The micronucleus assay in exfoliated human cells: a mini-review of papers from the CIS

The critical analysis of the data concerning micronucleus assay in exfoliated epithelial cells presented by the investigators from the CIS is carried out. Twenty two articles are evaluated, and shortcomings of some of them are discussed. Presented results are compared whenever possible with literature data. The aim of the mini-review is a criticism of shortcomings of the papersforfurther improvement of the presentation of the data on micronucleus assay which will give the possibility to compare the results with the data presented by foreign investigators.     

Lack of activity of estradiol in rodent bone marrow micronucleus assays

Estradiol has been evaluated in five independent rodent bone marrow micronucleus assays and has been found to be inactive. The dose-range evaluated extended from three daily doses of 20 μg/kg to the rat, a regimen that elicited a potent uterotrophic response from the animals, to single doses of between 10–150 mg/kg to the mouse. The mouse assays simulated and extended the conditions of test employed by earlier investigators who had found estradiol, and three structurally-related synthetic estrogens, to be active in mouse micronucleus assays over the dose range 1–10 mg/kg. It is concluded that estradiol is not genotoxic to the bone marrow of rodents. The top dose-level used in the present micronucleus assays (150 mg/kg) represented 150 000 times the minimum estrogenic dose of this chemical to rodents, and that was considered to be above the dose at which useful genetic toxicity data would be generated for this potent estrogen. The maximum tolerated dose (MTD) of estradiol to rodents remains to be established.     

Activation status of the X chromosome in human micronucleated lymphocytes

The frequency of X chromosome aneuploidy in human female peripheral blood lymphocytes has been reported by several investigators to be significantly higher than expected based upon chance alone. Studies in our laboratory showed that 72% of the micronuclei in the peripheral blood of human females contained the X chromosome. Such a high frequency of X chromosome loss suggests that some unique mechanism may be responsible for this phenomenon. The present study was carried out to test the hypothesis that the lost or micronucleated chromsome is the inactive and not the active X. Blood samples were obtained from two unrelated females, 36 and 33 years of age, each with a different X; 9 reciprocal translocation. In each, the normal X chromosome is inactive and the translocated X is active. Isolated lymphocytes were cultured according to standard techniques and blocked with cytochalasin B. Using a modified micronucleus assay, we scored 10,000 binucleated cells from the 36 year old, while 9,500 binucleated cells were scored from the 33 year old. The slides were first labeled and the kinetochore status of each micronucleus was determined. This was followed by simultaneous hybridization with a 2.0 kilobase centromeric X chromosome-specific probe and a chromosome 9 specific whole chromosome painting probe. All micronucleated cells were relocated and scored for their probe status. A total of 217 micronuclei were scored from the two subjects, of which 96 (44.2%) contained the X chromosome. Of these 96 micronuclei, 80 (83.3%) contained the inactive X, based on the absence of chromosome 9 material in the micronucleus. These results support our hypothesis that the inactive X chromosome is preferentially included in the micronuclei, and suggest that the X chromosome hypoploidy observed at metaphase in aging women is a related phenomenon. Received: 5 May 1995 / Revised: 15 July 1995     

Slide preparation and sampling as a major source of variability in the mouse micronucleus assay

This paper describes the results of a study in which mouse bone marrow micronucleus assay slides were assessed for homogeneity of micronucleated polychromatic erythrocytes (MPE) among polychromatic erythrocytes (PE). The slides were prepared by 3 distinct methods and several methods of slide reading were assessed. Observations made using our slides were confirmed by re-analysis of slides from 3 independent laboratories. It is concluded that the method of slide preparation and assessment can significantly influence the variability of data obtained from a study. The extent of this variability casts doubt upon the validity of certain assumptions concerning this assay--such as sex differences in MPE incidence, responder variability, etc. Results are discussed within the context of the very recent literature for this assay. Some laboratories appear to have adequate methods of slide preparation and data accumulation, while others do not. Methods to improve the sensitivity of this assay are suggested within the context of the recommendations made by the Gene-Tox review group. In particular, it is suggested that individual investigators present evidence of the adequacy of their data accumulation techniques in order to enhance the value of future studies.     

Micronucleus evaluation in peripheral blood reticulocytes of mice treated with procarbazine hydrochloride or mitomycin C.

The usefulness of the acridine orange (AO) supravital staining technique for the mouse peripheral blood reticulocyte micronucleus test was investigated independently by three laboratories using the known clastogens procarbazine hydrochloride (PCZ) and mitomycin C (MMC). In all three laboratories the highest frequencies of micronucleated peripheral blood reticulocytes were observed 48 h after treatment of mice with a single dose of either MMC or PCZ. The animals responded to both chemicals in a dose-dependent manner. Although similar qualitative results were observed, mean micronucleus frequencies induced by a particular dose of a given test chemical did vary quantitatively among the three laboratories. This was most probably due to the use of slightly different scoring criteria by each examiner. This aspect needs special attention. To minimize inter-laboratory variability, therefore, we recommend establishing unequivocal criteria to distinguish the subclass of reticulocytes. These should then be used consistently by all investigators using this method. The most striking advantages of the AO supravital staining technique were the ease of slide preparation, the ease with which reticulocytes and mature erythrocytes could be distinguished by the examiners, and the occurrence of numerous scorable reticulocytes in each microscopic field, which greatly speeded up the manual counting process. The disadvantages of the staining technique were the limited scoring time due to the rapid fading of the fluorescence stain, the degradation of the cells with time, and the frequent need to search for adequate scoring areas within a microscopic field. Based on the data of this study the authors conclude that the AO supravital staining technique is highly suitable for the micronucleus assay in erythrocytic cells of mouse peripheral blood. In addition, we consider the mouse peripheral blood reticulocyte micronucleus test to be a useful tool with which to investigate the clastogenic potential of chemicals in vivo. As pretreatment of mice with Aroclor 1254 markedly increased the effect of PCZ on micronucleus induction, we suggest that the inclusion of inducers of drug metabolizing enzymes in the micronucleus test would be useful for the detection of the clastogenic potential of promutagenic chemicals.     

Factors distinguishing couples at risk for nondisjunction

Micronucleus frequencies were determined on 24 young parents of trisomic infants, 21 individuals with recurrent unexplained abortions, and 42 control individuals with proven reproductive success. In addition to measurements of spontaneous micronucleus frequencies, mitomycin C induced frequencies were determined at two doses (2.5 and 5.0 ng/mL). Using the 95% confidence limits established from control data as an arbitrary upper limit, 16 of 24 parents of trisomics and 5 of 21 recurrent aborters were detected by the micronucleus assay to above this cutoff. The effects of sex, age, pregnancy status, and a variety of environmental exposures were studied by comparing the micronucleus frequencies of the exposed and unexposed populations. The data suggested that vitamins were associated with a lower micronucleus frequency and tea drinking with an increased micronucleus frequency in parents of trisomics, an effect not seen in controls. These results suggest that a biologic basis for nondisjunction may be associated with elevation in spontaneous and induced micronuclei. In addition, tea and vitamins may modulate micronucleus frequencies in parents of trisomics who appear to be more sensitive to these influences than the controls.     

The automated bone marrow micronucleus test

A new technology is presented which offers high-quality slides enabling the fully automated scoring of large quantities of erythrocytic cells for micronuclei by computerized image analysis. The techniques are applicable to bone marrow specimens as well as to peripheral blood obtained from various species of laboratory animals as well as from man. The key steps leading to this improved slide quality are the total removal of nucleated hematopoietic cells and the production of 'flat' cells by cytocentrifugation on polylysine-coated slides. The new procedures also allow the quantitative elimination of artifact-producing leukocytic granules from the rat bone marrow, even for the Fischer-344 strain, thus making the rat micronucleus test an attractive system for routine purposes in genetic toxicology. In addition, the proportion of immature erythrocytes can, if desired, be increased to more than 90% by using a Percoll step-gradient. This greatly facilitates the peripheral blood micronucleus test in laboratory animals as well as in (splenectomized) humans. First results, using peripheral blood from 2 rat strains, indicate that the immature erythrocyte population is very useful for micronucleus analysis, which encourages the development of a rat peripheral blood micronucleus test. This is an interesting application because it allows repeated testing in the same animals, resulting in fewer rats being needed, as no separate control groups are necessary. A further advantage is the possibility of concomitantly using rats from an ongoing toxicological study for micronucleus testing. The present results demonstrate that the new methodology is a valuable tool for improved micronucleus testing. Possible consequences in the field of genetic toxicology are discussed.     

Sensitivity of the erythrocyte micronucleus assay: dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., < or =0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

应用蝌蚪快速检测环境变异的两种方法--微核试验和单细胞凝胶电泳

本文介绍了应用无尾两栖类动物的蝌蚪进行环境监测的两种方法——微核试验和单细胞凝胶电泳。此两种环境检测的方法与其他环境检测方法相比具有快速,简便,易操作,适于检测现场应用,可大面积推广等优点。     

Sensitivity of the erythrocyte micronucleus assay: Dependence on number of cells scored and inter-animal variability

Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., ≤0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.     

In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test

The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size. The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.     

The mouse bone marrow micronucleus test: evaluation of 21 drug candidates

The mouse bone-marrow micronucleus test is one of the most widely used genetic toxicology assays. In this report the results of testing 21 compounds in the micronucleus test are presented. Of the 21 compounds tested, 3 potential chemotherapeutic agents were identified as strongly clastogenic. In addition, one compound was identified as a weak inducer of micronuclei in the assay. Further testing of this compound in an in vivo bone marrow metaphase analysis failed to confirm this material as clastogenic. The remaining 17 compounds were classified as negative in the assay. In general the results of the micronucleus test agreed with the results of other genetic toxicology assays on this group of compounds.     

Elimination of germ-line tandemly repeated sequences from the somatic genome of the ciliate Oxytricha fallax

The ciliated protozoa exhibit nuclear dimorphism. The genome of the somatic macronucleus arises from the germ-line genome of the micronucleus following conjugation. We have studied the fates of highly repetitious sequences in this process. Two cloned, tandemly repeated sequences from the micronucleus of Oxytricha fallax were used as probes in hybridizations to micronuclear and macronuclear DNA. The results of these experiments show: (1) the cloned repeats are members of two apparently unrelated repetitious sequence families, which each appear to comprise a few percent of the micronuclear genome, and (2) the amount of either family in the macronuclei from which our DNA was prepared is about 1/15 that found in an equal number of diploid micronuclei. Most, if not all, of the apparent macronuclear copies of these repeats can be accounted for by micronuclear contamination, which strongly suggests that these sequences are eliminated from the macronuclei and have no vegetiative function.     

Methods of diagnosis and differentiation of plague pathogen: approaches to detection of atypical strains of Yersinia pestis by molecular biology. Part I

A review of data on the modern methods of detection of typical and atypical strains of the plague agent Y. pestis is given. The history of the development of the molecular-biological tests for the differentiation of Y. pestis from the related microorganisms is presented. The problems facing investigators during the development of these tests are discussed.     

Risk factors for birth defects: data from the Atlanta Birth Defects Case-Control Study

The Atlanta Birth Defects Case-Control Study data comprises information obtained from interviews with parents of 4,900 babies born with major birth defects and with the parents of 3,000 babies born without defects. The source of cases is the Centers for Disease Control's Metropolitan Atlanta Congenital Defects program; the case-control study is population-based. Birth defects are classified into 92 groups and cross-tabulated by 105 exposure/risk factor variables; data from selected cross-tabulations are presented. The associations of each of the 105 exposure variables with all types of defects combined are presented, as are the associations of each of the 92 defect groups with the specific exposure variable, maternal diabetes. These data can be used to evaluate hypotheses arising from other sources, and for the purpose of "generating" hypotheses. The data describing all 92 x 105 cross-tabulations are available to other investigators on floppy disk; write to Chief, Birth Defects and Genetic Diseases Branch, Centers for Disease Control, Atlanta, Georgia 30333.     

Sequence characterization of Tetrahymena macronuclear DNA ends.

Tetrahymena is a ciliated protozoan which has two nuclei: a micronucleus, which maintains the genetic continuity of the cell, and the macronucleus which is derived from the micronucleus after sexual conjugation. A macronuclear DNA library was constructed to contain DNA ends. A probe containing C4A2 repeats which are known to be present at macronuclear DNA ends (1) was used to screen the library. Three clones were characterized by sequencing, restriction enzyme mapping and Bal 31 digestion. The data indicate that these three clones represent macronuclear DNA ends which were generated by DNA fragmentation during macronuclear formation. The sequencing data at the C4A2 repeat junction show a conserved sequence of five nucleotides, TTATT. Sequences further away show no obvious homologies except that they are highly enriched in AT. This structure is quite different from the subtelomeric sequences of other organisms.     

Development of a method for assessing micronucleus induction in a 3D human skin model (EpiDerm)

To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetics ingredients will not be allowed. Skin is a target area of interest for many cosmetic products because of its relatively high exposure. Therefore, it would be beneficial to have a non-animal, skin-based genotoxicity assay, especially one that utilized human skin in vitro. In this paper, we describe the development of a reproducible micronucleus assay that uses EpiDerm engineered human skin constructs (MatTek Corp., Ashland, MA). We describe methods for isolating single cells from the 3D skin model and for processing the cells for microscopic analysis of micronuclei (MN). In addition, since little was known about the kinetics of the dividing keratinocytes in the EpiDerm model, we evaluated whether cytochalasin B (Cyt-B) could be used to distinguish the population of dividing cells allowing the development of a micronucleus assay in binucleated cells. We found that the frequency of binucleated cells increased both with time and with increasing concentration of Cyt-B. After a 48-h exposure, 30-50% binucleated cells were reproducibly obtained. Finally, we evaluated micronucleus induction using the model genotoxicants mitomycin C (MMC) and vinblastine sulfate (VB). The background frequency of MN is very low and reproducible in this model, and statistically significant increases in the frequency of micronucleated cells were induced by both MMC and VB. These are initial steps in developing a routine "in vivo-like" assay for chromosomal damage in human tissue. It is hoped that other investigators utilize these methods to further the understanding of this potentially valuable new non-animal method.     

Resolution of the potassium pool size controversy in insect midgut

The isolated midgut of Lepidopteran larvae actively transports potassium from the hemolymph side to the lumen side when chamber-mountedin vitro. Active potassium transport is not affected by ouabain and is not dependent on sodium. A long controversy has existed over the fraction of the midgut cells that participate in active transport of potassium. One set of investigators demonstrated that only a small fraction of the tissue potassium was involved in the pool of potassium involved in active transport whereas a separate set of workers found that virtually all of the exchangeable potassium was thus involved. The results presented in this paper show that the insect's diet affects whether some or all the cells are in the pool, known formally as a transport pool. Leaf-reared insects are characterized by a small pool whereas diet-reared insects are characterized by a large pool. These results are shown to correlate with the pool size results of previous investigators.     

Vicia faba root tip micronucleus test on the mutagenicity of water-soluble contents of cigarette smoke

The possible mutagenicity of the water-soluble contents of cigarette smoke (WSCS) was evaluated by using the Vicia faba root tip micronucleus test. The results showed significant changes in micronucleus frequency which were caused by each different concentration of WSCS. This indicates that the Vicia faba root tip micronucleus test might be used as one kind of mutagenic detection method for cigarette smoke. A comparative evaluation on the mutagenicity of 10 brands of cigarettes was carried out. Results confirmed that various degrees of mutagenicity were found for all of the brand cigarettes, among them, Huaihai was the highest, while Camellia was the lowest. The micronucleus frequencies were reduced by adding tea polyphenol, nicotinamide adenine, vitamin C and sodium selenite to the WSCS. The results suggest that these added substances might reduce the genetic injury induced by cigarette smoke.     

Maintaining data integrity in microarray data management

Gene expression microarrays are a relatively new technology, dating back just a few years, yet they have already become a very widely used tool in biology, and have evolved to a wide range of applications well beyond their original design intent. However, while the use of microarrays has expanded, and the issues of performance optimization have been intensively studied, the fundamental issue of data integrity management has largely been ignored. Now that performance has improved so greatly, the shortcomings of data integrity control methods constitute a greater percent of the stumbling blocks for investigators. Microarray data are cumbersome, and the rule up to this point has mostly been one of hands-on transformations, leading to human errors which often have dramatic consequences. We show in this review that the time lost on such mistakes is enormous and dramatically affects results; therefore, mistakes should be mitigated in any way possible. We outline the scope of the data integrity issue, to survey some of the most common and dangerous data transformations, and their shortcomings. To illustrate, we review some case studies. We then look at the work done by the research community on this issue (which admittedly is meager up to this point). Some data integrity issues are always going to be difficult, while others will become easier-one of our goals is to expedite the use of integrity control methods. Finally, we present some preliminary guidelines and some specific approaches that we believe should be the focus of future research.