The influence of dietary flaxseed and other grains, fruits and vegetables on the frequency of spontaneous chromosomal damage in mice.

Spontaneous genetic damage, whether mutations or chromosomal aberrations, undoubtedly arise from a variety of sources including replication errors, oxidative damage, background radiation, and chemical exposure. Given the numerous correlations between diet and cancer, it seemed possible that diet could influence the spontaneous rate of DNA damage and its genetic consequences. Since diets high in vegetables, fruits, and grains are associated with lower rates of cancer, we supplemented the diets of mice and measured the frequency of micronuclei in the peripheral blood. Micronuclei arise from broken chromosomes or chromosome loss in the erythroblast. They are first seen in the short reticulocyte stage of the red blood cell but persist for the entire 30-day lifespan of the cell in mice. C57Bl mice were placed on a defined diet (AIN-93G) supplemented to 20% final dry weight with grains or freeze-dried fruits or vegetables. The micronucleus frequency was measured in a pre-exposure blood sample and every 2 weeks thereafter for 6 weeks. This was possible in spite of the low spontaneous frequency of 1/1000-2/1000 cells by the use of a novel flow cytometric method, which permitted the analysis of both the mature red blood cells and reticulocytes. Of the foods tested, flaxseed proved to be the most protective by reducing the incidence of micronuclei in both the reticulocyte and normochromatic erythrocyte cell populations by 30 and 11%, respectively. The results show that at least one class of spontaneous genetic damage can be modified by diet and suggests that short-term experiments with small numbers of animals can be used to identify dietary anticarcinogens that may influence human cancer rates.

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.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Induction and time-dependent accumulation of micronuclei in peripheral blood of transgenic p53+/- mice, Tg.AC (v-Ha-ras) and parental wild-type (C57BL/6 and FVB/N) mice exposed to benzene by inhalation.

In this study, we determined the induction and time-dependent accumulation of micronuclei in the peripheral blood of transgenic C57BL/6 p53+/- mice (p53+/- mice), FVB/N Tg.AC v-Ha-ras mice (Tg.AC mice) and their isogenic parental strains, FVB/N and C57BL/6 following inhalation exposure to benzene. Our objective was to determine the impact of p53 heterozygosity in p53+/- mice and the v-Ha-ras transgene in Tg.AC mice on micronuclei induction following exposure to inhaled benzene. A flow cytometric technique that distinguishes micronucleated red blood cells (MN-RBC) from micronucleated reticulocytes (MN-RET) was used. Mice were exposed to 0, 100 or 200 p.p.m. benzene using three different exposure regimens that resulted in an equal weekly cumulative exposure (3000 p.p.m.x hours) to benezene: 100 p.p.m. for 6 h/day, 5 days/week, Monday to Friday (M-F); 100 p.p.m. for 10 h/day, 3 days/week, Monday, Wednesday, Friday (MWF); and 200 p.p.m. for 5 h/day, 3 days/week MWF. Significant elevations of MN-RBC and MN-RET were observed from 1 week exposure in all of the benzene-exposed groups that increased in a time-dependent manner for up to 13 weeks exposure. Fewer MN-RBC and MN-RET were induced in the 200 p.p.m. benzene exposure group than in mice exposed to 100 p.p.m. The reduction in the frequency of MN-RBC in the 200 p.p.m.x5 h benzene exposure group is probably due to metabolic saturation resulting in a lower bone marrow dose (concentration x time) than in the 100 p.p.m. exposure groups. No differences were observed in the frequency of MN-RBC or MN-RET in Tg.AC compared with the FVB/N isogenic controls. At certain time points the frequency of micronuclei was less in the heterozygous p53+/- mice than determined in the wild-type C57BL/6 isogenic parental strain. These results indicate that the heterozygous state in p53+/- mice, but not the v-Ha-ras transgene in Tg.AC mice can influence the induction of micronuclei by benzene.

Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

Flow cytometry assay for counting micronucleated erythrocytes: development process.

Development of any new assay proceeds in several phases. When an assay is intended for regular use to support regulatory decision-making, there are significant additional stages in the development process beyond the initial description of the method. In this paper we discuss some of the studies related to the development of a flow cytometric method for counting micronuclei in rodent erythrocytes. Studies related to fixation methods and conditions, standardization of DNA staining, and antibody staining are discussed. These studies, while not part of the formal description of the method, are needed as part of the preparation for the formal validation of the method. In addition, the lessons learned in transferring the method to other laboratories are briefly discussed in relation to defining the final protocol.

Enumeration of micronucleated reticulocytes in rat peripheral blood: a flow cytometric study.

Micronuclei (MN) are routinely enumerated in mouse peripheral blood to index genotoxicity. Recent data from the Collaborative Study Group for the Micronucleus Test (CSGMT) [CSGMT (The Collaborative Study Group for the Micronucleus Test), Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS MMS, Environ. Mol. Mutagen. 32 (1998) 84-100] suggest that rat peripheral blood may also be appropriate for the enumeration of MN, if scoring is limited to the youngest fraction of reticulocytes. The experiments described herein were designed to test whether modifications to a flow cytometric scoring procedure for measuring micronucleated reticulocytes (MN-RET) in mouse peripheral blood could be extended to accurately enumerate MN in rat peripheral blood. Rats were treated with saline or one of three genotoxic agents (6-mercaptopurine, ethyl methanesulfonate or propane sultone) in an acute dosing protocol. Peripheral blood samples were subsequently collected for both microscopic and flow cytometric analysis. Micronucleus frequencies were scored in the youngest fraction of reticulocytes: scoring by microscopy was restricted to the types I and II reticulocytes based on RNA content utilizing acridine orange supravital staining; flow cytometric measurements were restricted to the youngest fraction of reticulocytes based on transferrin receptor (CD71) staining. A statistically significant dose-related increase in the incidence of MN was observed, irrespective of scoring method. A higher level of statistical discrimination between control and genotoxin-treated groups was observed for the flow cytometric data and can most likely be explained by the increased number of cells scored (10x more than microscopy) and the lower scoring variability. Together, these data suggest that (i) rat peripheral blood represents an appropriate compartment for evaluating genotoxin-induced MN when the analysis is restricted to young reticulocytes, and (ii) the measurement of MN in rat peripheral blood reticulocytes benefits from the high throughput methodology of flow cytometry.

Malaria-infected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry.

A procedure for optimizing the configuration of flow cytometers for enumerating micronucleated erythrocytes is described. The method is based on the use of a biological model for micronucleated erythrocytes, the malaria parasite Plasmodium berghei. P. berghei endows target cells of interest (erythrocytes) with a micronucleus-like DNA content. Unlike micronuclei, parasitized red blood cells have a homogenous DNA content, and can be very prevalent in circulation. These characteristics make malaria-infected erythrocytes extremely well suited for optimizing instrument setup on a daily basis. The experiment described herein was designed to test the hypothesis that malaria-infected erythrocytes can greatly enhance the consistency with which flow cytometers are configured for micronucleus analyses, and thereby minimize intra- and interexperimental variation. Data collected over the course of several months, on two different flow cytometers, supports the premise that malaria-infected blood represents a useful biological standard which helps ensure reliable and consistent flow cytometric enumeration of rare micronucleated erythrocytes.

Impact of p53 status on heavy-ion radiation-induced micronuclei in circulating erythrocytes.

Transgenic mice that differed in their p53 genetic status were exposed to an acute dose of highly charged and energetic (HZE) iron particle radiation. Micronuclei (MN) in two distinct populations of circulating peripheral blood erythrocytes, the immature reticulocytes (RETs) and the mature normochromatic erythrocytes (NCEs), were measured using a simple and efficient flow cytometric procedure. Our results show significant elevation in the frequency of micronucleated RETs (%MN-RETs) at 2 and 3 days post-radiation. At 3 days post-irradiation, the magnitude of the radiation-induced MN-RET was 2.3-fold higher in the irradiated p53 wild-type animals compared to the unirradiated controls, 2.5-fold higher in the p53 hemizygotes and 4.3-fold higher in the p53 nullizygotes. The persistence of this radiation-induced elevation of MN-RETs is dependent on the p53 genetic background of the animal. In the p53 wild-type and p53 hemizygotes, %MN-RETs returned to control levels by 9 days post-radiation. However, elevated levels of %MN-RETs in p53 nullizygous mice persisted beyond 56 days post-radiation. We also observed elevated MN-NCEs in the peripheral circulation after radiation, but the changes in radiation-induced levels of MN-NCEs appear dampened compared to those of the MN-RETs for all three strains of animals. These results suggest that the lack of p53 gene function may play a role in the iron particle radiation-induced genomic instability in stem cell populations in the hematopoietic system.

New approaches to antimutagenesis.

One of the most worrisome possibilities in the field of antimutagenesis and anticarcinogenesis is that factors that protect against one mutagen or carcinogen will not be effective against another. Indeed, such specificities are known. Consequently, protective agents found in experimental screens and systems may not be relevant to the human situation. Additionally, the question of dose is also problematic, because factors that can protect against the levels of mutagens or carcinogens present in the human environment may be overwhelmed by the large dose of the agent used in the experiment, a dose necessitated by the need to have an effect against which protection can be judged. We suggest here that the new technologies available for the measurement of somatic mutation and chromosomal damage can be used to study spontaneous events, thus avoiding both problems.

An automated method for discriminating aneugen- vs. clastogen-induced micronuclei.

A flow cytometric (FCM) procedure for quantitating micronucleated reticulocytes in mouse peripheral blood samples was evaluated for its ability to discriminate between aneugen- and clastogen-induced micronuclei (MN). In this experiment, BALB/c mice were injected with 0.9% saline, the model clastogen methyl methanesulfonate (100 mg/kg bw) or the aneugen vincristine (0.2 mg/kg bw). Peripheral blood samples were collected 48 hr after injection and were subsequently fixed and stained for flow cytometric analysis. The staining method utilized FITC-conjugated anti-CD71 to differentially label reticulocytes, and the nucleic acid dye propidium iodide to resolve erythrocyte populations with and without micronuclei. The frequency of micronucleated reticulocytes was determined by analyzing 10,000 total reticulocytes per blood sample. A second analysis was performed on each sample whereby the propidium iodide associated fluorescent signals of 250 MN were collected and graphed as a single-parameter histogram. The histogram statistic "median channel" was recorded for each sample and provided a quantitative description of MN distribution according to DNA content. Cumulatively, the results of this study suggest that 1) flow cytometry can be employed to measure the incidence of MN resulting from clastogenic or aneugenic activity, and 2) MN resulting from aneugens can be discriminated from those arising spontaneously or from clastogen treatment based on flow cytometric analysis of DNA content.

Flow cytometric analysis of micronucleated reticulocytes in mouse bone marrow.

This laboratory has previously reported a flow cytometric procedure for quantitatively analyzing mouse peripheral blood reticulocytes for micronucleus content. The current study extends this line of investigation by evaluating whether these same flow cytometric scoring procedures can be applied to the analysis of mouse bone marrow samples. To validate the method, three groups of male BALB/c mice were treated with 100 mg/kg b.wt. methyl methanesulfonate. Bone marrow samples were collected 20, 40 or 60 h after administration. A set of 5 untreated animals was included to provide an indication of spontaneous micronucleus frequencies. The cells were fixed with ultracold methanol, treated with ribonuclease, and labeled with anti-CD71 antibody (FITC conjugate) and propidium iodide. This fixing and labeling procedure resulted in the resolution of the micronucleated reticulocyte population and facilitated high-speed acquisition and enumeration via flow cytometry. The number of micronucleated reticulocytes was determined flow cytometrically by the analysis of 10,000 total reticulocytes per bone marrow sample. In addition to these automated measurements, slides stained with acridine orange were prepared and the number of micronuclei per 1000 reticulocytes was determined microscopically for each sample. The resulting data demonstrate that flow cytometry can effectively enumerate micronucleated reticulocytes in mouse bone marrow. The advantages associated with an objective, high throughput scoring methodology are also clearly indicated.

Simple and reliable enumeration of micronucleated reticulocytes with a single-laser flow cytometer.

A flow cytometric procedure for scoring micronuclei in mouse peripheral blood erythrocytes, especially reticulocytes, is described. The methods reported herein were developed in an effort to simplify the techniques and to reduce the equipment requirements associated with automated micronucleus analyses. With this procedure, fluorescein-conjugated monoclonal antibodies which bind to the CD71-defined antigen (the transferrin receptor) are used to label reticulocytes. The nucleic acid dye propidium iodide is used to identify cells with micronuclei. Given 488 nm excitation, four populations of erythrocytes are clearly resolved: normochromatic erythrocytes with and without micronuclei, and reticulocytes with and without micronuclei. Since the method is capable of simultaneously providing the incidence of micronuclei in both mature and immature erythrocyte populations, it is compatible with either chronic or acute treatment regimens. To demonstrate cell handling and flow cytometric procedures for quantitatively analyzing peripheral blood micronuclei, an experiment with the model clastogen methyl methanesulfonate is described. Additionally, a reconstruction experiment was performed whereby three mouse blood samples were spiked with successively greater volumes of blood from a clastogen-treated animal so each preparation differed slightly, but definitely, in micronucleus content. Each sample was scored six times by conventional microscopy and by flow cytometry so that the two methods could be directly compared. Collectively, the results from the methyl methanesulfonate experiment and the reconstruction study demonstrate the accuracy and reliability of the flow cytometric method. Furthermore, advantages associated with objective, high throughout scoring methodology are clearly indicated.

Induction of micronuclei by low doses of azidothymidine (AZT).

The dideoxynucleoside azidothymidine (AZT; Zidovudine) was assessed for its ability to induce micronuclei in mouse erythrocytes at a low (therapeutic) dosage. Specifically, male and female BALB/c mice were treated via intraperitoneal injection 5 days a week for 2 weeks with saline or 17 mg AZT/kg body weight per day. Each animal was monitored for chemical-induced micronucleus formation over the course of the treatment regimen through the flow cytometric analysis of one million pre-dosing and one million post-dosing peripheral blood erythrocytes. No significant change in micronucleus frequencies was observed for the vehicle control group as micronuclei continued to enter the peripheral blood pool at background levels. Conversely, the AZT-treated mice exhibited a statistically significant net increase in micronucleated cells over the course of dosing as erythrocytes with a high incidence of micronuclei entered the peripheral blood pool. The advantages of high throughput scoring protocols utilizing flow cytometry are discussed.

Modulation of phorbol ester-induced HL-60 differentiation by prostaglandin E2.

When treated with phorbol tumor promoters, HL-60 cells undergo terminal differentiation evidenced by a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture with high phagocytic activity. Internalization of fluorescent particles by cells exhibiting the phagocytic positive phenotype (phag+) provides a sensitive indication of promoter-induced differentiation, and the resulting fluorescent cells can be quantitatively analyzed by flow cytometry. The current study was initiated to further test the predictive power of a flow cytometry based HL-60 differentiation assay in the detection of agents associated with tumor promotion. Specifically, experiments were designed to assess the sensitivity of the test system to co-promoters which enhance promoter activity in vivo. Prostaglandin E2 (PGE2) was chosen as a model co-promoter since it has been shown to potentiate phorbol ester (i.e. 12-O-tetradecanoyl phorbol-13-acetate; TPA) induced biological effects in vivo. Results detailed in the current report indicate that PGE2 enhances TPA-induced differentiation of HL-60 cells in a dose-dependent manner. As with in vivo co-promotion experiments, PGE2 exhibited a maximum potentiating effect when administered prior to TPA. These data indicate that HL-60 cells are not only sensitive to phorbol promoters, but also to the co-promoter PGE2. These experiments support the hypothesis that a flow cytometry based HL-60 assay may prove useful for studying chemical agents or intrinsic cellular factors that are involved in the tumor promotion phase of carcinogenesis.

Development of a sensitive in vitro method for identifying tumor promoters.

The identification and characterization of nongenotoxic carcinogens represents a significant challenge to toxicologists. In vitro methods for identifying tumor promoters with suitable sensitivity and specificity have been particularly elusive. Experiments are described which suggest that the human promyelocytic leukemia cell line HL-60 provides a sensitive indicator of promoter-induced changes to gene regulation and expression. As a result of differentiation these cells undergo a transition from a non-phagocytic suspension culture to an attached fibroblast-like culture which exhibits high phagocytic activity. Fluorescent latex particles were used as sensors to highlight the phagocytic phenotype and permitted the use of flow cytometry to automatically quantitate particle internalization. To evaluate specificity, HL-60 cells were treated with a series of phorbol esters covering a range of in vivo tumor promoting activity. Results indicate that this family of compounds induces HL-60 cells to differentiate in proportion to their in vivo promoting activity. To closely assess the sensitivity of the phagocytic endpoint, HL-60 cells were treated with picogram levels of 12-O-tetradecanoyl phorbol-13-acetate (TPA), whereupon increments as low as 50 pg of TPA per ml caused statistically significant increases in phagocytic activity. The experiments described herein suggest that in vitro differentiation of HL-60 cells may reflect the promoter-dependent modifications to gene expression that are observed in vivo during the promotion phase of carcinogenesis. The described method may represent a sensitive promoter screening assay which is both rapid and economical.

Analysis of micronucleated cells by flow cytometry. 4. Kinetic analysis of cytogenetic damage in blood.

Micronucleated cells (MN cells) generated spontaneously or by clastogen action accumulate in the peripheral blood of the mouse, and their presence reflects the level of chromosome damage. Traditionally, micronucleated cells have been scored by visual inspection. With the development of flow cytometry based scoring procedures, vast numbers of cells can be analyzed, making it possible to determine the change in the number of MN cells in the total peripheral blood pool. This report describes experiments whereby initial blood samples were obtained before dosing, providing mouse-specific controls for measuring subsequent changes in MN cells. Mice were then dosed with saline (solvent control), methyl methanesulfonate, cyclophosphamide or colchicine every 48 h and bled every 96 h for 12 days. For each blood sample, one million fixed erythrocytes (RBCs) were interrogated for the presence of micronuclei, and regression analysis was used to determine the rate of MN cell influx per day for each animal or sets of animals. To evaluate the effect of treatment on MN induction, the mean slopes of solvent and chemically treated animals were compared using t-tests. The results of these experiments indicate that the kinetics of MN induction continues near the background frequency for saline dosed mice, whereas clastogenic agents or spindle poisons cause a significant influx of MN events into the blood. The results suggest that some studies may benefit from a flow cytometry based analysis of multiple blood samples, especially when the number of mice is limited, or when a weak clastogen is being investigated.

Analysis of micronucleated cells by flow cytometry. 2. Evaluating the accuracy of high-speed scoring.

Micronucleated blood cells--whether generated spontaneously or by clastogen treatment--are present in the blood and bone marrow as rare events. Historically they have been scored manually by microscopic inspection which is labor-intensive and tedious. It has been recognized by investigators that a need exists for an automated method which can accurately, objectively and quantitatively score rare micronucleated cells. In order to improve assay statistics more cells must be processed, making high-speed scoring an important objective of any automated procedure. Flow cytometry can provide the means to quantitatively analyze micronucleated cells at high speeds and with great accuracy once the chemical, biological and instrumentation conditions are optimized. Recent literature suggests that noise and fidelity of the data, as well as the sensitivity of present flow cytometers, are major obstacles that still must be overcome. Experiments are described herein which demonstrate that flow cytometry is able to score micronucleated cells under conditions where noise levels are low, and the fidelity and accuracy are high. In addition, the accuracy of scoring rare events is maintained at high speeds (e.g. 1,000,000 cells/min). A major emphasis of this manuscript is to demonstrate the means for evaluating the accuracy and sensitivity of the flow cytometer in scoring rare events. Both computer simulation and reconstruction experiments were used to gauge scoring accuracy and guided optimization experiments. These experiments demonstrate that when optimum conditions are used in conjunction with a suitable flow cytometer, it is possible to score micronucleated cells at high speeds with great precision.

Analysis of micronucleated cells by flow cytometry. 1. Achieving high resolution with a malaria model.

Micronucleated cells (MN cells) are present in the blood as rare events (i.e. about 2 MN cells/1000 total). Scoring MN cells by hand is both time-consuming and tedious, which is the primary reason why only 1000-2000 total cells (PCEs) are routinely scored for each sample. It is generally recognized that scoring larger numbers of cells would improve assay statistics and is desirable, but impractical with hand-scoring. In contrast, automated scoring methods can process large numbers of cells, thus improving statistical analysis. In order to accurately and quickly evaluate clastogenic activity, we have developed a flow cytometry based method of scoring micronucleated cells. One of the first steps in developing an automated assay is to demonstrate the ability of the method to resolve the cells of interest. In this case, micronucleated cells must be resolved from DNA-deficient red blood cells (RBCs). Since micronuclei are heterogeneous rare events which vary in both size and DNA content, we chose to use a more enriched and homogeneous biological model for optimizing the experimental variables of this assay, leading to high resolution of the rare cells. Experiments are described in which the murine malaria parasite, P. berghei, served as a micronucleus model and facilitated the development of an accurate flow cytometry based scoring method. This parasite resides in the red blood cell population and endows the cells with a homogeneous (genetically determined) DNA component in the micronucleus size range. The conditions developed with the malaria parasite are readily applied to the analysis of micronucleus events in blood samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of micronucleated cells by flow cytometry. 3. Advanced technology for detecting clastogenic activity.

Under optimum conditions, flow cytometry (FCM) can provide a powerful technology for analyzing rare micronucleated cells in the peripheral blood. Our efforts in this endeavor have been directed toward a careful and meticulous optimization of experimental conditions, in order to achieve high resolution and high accuracy before introducing biological variation. We have achieved high resolution (Tometsko et al., 1993a) wherein the micronucleus signal is moved 100-fold upfield and away from the DNA deficient red blood cell (RBC) peak. In addition, we have demonstrated the high accuracy of our flow cytometry method in scoring rare micronucleated cells (Tometsko et al., 1993b). In the course of our studies, we rigorously pursued conditions which minimized experimental noise, demanding that FCM-scoring accuracy should approach theoretical limits. Thus, we laid the foundation for detecting clastogen activity with great sensitivity. The experiments described herein extend the previous studies by using high-speed flow cytometry to detect a clastogen-induced increase in MN cells in the total erythrocyte population. Methyl methanesulfonate (MMS) served as a model clastogen in these studies. This manuscript describes our development of a suitable blood-sampling regimen, the advantages of obtaining initial blood samples before dosing, the sex-linked difference in background micronucleus levels in BALB/c mice, and the analysis of a clastogen-induced biological response in male and female mice. As described, our flow cytometry method is able to provide a quantitative analysis of the net change in micronucleated cells (delta MN) for each mouse.