Micronucleus Technique Research Articles

Testing the mutagenicity of five pesticides with micronucleus technique ofTradescantia paludosa

Applying the micronucleus techniqueTradescantia paludosa, the mutagenicity of five pesticides, Decis, Sumicid, Cypermethrin, Trichlorphon and Diazinon were tested. The testing results showed that 0.64%, 0.32%, 0.16%, 0.08% of Decis, 0.64%, 0.32%, 0.16%, 0.04% of Trichlorphon and 0.32%, 0.16%, 0.08%, 0.04% of Diazinon all had mutagenicity onTradescantia paludosa, but Sumicid and Cypermethrin had no such characteristic.

Mouse and human micronucleus models for assessing genotoxicity of whole foods in intervention studies

There is an emerging need to apply classical genotoxicity assays to assess, in vivo, the carcinogenic or anti-carcinogenic potential of traditional and novel foods. The micronucleus techniques in peripheral blood erythorcytes and lymphocytes are two of the methods that have been used for assessing chromosome-damaging effects following and during dietary interventions with whole foods. The advantage of this approach stems from the practicality of the methods and the possibility of using these techniques in both human and rodent models. The potential application of these methods is illustrated by comparison of recent examples in the literature and from ongoing research.

The cytokinesis-block micronucleus technique and its application to genotoxicity studies in human populations.

The development of the cytokinesis-block (CB) technique has made the human lymphocyte micronucleus assay (MN) a reliable and precise method for assessing chromosome damage. Recent studies in our laboratory have confirmed that this method is a sensitive indicator of in vivo radiation exposure in patients undergoing fractionated partial-body radiotherapy and rodents exposed to uniform whole-body irradiation, thus supporting the application of the cytokinesis-block micronucleus (CBMN) assay for biological dosimetry. To further define the use of this assay in biomonitoring, we have also undertaken extensive studies to determine the spontaneous level of MN in normal human populations and its relationship to various lifestyle factors. During the past year, we have also developed a new variation to the CBMN assay that enables the conversion of excision-repairable lesions to MN within one cell-cycle using cytosine arabinoside. With this method the slope of the in vitro dose-response curves was increased by a factor of 1.8 for X-rays, 10.3 for ultraviolet (254 nm) radiation, and approximately 40-fold for methylnitrosourea. Consequently, the CBMN assay can now be used not only to measure whole chromosome loss or chromosome breaks but also excision repair events. The versatility and simplicity of the CBMN assay together with new developments in automation should enable its successful application in monitoring exposed populations as well as identifying mutagen-sensitive individuals within a population.

The Cytokinesis-Block Micronucleus Technique and Its Application to Genotoxicity Studies in Human Populations

The Cytokinesis-Block Micronucleus Technique and Its Application to Genotoxicity Studies in Human Populations

Micronucleus incidence and hematological effects in bullfrog tadpoles (Rana catesbeiana) exposed to 2-acetylaminofluorene and 2-aminofluorene

The micronucleus technique used with Rana catesbeiana tadpoles is useful for the evaluation of aromatic amine toxicity. When exposed to increasing concentrations of the aromatic amine 2-acetylaminofluorene (2-AAF 1 x 10(-8) M to 5 x 10(-5) M), R. catesbeiana tadpoles responded with an increasing frequency of micronuclei (MN) in the peripheral blood. Micronuclei increased fourfold with the 5 x 10(-5) M 2-AAF exposure. Micronuclei frequency significantly increased with concentrations as low as 5 x 10(-8) M. The time from initial mutagen exposure to the time of maximum number of MN present in the peripheral blood occurred at two separate times: 2 d and 14 d after exposure. The occurrence of two MN frequency peaks may be due to the presence of two erythrocyte populations from two hemopoietic sources (liver and kidneys) in young tadpoles. The occurrence of immature erythrocytes, as well as general degenerative changes [such as fragmentation (schistocytosis)] in the erythrocytes and vacuolated cytoplasms suggested 2-AAF may contribute to hemolytic anemia effects. 2-AAF also increased the frequency of rubricytes found in the peripheral blood, but had no significant effect on the hematocrit values of animals exposed to concentrations up to 1 x 10(-5) M. The tadpoles may have been producing rubricytes to replace the damaged red blood cells (RBCs). The direct-acting mutagen 2-aminofluorene (2-AF) was considerably more toxic to peripheral RBCs than the indirect-acting 2-AAF.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptive response to radiation damage in human lymphocytes conditioned with hydrogen peroxide as measured by the cytokinesis-block micronucleus technique

We have made use of the cytokinesis-block micronucleus method to evaluate the adaptive response in human lymphocytes from two donors conditioned with low doses of hydrogen peroxide before irradiation with either 1.5 or 3.0 Gy of X-rays. A protective effect of pre-exposure to H 2O 2 against radiation damage detected as micronuclei in binucleate cells was evident in cells allowed to recover for different periods after X-ray exposure, though cells challenged with 3.0 Gy of X-rays showed the adaptive response at later fixation time than those exposed to the lower dose (1.5 Gy). We propose this protocol as an interesting alternative to the single fixation method to score chromosomal aberrations at metaphase for the study of the adaptive response.

The cytokinesis-block micronucleus technique: A detailed description of the method and its application to genotoxicity studies in human populations

The development of the cytokinesis-block (CB) technique has transformed the human-lymphocyte micronucleus assay (MN) into a reliable and precise method for assessing chromosome damage. Recent studies in our laboratory have confirmed that this method is a sensitive indicator of in vivo radiation exposure in (a) patients undergoing fractionated partial-body radiotherapy and (b) rodnets exposed to uniform whole-body irradiation, thus supporting the application of the cytokinesis-block micronucleus (CBMN) assay for biological dosimetry. To further define the use of this assay in biomonitoring we performed extensive studies to determine the spontaneous level of MN in normal human populations and its relationship to various life-style factors. We have also developed a new variation to the CBMN assay that permits the conversion of excision-repairable lesions to MN within one cell-cycle using cytosine arabinoside. With this method the slope of the in vitro dose-response curves was increased by a factor of 1.8 for X-rays, 10.3 for ultraviolet (UV, 254 nm) radiation and approximately 40-fold for methylnitrosourea. Consequently the CBMN assay can now be used to measure not only whole chromosome loss or chromosome breaks but also excision-repair events. The versatility and simplicity of the CBMN assay together with new developments in automation should ensure its successful application in monitoring exposed populations as well as in identifying mutagen-sensitive individuals within a population.

Damage to lymphocytes by X-ray and bleomycin measured with the cytokinesis-block micronucleus technique

Chromosome damage induced by X-irradiation or bleomycin was measured using the cytokinesis-block micronucleus assay in the peripheral blood lymphocytes of 6 newborn, 8 young and 10 elderly individuals. An increase in the frequency of spontaneous micronuclei with age was observed. There was no difference in the X-irradiation-induced micronucleus frequency between the 3 groups. There was a significant increase with age in the number of micronuclei induced by bleomycin. Kinetochore-labelling studies revealed that the percentage of kinetochore-positive induced micronuclei was higher for bleomycin (36.2-43.3%) than for X-irradiation (17.1-19.7%). The age-related increase in frequency of spontaneous or bleomycin-induced micronuclei was due to increases in both kinetochore-positive and kinetochore-negative micronuclei. The frequency of kinetochore-positive or -negative micronuclei induced by X-irradiation was not different between the 3 age groups. These results suggest that bleomycin is more potent in inducing whole-chromosome loss than X-rays, and that lymphocytes from aged individuals are more sensitive to bleomycin in terms of both chromosome breakage and whole chromosome loss.

Mutagenicity studies on paracetamol in human volunteers: III. Cytokinesis block micronucleus method

As paracetamol (PC) affected the frequency of chromosome aberrations and unscheduled DNA synthesis in human volunteers, the genotoxic effects of PC were studied in a group of 12 healthy volunteers (9 females, 3 males, aged 37.8 ± 8.7 years) using the cytokinesis block micronucleus method in human peripheral lymphocytes. As a positive control a group of elderly people was used, 20 females and 10 males, aged 79.9 ± 9.5 years. PC was administered orally 3 times in a dose of 1000 mg during 8 h. Blood samples were taken at intervals of 0, 24, 72 and 168 h after the first dose of PC. Cytochalasin B was added to the cultures 44 h after the beginning of the 72-h cultivation at a concentration of 3.0 μg/ml. The frequency of cells with micronuclei in the group of volunteers was not significantly increased after PC administration. Using the cytokinesis block micronucleus method, the frequency of micronuclei was stable and the interindividual variability was low. The application of the micronucleus technique in genetic monitoring, e.g., for the occupational exposure to mutagens, is questioned.

Micronuclei in human lymphocytes as a biological dosemeter. Preliminary data following beta irradiation in vitro

The cytokinesis-blocked micronucleus technique has the potential of being a simple method of biological dosimetry for the rapid screening of individuals following large scale radiation accidents and for the assessment of suspected radiation over exposures. This technique was used to study the induction of micronuclei in human lymphocytes following irradiation in vitro by strontium/yttrium-90 beta rays. The lymphocytes were obtained from eight individuals aged between 23 and 57 years. The wide variation in dose-response for this limited sample suggests that this technique requires prior routine measurements on unirradiated personnel if the sensitivity of the technique is to be maximised. Beta irradiation in vitro appears to be less effective at inducing micronuclei than X-rays.

Radiation induction of micronuclei in human lymphocytes

An examination of the cytokinesis-blocked micronucleus technique confirmed its potential usefulness as a method of biological dosimetry for radiation accidents. Several advantages and disadvantages of the system are discussed. It has been demonstrated that under these conditions of these experiments, the blocking agent, cytochalasin B does not induce micronuclei or unstable chromosome aberrations. The induction of sister-chromatid exchanges proved just significant. Analysis of the dose response for 250 kVp X-rays indicates that although the Y = αD + βD 2 model fits the data, the relationship does not correspond to that for total aberration induction as might have been expected. The background frequency of micronuclei and the value of the α coefficient are higher than for total aberrations and the β term is lower. This indicates that simple incorporation of acentric chromosome fragments into micronuclei may not wholly account for the phenomenon.

Radiation induction of micronuclei in human lymphocytes

An examination of the cytokinesis-blocked micronucleus technique confirmed its potential usefulness as a method of biological dosimetry for radiation accidents. Several advantages of the system are discussed. It has been demonstrated that under the conditions of these experiments, the blocking agent, cytochalasin B does not induce micronuclei or unstable chromosome aberrations. The induction of sister-chromatid exchanges proved just significant. Analysis of the dose response for 250 kVp X-rays indicates that although the Y = αD + βD 2 model fits the data, the relationship does not correspond to that for total aberration induction as might have been expected. The background frequency of micronuclei and the value of the α coefficient are higher than for total aberrations and the β term is lower. This indicates that simple incorporation of acentric chromosome fragments into micronuclei may not wholly account for the phenomenon.

Induction and survival of micronuclei in rat spermatids. Comparison of two meiotic micronucleus techniques using cyclophosphamide

The induction and survival of micronuclei (MN) in rat spermatids was studied by two different methods, the dissection method (DM) and the suspension method (SM). It was observed that MN are induced by cyclophosphamide in the S phase of meiosis, in preleptotene spermatocytes, and in an earlier cell stage, the type B spermatogonia. Both techniques showed that MN survive in spermatids at least 5 days. Advantages of the DM include the use of a DNA-specific fluorochrome for staining of MN, higher MN frequencies observed, and the possibility to gain detailed information of the kinetics of induction of MN. In the SM, slide preparation is simpler than in the DM, and several samples can be prepared simultaneously but the scoring of slides is time consuming. Improvements of the sampling system of the DM are suggested. For evaluation of clastogenic action of chemicals on male germ cells both techniques provide a simple and rapid approach.

A search for radiosensitive mouse mutants by use of the micronucleus technique

In order to identify radiosensitive mutations in mice, 26 genetically well defined mutations in 26 different combinations of homozygous, hemizygous or heterozygous conditions, together with normal mice and mutagen-sensitive MS/Ae mice were analysed for the induction of micronuclei by X-rays in bone-marrow cells. For each mutant two doses of 0.5 and 1.0 Gy, two sampling times of 18 and 27 h after irradiation and unirradiated controls were studied. Using our criteria, homozygous contrasted allele of steel ( Sl con), scabby ( scb), viable dominant spotting ( W v), quaking ( qk). fidget ( fi) and postaxial hemimelia ( px), heterozygous lurcher ( Lc), hemizygous gyro ( Gy), the compounds Sl con/grizzle-belly ( Sl gbH) and W v/rumpwhite ( Rw) and MS/Ae mice, were regarded as radiosensitive, with Sl con/ Sl con the highest in rank order. Homozygous wabbler-lethal ( wl) and wasted ( wst) showed hyposensitivity which for the latter may be connected with enhanced cell killing.

The present state and perspectives of micronucleus assay in radiation protection. A review

The occurrence of micronuclei proved to be a sensitive biological indicator of clastogenic effects of many chemical and physical agents. The possibility of using the micronucleus technique in radiation protection as an alternative to the traditional chromosomal analysis has recently been followed with increasing interest. This review outlines the main biological and practical aspects of the micronucleus assay and discusses its potential to serve as a rapid and reliable measure of radiation overexposures and hypersensitivities.

Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation

The cytokinesis-block micronucleus technique was developed to overcome the kinetic problems inherent in the use of human lymphocytes for micronucleus assays. Using this technique the number of spontaneous micronuclei in lymphocytes from 42 individuals aged between 20 and 85 years was studied and was found to increase at a rate of 4.3% per year. Comparison with the results obtained with the conventional micronucleus assay confirmed that the conventional method markedly underestimates this age effect. The sensitivity of the cytokinesis-block method was determined by studying the effect of low-dose (< 50 rad) X-irradiation. The results indicated that the dose-response was linear and a single in vitro exposure to 5 rad of X-rays could be unequivocally detected. We concluded that the cytokinesis-block micronucleus method is more sensitive and precise than the conventional micronucleus method and classical metaphase analysis, and that it will be of value for detecting chromosome damage induced in vivo by genotoxic agents.

Measurement of micronuclei in lymphocytes

The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [ 3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 μg/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the ‘in vivo’ micronucleus frequency in normal individuals was 4.4 ± 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.

Short-term tests for transplacentally active carcinogens: I. Micronucleus formation in fetal and maternal mouse erythroblasts

A cell-kinetic model for the application of the micronucleus test to polychromatic erythrocytes in mouse fetal liver, fetal blood, and maternal bone marrow after exposure to clastogenic agents is described. The time of expression and dose-response relationships obtained with γ-radiation, methyl methanesulphonate, procarbazine, mitomycin C and benzo[ a]pyrene are analysed in terms of this model. The numbers of target cells damaged per unit dose has been calculated and the dose equivalents obtained. Maternal and fetal cells show similar sensitivity to γ-radiation, but fetal cells are markedly more sensitive to MMS and procarbazine. This probably due to differences in tissue distribution and metabolism. Maternal and fetal erythroid tissues can show linear and exponential dose-response relationships, which may not coincide (e.g. with MMS). It is concluded that risks from fetal exposure to genotoxic agents cannot be reliably predicted from in vivo tests restricted to adult animals. However, the micronucleus technique appled to fetal erythroid cells proveds a rapid and reliable short-term test, appropriate to minimising risks of genome damage during prenatal development.

A micronucleus technique for detecting clastogenic effects of mutagens/carcinogens (DEN, DMN) in hepatocytes of rat liver in vivo

A micronucleus assay in vivo has been developed that utilizes freshly isolated hepatocytes from livers of hepatectomized rats. In a small validation study, increased frequencies of micronuclei were detected in rats exposed to DEN and DMN before or after hepatectomy. The method is suitable for the detection of clastogenic effects of compounds or their metabolites that are too short-lived to reach the classical target cells used in cytogenetic studies. Because the non-hepatectomized liver shows very low levels of mitosis, the liver-micronucleus assay can also be used for the study of storage effects and the clastogenic effects of chronic exposures to mutagens/carcinogens.