–2 Kubogaoka, Moriya, Kitasoma, Ibaraki 302–01, Japan     

Pharmacodynamic monitoring of molecular-targeted agents in the peripheral blood of leukemia patients using flow cytometry

The introduction of specific, molecular-targeted drugs is radically changing cancer treatment. Pharmacodynamics, which measures drug effects on the host, is key during early-phase clinical trials of novel agents to determine the relations between drug dose and target inhibition as well as measure the downstream effects of target inhibition on the cancer. In this article, we describe the application of flow cytometry to the pharmacodynamic monitoring of molecular-targeted agents in leukemia patients. The methods are based on current clinical flow-cytometry applications, with the addition of phosphospecific antibodies to measure the activation states of intracellular signaling elements and the introduction of techniques that maintain drug-target equilibrium during sample preparation. Using this approach, we successfully showed dose-dependent inhibition of c-Kit during a phase I clinical trial treating acute leukemia patients with the novel agent sorafenib. Further refinements identify considerable interpatient variation in signaling activity within leukemic blast populations, suggesting that an individualized approach to treatment based on flow cytometric monitoring might be advantageous. Improvements in sample turnaround offer the potential to introduce real-time pharmacodynamic monitoring during early-phase clinical trials.     

Stability of immunophenotypic markers in fixed peripheral blood for extended analysis using flow cytometry

The analysis of whole blood samples by flow cytometry for pharmacodynamic and biomarker assessments in clinical studies has been limited by the necessity to test these samples within a short time frame after blood collection. In most clinical studies, blood specimens are shipped to a centralized testing facility; it is critical to demonstrate specimen stability over a period of time which will encompass the time elapsed between specimen collection and testing. A possible solution to overcome this limitation is the use of a fixative to preserve the cell surface antigen stability in whole blood. We examined the stability of markers for T lymphocytes (CD3, CD4, CD8, CD45RA, and CD45RO), B lymphocytes (CD19), NK cells (CD16 + CD56), activation (CD25 and HLA-DR), chemokine receptors (CCR5 and CXCR3) and skin homing (CLA) in fixed blood over 7 days and used this information to select the markers for global clinical studies. These assays with selected markers were further validated using fit-for-purpose approach (Lee et al., 2006) and to set the sample acceptability criteria for use in clinical sample testing. Most of the markers examined were stable when collected in Cyto-Chex® BCT for one week with the exception of the activation markers on T cells.     

An optimal, generalized sampling time of 30{+/-} 6 h after double dosing in the mouse peripheral blood micronucleus test

Double dosing and single sampling seems to be the simplest andmost reliable method for detecting clastogens in the mouse peripheralblood micronucleus test. Optimal sampling times after doubledosing are studied here. Eleven clastogens (water soluble: colchicine,cyclophosphamide, cytosine arabinoside, 5-fluoro-2-deoxyuridine,5-fluorouracil (5-FU), 6-mercaptopurine, methotrexate (MTX),mitomycin C; water insoluble: N-2-acetylaminofluorene, diethylsulfate, 7, 12-dimethyl-benz(a)anthracene (DMBA) were giveni.p. twice to MS/Ae mice and peripheral blood samples were collectedat 6 h intervals starting 24 h after the second treatment. Samplescollected at 30 ± 6 h were nearly optimal for all 11chemicals. When DMBA, 5-FU and MTX were given, elevated micronucleusfrequencies tended to last longer relative to those inducedby direct-acting chemicals. Since samples collected 30 ±6 h after the second treatment with these three chemicals showedalmost the same micronucleus frequencies observed in later samples,the time 30 ± 6 h could be applied for these long-actingchemicals. Micronucleated reticulocytes persist in the peripheralblood (PB) longer than micronucleated polychromatic erythrocytesare retained in the bone marrow, which thus provides a simple,effective and generalized protocol for the mouse short-termPB micronucleus test, namely double dosing and sampling 30 ±6 h after the second dose. When one sample time has to be selected,30 h after the second treatment is recommended. ¹To whom correspondence should be addressed     

Performance of flow cytometric analysis for the micronucleus assay--a reconstruction model using serial dilutions of malaria-infected cells with normal mouse peripheral blood

To confirm the performance and statistical power of a flow cytometric method for scoring micronucleated erythrocytes, reconstruction experiments were performed. For these investigations, peripheral blood erythrocytes from untreated mice, with a micronucleated erythrocyte frequency of approximately 0.1% were combined with known quantities of Plasmodium berghei (malaria) infected mouse erythrocytes. These cells had an infected erythrocyte frequency of approximately 0.7%, and mimic the DNA content of micronuclei (MN). For an initial experiment, samples with a range of MN/malaria (Mal) content were constructed and analysed in triplicate by flow cytometry until 2000, 20,000 and 200,000 total erythrocytes were acquired. In a second experiment, each specimen was analysed in triplicate until 2000, 20,000, 200,000 and 1,000,000 erythrocytes were acquired. As expected, the sensitivity of the assay to detect small changes in rare erythrocyte sub-population frequencies was directly related to the number of cells analysed. For example, when 2000 cells were scored, increases in MN/Mal frequencies of 3.9- or 2.7-fold were detected as statistically significant. When 200,000 cells were analysed, a 1.2-fold increase was detected. These data have implications for the experimental design and interpretation of micronucleus assays that are based on automated scoring procedures, since previously unattainable numbers of cells can now be readily scored.     

A proposal for a simple way to distinguish aneugens from clastogens in the in vitro micronucleus test.

In our previous in vitro micronucleus (MN) study, we showed that aneugens, in addition to inducing micronuclei, induce a higher frequency of polynuclear (PN) and mitotic (M) cells than clastogens. We hypothesized that the frequency of PN and M cells induced can distinguish aneugens from clastogens. To test the hypothesis, we conducted the micronucleus tests with mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), vincristine (VINC) and diazepam in a Chinese hamster cell line (CHL) and VINC, benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) in a subclone of V79 cells (V79-MZ). All chemicals increased the frequency of M cells with statistical significance. All chemicals except diazepam increased the frequency of PN cells with statistical significance. Three of the aneugens (VINC, BP and DMBA) induced >/=200 PN cells/1000 cells while the clastogens (MNNG and MMC) induced 100 PN cells at most. All the aneugens but no clastogens significantly increased the frequency of M cells. We propose that micronucleus test-positive chemicals that induce >/=200 PN cells/1000 cells and significantly increase the frequency of M cells are aneugens and those that induce at most 100 PN cells/1000 cells and do not significantly increase the frequency of M cells in our MN test protocol are clastogens. Diazepam, however, did not induce PN cells, although it increased the frequency of M cells dose dependently. We explain this fact in relation to diazepam's mode of action. Our proposal suggests a quick, easy and practical way to distinguish aneugens from clastogens for screening purposes.     

流式细胞术在外周血嗜酸性粒细胞及其相关分子检测中的应用

目的 :应用流式细胞术 ,建立一种准确、外周血嗜酸性细胞及其相关分子的快速测定方法。方法 :采集正常人外周血 ,用茶碱 (10 -4mol/L)、地塞米松 (10 -4mol/L)和rhIL 5 (10 -8mol/L)预处理 ,用抗CD16 PEmAb与FITC标记的抗相关细胞分子的进行双标记染色 ,并以CD16 FL2辅助设门 ,准确找到嗜酸性粒细胞群 ,然后对其相关分子进行分析。结果 :嗜酸性粒细胞定位准确 ,茶碱和地塞米松能够抑制IL 5引起的Eos的活化并使其表面的CD6 2L脱落。结论 :应用流式细胞术与二色荧光mAbCD16 PE阴性细胞法设门 ,可准确快速地检测外周血中嗜酸性粒细胞及分子的表达率 ,血液用样量小 ,人为影响因素少 ,是免疫学基础研究和临床检验较理想的测定方法。     

Analysis of eight known or suspected aneugens by the in vitro human lymphocyte micronucleus test.

In the light of the CEC aneuploidy programme eight known or suspected aneugens were evaluated with the human lymphocyte 'in vitro' micronucleus test using the cytochalasin-B technique. Only colchicine, chloral hydrate and hydroquinone induced dose-dependent increases in micronucleus frequencies. The other five chemicals (cadmium chloride, econazole, pyrimethamine, thiabendazole and thimerosal) were all negative, both with and without S9, and treated in G1 or G2 phase. These data are in good agreement with results from different 'in vivo' studies. However, discrepancies were found between the results from this study of human lymphocytes and those of other cultured cell types where econazole, cadmium chloride, pyrimethamine and thiabendazole induce a significant increase of micronuclei.     

Etoposide (VP-16) is a potent inducer of micronuclei in male rat meiosis: Spermatid micronucleus test and DNA flow cytometry after etoposide treatment

The genotoxic and cytotoxic effects of etoposide (VP-16), a topoisomerase II inhibitor, on male rat spermatogenic cells were studied by analysing induction of micronuclei during meiosis. Micronuclei (MN) were scored in early spermatids offer different time intervals corresponding to exposure of different stages of meiotic prophase. Etoposide had a strong effect on diplotene-diakinesis I cells harvested 1 day after exposure, and a significant effect also on late pachytene cells harvested 3 days after exposure. The effect at 18 days corresponding to exposure of preleptotene stage of meiosis (S-phase) was weaker but also statistically significant. Adriamycin was used as a positive control in this study. The results indicate a different mechanism of action of etoposide compared with adriamycin and other chemicals studied previously with the spermatid micronucleus test. DMA flow cytometry was carried out to assess cytotoxic damage at the same time intervals (1, 3, and 18 days after treatment) at stages I and VII of the seminiferous epithelial cycle allowing a study of cytotoxicity to different spermatogenic cell stages. Damage of differentiating sper-matogonia was observed by a decrease in the cell numbers of the 2C peak 1 and 3 days after treatment and by a reduction of the number of 4C cells (primary spermatocytes) 18 d after etoposide treatment. Adriamycin also killed differentiating spermatogonia. Since the cell population which showed a high induction of MN by etoposide was not reduced in number, the genotoxic effect is remarkable. We conclude that etoposide is a potent inducer of genotoxicity and patients treated with this agent during cancer chemotherapy are at a risk of genetic damage. © 1994 Wiley-Liss, Inc.     

Detection of histidine decarboxylase in peripheral blood leukocytes by flow cytometry

Inflammation Research -     

1,4-Dioxane is not mutagenic in five in vitro assays and mouse peripheral blood micronucleus assay,but is in mouse liver micronucleus assay

1,4-Dioxane, an animal carcinogen, was not previously genotoxic in in vitro assays. We reevaluated the compound's genotoxic potential in five in vitro genotoxicity tests in the presence and absence of S9 mix using recommended new protocols. We used the bacterial reverse mutation assay with Salmonella TA and E. coli WP2 strains, including the plate and preincubation methods, the CHO chromosomal aberration assay, including examination of polyploid induction and extended sampling time, the CHO sister-chromatid exchange assay with short and long treatment time, the mouse lymphoma tk assay (microtiter method), including longer treatment time (24 hr), and the CHO micronucleus assay with short and long treatment times. The highest concentration we used was five mg/ml or plate. We also evaluated the genotoxic effect of 1,4-dioxane in vivo by conducting peripheral blood and liver micronucleus assays in the same mice after single oral administration of up to 3,000 mg/kg. All in vitro assays and the peripheral blood micronucleus assay were negative. The mouse liver micronucleus assay, on the other hand, was positive, indicating that 1,4-dioxane might be genotoxic. It is also conceivable that the positive result in mouse liver micronucleus assay was due to a nongenotoxic mechanism, i.e., errors in genetic repair following enhancement of hepatocyte proliferation. Environ. Mol. Mutagen. 32:269–280, 1998 © 1998 Wiley-Liss, Inc.     

Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.     

Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay

Glyphosate is a widely used broad-spectrum weed control agent. In the present study, an in vivo study on the genotoxic effects of a technical herbicide (Roundup) containing isopropylamine salt of glyphosate was carried out on freshwater goldfish Carassius auratus. The fish were exposed to three doses of glyphosate formulation (5, 10 and 15 ppm). Cyclophosphamide at a single dose of 5 mg/l was used as positive control. Analysis of micronuclei, nuclear abnormalities and DNA damage were performed on peripheral erythrocytes sampled at intervals of 48, 96 and 144 h posttreatment. Our results revealed significant dose-dependent increases in the frequencies of micronuclei, nuclear abnormalities as well as DNA strand breaks. Our findings also confirmed that the alkaline comet assay and nuclear deformations in addition to micronucleus test on fish erythrocytes in vivo are useful tools in determining the potential genotoxicity of commercial herbicides.