Comparative Scoring of Micronucleated Reticulocytes in Rat Peripheral Blood by Flow Cytometry and Microscopy

Dorothea K. Torous, Nikki E. Hall, Francis G. Murante, Sarah E. Gleason, Carol R. Tometsko and Stephen D. Dertinger1

Litron Laboratories, 1351 Mount Hope Avenue, Rochester, New York 14620

Received March 31, 2003; accepted April 30, 2003

ABSTRACT

A flow cytometric technique for scoring the incidence of micronucleated reticulocytes in rat peripheral blood was compared to a standard microscopy-based procedure. For these studies, groups of five male Sprague-Dawley rats were treated with vehicle or a broad range of chemical genotoxicants: 6-thioguanine, N-methyl-N‘-nitro-N-nitrosoguanidine, vincristine, methylaziridine, acetaldehyde, methyl methanesulfonate, benzene, monocrotaline, and azathioprine. Animals were treated once a day for up to 2 days, and peripheral blood was collected between 24 and 48 h after the final administration. These samples were processed for flow cytometric scoring and microscopy-based analysis using supravital acridine orange staining, and the percentage of reticulocytes and micronucleated reticulocytes was determined for each sample. The resulting data demonstrate good agreement between these scoring methodologies, although careful execution of the flow cytometric method was found to enhance the micronucleus assay by reducing both scoring time and scoring error. These data add further support to the premise that the peripheral blood compartment of rats can be used effectively to detect genotoxicant-induced micronuclei.

Key Words: micronuclei; genotoxicity; flow cytometry; rats; CD71 antigen.

The micronucleus (MN) assay (Hayashi et al., 2000Go; Heddle, 1973Go; Schmid, 1975Go) is the most widely utilized in vivo system for evaluating chemicals’ potential to induce chromosome breaks or to poison mitotic spindle apparatus. The test is based on the observation that replicating cells with chromatid breaks or dysfunctional mitotic apparatus exhibit disturbances in the anaphase distribution of their chromatin. After telophase, this displaced chromatin can be excluded from the nuclei of the daughter cells and is found in the cytoplasm as a micronucleus. Micronuclei therefore represent chromosome fragments or whole chromosomes resulting from clastogenic or aneugenic events. Erythrocytes are particularly well suited for evaluating MN events since erythroblast precursors are a rapidly dividing population of cells, and their nucleus is expelled a few hours after the last mitosis, making MN-associated chromatin relatively simple to detect.

The MN assay was originally devised to score chromosome damage in mouse bone marrow. MacGregor et al.(1980)Go demonstrated that micronuclei formed in the bone marrow of mice persist in the peripheral blood. Therefore, assay sensitivity is retained when studying gentoxicant-induced micronucleated erythrocytes in the peripheral blood of mice (CSGMT, 1992Go; Hayashi et al., 1990Go). However, in the area of product safety assessment, the mouse is not the preferred test species. Rather, the majority of studies are performed with rats (e.g., acute and subchronic toxicology and pharmacokinetic studies). This being the case, a rat peripheral blood-based MN test has tremendous potential for providing investigators with concurrent in vivo endpoints. The peripheral blood compartment is ideal for such integrated studies, since it can be readily sampled at any point during subchronic studies (Asanami et al., 1995Go). Thus, if the MN endpoint proves to be an appropriate and sensitive index of genotoxicity in rat peripheral blood, it may be possible to significantly reduce animal usage by eliminating dedicated rodent micronucleus tests.

To date, erythrocyte-based MN studies involving the rat blood compartment have been qualified because it has been assumed that the high efficiency in which the spleen eliminates MN erythrocytes would severely limit assay sensitivity. Even so, data are accumulating which suggest that rat blood may be used effectively to study chemical-induced genotoxicity (Abramsson-Zetterberg et al., 1999Go; Asanami et al., 1995Go; Hamada et al., 2001Go; Hayashi et al., 1992Go; Hynes et al., 2002Go; Torous et al., 2000Go; Wakata et al., 1998Go). These studies have typically restricted analysis of MN to the youngest fraction of reticulocytes (RETs). The premise is that the impact of spleen function would be reduced by scoring MN in these cells. The flow cytometric (FCM) system for scoring rodent MN-RET frequency developed by this laboratory is based on anti-CD71-FITC staining of RETs (MicroFlow®, Dertinger et al., 1996Go; Torous et al., 2000Go). By using CD71-based fluorescence as an index of RET age, this system has the potential to focus analysis of MN in the youngest RET population (in analogy to microscopists who rely on RNA content).

Previously, Schlegel and MacGregor (1984)Go estimated that rat peripheral blood studies would require the analysis of six to eight times the number of RETs in comparison to bone marrow to achieve equivalent statistical power. By analyzing blood samples for the presence of MN at rates up to several thousands of cells/second, a much greater number of cells can be analyzed in comparison to microscopic scoring. Flow cytometry therefore represents a technology that allows MN-RET scoring to be restricted to certain (e.g., youngest) RET cohorts, and also allows for eightfold (or greater) numbers of cells to be scored compared to conventional methodologies. Experiments were initiated to test the hypothesis that rat peripheral blood-based MN tests would benefit from automated scoring which allows for (1) analysis to be restricted to the most immature fraction of RETs, and (2) analysis of 20,000 RETs per animal compared to the standard value of 2000. Dose response data for nine well-characterized genotoxicants is presented herein, both from microscopy- and FCM-derived measurements.

MATERIALS AND METHODS

Animals and treatment regimen.
Six-week-old male Sprague-Dawley rats were purchased from Taconic (Germantown, NY). Animals were group housed and randomly assigned to treatment groups. The animals were acclimated for one week before experiments were initiated. Food and water were available ad libitum throughout the acclimation and experimentation periods. Each treatment group consisted of five rats. The nine genotoxicants studied were evaluated in a total of four separate experiments. Each of these experiments incorporated a concurrent vehicle control. CAS numbers and choice of solvents, as well as administration/bleeding details, can be found in Table 1Go. The choice of nine chemicals, representing both clastogenic and aneugenic mechanisms of action, was designed to test the robustness of the system for identifying diverse classes of genotoxicants. Note that choice of solvents, dose levels, and blood harvesting times were derived from literature, especially Wakata et al., 1998Go, and Abramsson-Zetterberg et al., 1999Go.


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TABLE 1 Chemical and Dosing Information
 
Blood sample preparation.
Between 24 and 48 h after the last administration, blood samples were collected from the tail vein after a brief warming period under a heat lamp. Approximately 10 µl of blood was applied to acridine orange (AO) coated slides. These slides were used to accomplish the microscopic scoring portion of these experiments according to the supravital staining method of Hayashi et al.(1983)Go. After applying coverslips and allowing the coded slides to sit on a level surface, they were boxed and frozen at –20°C. Slides were maintained in this state until analysis. Approximately 120 µl of peripheral blood was obtained for flow cytometric analysis using procedures described in the Rat MicroFlowPLUS Micronucleus Analysis Kit (Litron Laboratories, Rochester, NY, or BD-Biosciences Pharmingen, San Diego, CA). Briefly, heparinized blood samples were maintained at room temperature for no more than 2 h before being fixed. Samples were added to ultracold fixative, and were stored at –80°C until analysis. On the day of analysis, blood samples were simultaneously treated with RNase A and anti-CD71-FITC and finally resuspended with 1 ml staining solution (propidium iodide; PI). Before analyzing vehicle or genotoxin-treated rat blood samples, a malaria-infected blood sample was used for instrument set-up as described below.

Instrument setup with malaria-infected blood.
All flow cytometric analyses were carried out with a FACSCalibur flow cytometer (Becton Dickinson; 488 nm excitation). The CD71-FITC and PI fluorescence signals were detected in the FL1 and FL3 channels, respectively. To reproducibly calibrate the instrument for the micronucleus scoring application, this laboratory has recommended analyzing rodent erythrocytes infected with the malaria parasite Plasmodium berghei (Tometsko et al., 1993Go; Torous et al., 2001Go). Fixed P. berghei-infected rodent cells were therefore stained in parallel with test samples on each day of analysis. By maximizing the fluorescent resolution of erythrocytes with and without malaria parasites, these MN-erythrocyte mimicking biostandards facilitated optimization of FL1 and FL3 PMT voltages. Blood rich in malaria-infected erythrocytes also guided FL3-%FL2 compensation settings, and the PI-associated fluorescent signal of single parasite-containing cells was used to set the boundary of the quadrant which differentiates erythrocytes with and without MN (see Fig. 1Go).



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FIG. 1. Bivariate graph of malaria-infected rat blood. Green fluorescence associated with CD71 expression is graphed on the Y axis, and red fluorescence associated with DNA content is graphed on the X axis. Note that nucleated cells, which fall in the forth decade of propidium iodide fluorescence, have been excluded from this plot based on their high (2n) DNA content. One malaria-infected blood sample was stained in parallel with test samples and was analzyed at the beginning of each day of analysis. These samples were used to set appropriate PMT voltages, as well as FL3–%FL2 compensation. As malaria-infected erythrocytes mimic the DNA content of micronucleated erythrocytes, they also served to guide the position of the red fluorescence demarcation line, which was used to distinguish erythrocytes with and without micronuclei. Since these blood samples were obtained from juvenile rats, a high proportion of reticulocytes exhibit a high and uniform CD71 expression level. The fluorescence of this CD71 positive population was used to guide the position of the green fluorescence demarcation line, which was used to distinguish the most immature fraction of reticulocytes from more mature reticulocytes and normochromatic erythrocytes.

 
Focusing analysis on young reticulocytes.
When utilizing rat peripheral blood for the MN assay, current recommendations indicate that analyses should be restricted to the most immature RETs (Hayashi et al., 2000Go). Therefore, for all AO-coated slides, MN measurements were accomplished by restricting analysis to Type I and II RETs (i.e., those with reticulum covering at least half the cytoplasm; CSGMT, 1992Go). For flow cytometric analysis, the lower green fluorescent threshold demarcating the most immature RETs (i.e., high CD71-staining cells) from the more mature RETs and normochromatic erythrocytes was set according to recommendations in the MicroFlowPLUS kit (see Fig. 1Go). Based on labeling with anti-CD71-FITC plus PI staining (without RNase treatment), it appears that between 30 and 50% of all RNA-positive RETs exhibit this high CD71 expression level (see Fig. 2Go).



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FIG. 2. Bivariate graph of rat blood labeled with anti-CD71-FITC and propidium iodide. When applied to propidium iodide stained cells (no RNase treatment), the anti-CD71-FITC reagent is shown to label those erythrocytes with the highest PI signal. Based on this and other analyses, the highest CD71-expressing cells represent approximately the youngest 30 to 50% of RNA-positive reticulocytes.

 
Scoring and statistical analyses.
AO-coated slides were scored blind for %MN-RET and %RET frequency using a fluorescence-equipped microscope (Olympus BH-2). To accomplish RET scoring, 1000 total erythrocytes were inspected and the number of all RNA-positive erythrocytes were tallied. For determination of MN-RET frequency, 2000 Type I and II RETs per sample were evaluated for MN. Flow cytometric scoring occurred after instrumentation was optimally configured with malaria biostandard samples. Data acquisition was performed with CellQuest software (v3.3), with the stop mode set so that 20,000 high CD71-expressing RETs were collected per blood sample. Since the number of normochromatic erythrocytes (NCEs) was also measured, an index of cytotoxicity (percentage of high CD71-expressing RETs) was simultaneously obtained.

Statistical analyses were performed with JMP Software (v5, SAS Institute, Cary, NC). For each treatment group, the mean and standard deviation for %RET and %MN-RET was calculated. ANOVA was used to assess whether there were treatment-related changes to RET and MN-RET frequencies (significance indicated by p < 0.05). Positive ANOVA results were followed by Dunnett’s pair-wise tests. A trend test was also utilized to evaluate whether a dose-related increase in MN-RET had occurred. This assessment was performed for each chemical using a linear regression model. A regression effect (i.e., a dose-related trend) was indicated when p < 0.05. We considered the MN-RET data sets to exhibit evidence of genotoxicity if either a pair-wise test or the trend test was positive.

To further investigate correspondence between scoring methodologies, the mean %RET value for each treatment group as measured by FCM was plotted against the corresponding mean %RET value obtained through microscopic inspection. A linear correlation coefficient (r value) was calculated for each of the nine chemicals examined. This same analysis was performed for %MN-RET frequencies.

RESULTS

The effect of chemical treatment on RET frequency is presented in Table 2Go. The frequency of RETs based on AO-coated slides represents all RNA-positive erythrocytes. The frequency of RETs measured by FCM is based on the expression of high levels of CD71 (Fig. 2Go). Therefore, while these measurements are closely related, the higher absolute microscopy-based values were expected. Even so, there was good agreement between scoring systems as evidenced by high linear correlation coefficient values. There were two exceptions that correspond to chemicals that produced modest (or no) change in RET levels. In the case of MNNG, it is likely that a reduction in very young RETs detected by FCM reflects an early indication of cytotoxicity. That is, we would expect the youngest cohort of RETs (high CD71 expressing erythrocytes) to be a leading indicator of cytotoxicity that the entire RNA-positive RET cohort (AO staining) will reflect at a somewhat later time point. Acetaldehyde showed no significant effect on RET levels, irrespective of scoring method. The low r value resulted from a best fit line algorithm that was based on very few data points (4), coupled with the fact that the mean %RET values plotted did not differ appreciably over the concentration range tested.


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TABLE 2 Reticulocyte and Micronucleated Reticulocyte Data
 
The frequency of MN-RET data is presented in Table 2Go. Each of the nine chemicals induced MN that could be detected in the peripheral blood compartment of rats. In general, the high linear correlation coefficients provide evidence of good correspondence between scoring methodologies. Even though high linear correlation was obtained in the case of vincristine and azathioprine data sets, it is interesting that MN-RET values were somewhat higher when scored by microscopy, even though both microscopy- and FCM-based procedures were based on analyses that were restricted to the youngest RETs. The reason for this is not completely understood at this time, but may reflect modest differences in the age cohort of RETs evaluated in each methodology, and/or the threshold size of MN detected by each system.

In addition to analyses of correlation, it was also informative to consider statistical tests that assess whether significant chemical-dependent MN formation had occurred. When criteria for a genotoxic response was defined as a significant pair-wise test or a significant trend test, then seven of nine chemicals were judged to be positive when based on microscopic inspection. When these same criteria are applied to each FCM-based data set, all nine chemicals were positive. The two discrepancies were with MNNG and benzene. These chemicals were judged positive in the report by Wakata et al.(1998)Go, and indeed the microscopy-based data approached statistical significance in the present study. In both cases, it seems likely that it was the more accurate depiction of the dose-response curve and the lower variability associated with FCM measurements that provided for a more sensitive trend test (Table 2Go and Fig. 3Go).



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FIG. 3. Percent micronucleated reticulcoyte (MN-RET) frequency histograms are plotted for vehicle control animals (top: FCM-based measurements; bottom: microscopy-based measurements). These graphs demonstrate a reduction to scoring error achieved with flow cytometry-based analyses compared to the conventional microscopy-based approach.

 
DISCUSSION

The induction of DNA damage and the resulting sequelae of mutations and chromosomal rearrangements are primary mechanisms by which cancers arise (Barrett, 1993Go; Bishop, 1991Go). These types of events have also been implicated in diseases such as atherosclerosis, and processes such as aging (Zwijsen et al., 1990Go). Therefore, there is an important need for sensitive methods that are capable of identifying chemical or physical agents that can permanently alter DNA. Given the tremendous cost of long-term chronic studies such as two-year bioassays, short- and medium-term systems for predicting DNA reactivity will continue to play a vital role in carcinogen identification, as well as in lead prioritization strategies. In fact, the need for short-term tests that have a high throughput capacity has never been greater (Gollapudi and Krishna, 2000Go). Advances in molecular biology and combinatorial chemistry have provided large numbers of potential targets and many novel compounds that may be useful for treating or preventing disease. This situation clearly calls for methods that are able to quickly and reliably determine toxicological profiles of compounds under consideration. In the case of in vivo micronucleus testing, flow cytometry technology may be of great assistance. For the experiments described herein, throughput was as high as 100 blood samples per day. Compared to microscopy, this represents roughly a tenfold higher rate of analysis (also consider that the FCM method evaluated tenfold more cells in this amount of time).

The greatly enhanced efficiency of FCM-based MN measurements may have implications for where in the drug development process the endpoint is first studied. As far as in vivo genotoxicity assessment, the rodent MN erythrocyte test is the most widely utilized system. Even so, it is often applied late in drug development, and often using mice. The rat has traditionally been more vigorously studied for product safety testing. There would be advantages to obtaining genotoxicity data in this species, as this would allow investigators to relate any observed effects to information regarding deposition, metabolism, and elimination. Since the peripheral blood compartment of rats is so amenable to sampling (even repeat sampling), it is conceivable that MN data could be generated in early toxicology investigations, such as during acute rat studies. This strategy could provide important information that might highlight problem chemicals early in the development process, and thereby help redirect resources to more promising leads.

Beyond confirming the appropriateness of the rat peripheral blood compartment for conducting erythrocyte-based MN analyses, the data presented herein are valuable for considering whether this endpoint may also be utilized to study chemical-induced genotoxicity in other species with robust spleen function. For instance, in regard to MN filtering, the human spleen behaves similarly to that of the rat. Therefore, data pertaining to the suitability of an FCM-based rat peripheral blood system may provide clues as to the feasibility of FCM-based human blood measurements (Abramsson-Zetterberg et al., 2000Go; Dertinger et al., 2002Go). Experiments designed to test this scenario are in progress.

ACKNOWLEDGMENTS

This work was supported by a grant from the National Institute of Environmental Health Sciences (NIEHS; grant number R44 ES 09578-03). The contents are the sole responsibility of the authors and do not necessarily represent the official views of NIEHS. The authors would like to thank Drs. James MacGregor and Makoto Hayashi for many valuable discussions.

NOTES

1 To whom correspondence should be addressed. Fax: 585-442-0934. E-mail: sdertinger{at}litronlabs.com. Back

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