Mutagenesis, Vol. 17, No. 1, 15-23,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
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
Genetic Toxicology, Preclinical Safety Sciences, GlaxoWellcome Park Road, Ware, UK and 1 Litron Laboratories, 1351 Mount Hope Avenue, Rochester, NY, USA
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Abstract |
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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.
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Introduction |
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The micronucleus (MN) test (Schmid, 1975
) in rodent bone marrow or peripheral blood is an internationally recognized and validated system for the detection of compounds that are potentially clastogenic/aneugenic. However, conventional manual analysis currently employed within most pharmaceutical industries is both time consuming and labour intensive. Flow cytometry (FCM) provides obvious advantages over manual scoring, i.e. high speed, larger sample sizes and, hence, the potential to improve sensitivity and consistency. Such an approach utilizing rat peripheral blood might also be more amenable to integration into routine toxicology assays, thus maximizing animal usage (Wakata et al., 1998). However, splenic removal of micronucleated cells within the rat can reduce the sensitivity of this procedure (Schlegel and McGregor, 1984). This is borne out by the negative/equivocal results reported for colchicine in this species compared with the positive effects seen in the mouse (Hayashi et al., 1989). Interestingly, a recent publication from Abramsson-Zetterberg et al. (1999), using dual beam FACS analysis, has shown that by restricting analysis to the youngest reticulocyte (RET) population within rat peripheral blood it was possible to considerably improve assay sensitivity, especially for aneugens such as vincristine. Also, work on human cytogenetic biomonitoring using flow cytometric analysis (Abramsson-Zetterberg et al., 2000) has shown that nascent human RET in peripheral blood can be analysed for the presence of micronuclei, even though in the rat these cells are subject to removal by the spleen.
In papers published by Dertinger et al. (1996) and Torous et al. (2000) a methodology was described that could resolve both micronucleated reticulocytes (MNRET) and normochromatic erythrocytes (NCE) within mouse or rat peripheral blood using a single laser flow cytometer. With this procedure, fluorescein-conjugated antibodies were used to bind to the CD71-defined antigen (the transferrin receptor) of RET, thus rendering them identifiable by flow cytometric analysis. By degrading the RNA content of RET and simultaneously labelling them with the antibody, propidium iodide could be used to label the DNA-containing cells, i.e. micronucleated cells, thus clearly resolving the different cell types. Accurate and reliable flow cytometer set-up was achieved using malaria-infected erythrocytes (Tometsko et al., 1993; Dertinger et al., 2000). These cells mimic micronucleated erythrocytes and are very prevalent (10–20%) among the cells of interest (compared with 0.2% in micronucleated erythrocyte control blood). This enabled comparison of data obtained on different days or from different experiments and, conceivably, between laboratories.
In this study further evaluation of the utility of this method was carried out and inter-laboratory variability was examined. Colchicine, urethane and acetaldehyde were tested in a time course study, tracking the incidence of MNRET frequency in the peripheral blood pool of rats and mice (acetaldehyde in rats only) over 24, 48 and 72 h. In the study using cyclophosphamide and methylmethanesulfonate (MMS) only mice were used and peripheral blood samples were taken at 48 h post-dosing only.
Earlier studies on colchicine using conventional microscopic analysis yielded inconclusive results in rat peripheral blood, although clearly positive effects were obtained in the mouse (Wakata et al., 1998). Splenic removal within the rat of reticulocytes containing large micronuclei derived from aneugenic events has been suggested as a possible reason for this weak/negative result (Wakata et al., 1998). FCM allows the analysis of a larger RET sample and, furthermore, by focusing on the more immature RET population this may enable micronuclei arising from aneugenic events to be clearly detected before removal by the rat spleen. This study may then provide a more conclusive result in rat peripheral blood after treatment with colchicine.
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Materials and methods |
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Test chemicals and reagents
The test chemicals colchicine (CAS no. 64-86-8) and urethane (CAS no. 51-79-6) were obtained from Sigma (Dorset, UK). Acetaldehyde (CAS no. 75-07-0), cyclophosphamide (CAS no. 6055-19-2) and MMS (CAS no. 66-27-3) were obtained from Aldrich (Dorset, UK).
For all samples tested a bicarbonate-buffered salt solution was supplied by Litron Laboratories as part of the MicroFlow Plus kit. The heparin anticoagulant was supplied by Sigma (CAS no. 9045-22-1).
The mouse anti-rat CD71–FITC conjugate was supplied by Serotec (Oxford, UK; product no. MCA155F, lot no. 0698) and the rat anti-mouse CD71–FITC conjugate by Pharmingen International–Becton Dickinson (product no. 01594D, lot no. M0320240698). RNase A was supplied by Sigma (product no. R5250, lot no. 20K7660). Propidium iodide (PI) staining solution was supplied by Pharmingen International–Becton Dickinson (product no. 66211E).
For studies in rats dosed with colchicine, urethane and acetaldehyde, MicroFlow Plus reagents were supplied by Litron Laboratories.
Animals
Adult male Wistar Han rats and adult male CD1 mice were purchased from Charles River Laboratory UK. All rats (18–20 weeks of age) and mice (7–10 weeks of age) were acclimatized for 5 days before the experiments were initiated. Rat and mouse No. 1 Expanded Diet, supplied by Special Diets Services, and water drawn from the normal domestic supplies were available to all animals ad libitum.
In the initial cyclophosphamide and MMS experiments the adult male CD1 mice were 7–9 weeks of age.
The practical aspects of all the animal work were carried out at GlaxoWellcome (Ware, UK) and duplicate samples were analysed at GlaxoWellcome, with coded samples also being sent to Litron Laboratories (Rochester, NY) for independent analysis.
Chemical treatment
In a preliminary study conducted in mice cyclophosphamide was administered at 10, 20 or 40 mg/kg body wt via oral gavage and MMS was administered at 100 mg/kg body wt via a single i.p. injection. For integrity, only samples at 48 h post-dose were analysed by Litron Laboratories to ensure that transport of samples did not compromise analysis.
In subsequent experiments four treatment groups consisting of five male CD1 mice were treated with colchicine at 1 or 2 mg/kg body wt or urethane at 750 or 1000 mg/kg body wt via a single i.p. injection. Seven treatment groups consisting of five male Wistar Han rats were treated with colchicine at 2, 4 or 6 mg/kg body wt via oral gavage and at 3 mg/kg body wt via a single i.p. injection. Urethane was administered at 750 or 1000 mg/kg body wt via a single i.p. injection. Acetaldehdye was administered at 125 or 250 mg/kg body wt via a single i.p. injection. The control groups at 0 h were not dosed in this study. For the rat studies, only one peripheral blood sample from each of the dosed groups was sent to Litron Laboratories for analysis.
All doses for colchicine, urethane and acetaldehyde were selected from earlier published results (Wakata et al., 1998). The doses of cyclophosphamide and the single dose of MMS were chosen based on in-house historical data obtained from experiments employing conventional manual slide analysis for micronucleus incidence.
Study design
Rats were housed at 2–3 animals/cage and the mice singly. At 24, 48 and 72 h after treatment peripheral blood samples were collected from the vena cava for mice and via a tail bleed for rats. Due to procedural constraints, the volume of blood taken was limited, therefore multiple bleeds could not be performed and single mice per dose and time point were assigned. Peripheral blood (200 µl) was collected from each animal and immediately placed in a 2 ml tube containing 500 µl of anticoagulant solution (500 USP units heparin/ml 0.9% saline). Blood samples were maintained at room temperature for no longer than 60 min. Each sample was then fixed by forcefully pipetting 180 µl into a 15 ml polypropylene tube containing 2 ml of methanol at –80°C. Each tube was then tapped sharply several times to break up any cell aggregates and stored in a polystyrene rack at –80°C for at least 48 h before analysis.
All samples sent to Litron Laboratories for analyses were maintained at –80°C for at least 7 days before dispatch. The samples were removed from the –80°C freezer, double bagged in plastic bags, sealed and immersed in sufficient dry ice for transport.
Flow cytometric analysis
The FCM analyses described herein were carried out with a BD FACScan (mouse sample analysis) or a BD FACSCalibur (rat sample analysis) flow cytometer (Becton Dickinson, Oxford, UK) at GlaxoWellcome. A BD FACSTARplus flow cytometer (Becton Dickinson, Sunnyvale, CA) was used at Litron Laboratories The lasers on the flow cytometers were set to provide 488 nm excitation. Gating on the forward light scatter and side light scatter parameters isolate the erythrocyte population and electronic compensation was adjusted to eliminate the longer wavelength emissions of the FITC signals by using separate samples with PI only and CD71–FITC only. The BD FACSCalibur used FL1 (BP 530/30 nm) for CD71–FITC fluorescence and FL3 (LP 670 nm) for PI, whereas the BD FACScan and the BD FACSTARplus used FL1 (BP 540/20 nm) and FL2 (LP 580 nm), respectively. Due to the differences in concentration of blood per sample, a RET count of 20 000 at an analysis rate of 60 µl/min was used throughout the study for all three instruments.
On the day of analysis the stored samples were removed from the –80°C freezer in pairs and placed on ice. Each tube in turn was then tapped sharply and 8 ml of bicarbonate-buffered saline was added. These tubes were spun down at 1200 r.p.m. for 5 min, the supernatant removed and the pellet resuspended in ~100 µl. A 20 µl aliquot of each fixed mouse blood sample was transferred to a separate tube with 80 µl of bicarbonate-buffered saline containing 10 µl/ml anti-mouse CD71–FITC conjugate and 1 mg/ml RNase A. This solution labels the RET with anti-CD71–FITC and removes any RNA from the sample. All samples were kept at 2–8°C for 30 min, after which the tubes were placed at room temperature for a further 30 min and then returned to 2–8°C until analysis. Immediately before each sample was analysed, 1 ml of ice-cold PI solution (1.25 µg/ml bicarbonate-buffered saline) was then added to each tube. Stained cells were kept at 2–8°C for a maximum of 15 min until analysis.
The rat samples were treated identically to the mouse samples except that the samples were labelled with anti-rat CD71–FITC conjugate.
The bivariate graphs for mouse (Figure 1) and rat (Figure 2) samples were generated by analysis of fixed mouse and rat erythrocytes infected with malaria (Plasmodium berghei) supplied by Litron Laboratories (Tometsko et al., 1993; Dertinger et al., 2000) (Table I). The negative control animals were not dosed and the positive control animals were dosed with MMS administered at 100 mg/kg body wt via a single i.p. injection. The frequencies of each cell population were automatically calculated by CellQuest software upon collection of 20 000 total RET. The dual labelling methodology employed for this study allows the resolution of five cell types in whole peripheral blood. Instrument settings and fluorescent compensations were adjusted accordingly to optimize the NCE, MNNCE, RET, MNRET and nucleated cell populations. The nucleated cells act as a biological standard and are set in the fourth decade, at the far end of the red fluorescence (FL3). A higher green fluorescence threshold is used for rat samples (relative to mouse) so that MN analysis is restricted to the youngest fraction of RET. Differences in per cent parasitaemia for both mouse and rat between the two laboratories can be attributed to slight differences in gate setting between the different flow cytometers, their optical and electrical systems and the laser optimization.
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In the mouse peripheral blood MN assay all RETs are analysed for the presence of micronuclei. The boundary distinguishing CD71-positive RET from CD71-negative NCE can best be determined by visualizing 1–5% of the total cells collected when setting up with the malaria-infected standard. In this manner, the location of an appropriate boundary which discriminates RET from the major NCE population becomes apparent. However, when analysing rat peripheral blood for MN it is important to restrict the analysis to the most immature RET (Wakata et al., 1998
; Torous et al., 2000). In order to rationally define a lower green fluorescence threshold which defines the most immature RET from the more mature RET and NCE, bone marrow samples can be utilized, since most cells in this compartment are newly formed and immature. In this manner, FCM measurements are restricted to the youngest fraction of reticulocytes based on transferrin receptor (CD71) staining.
Data collection and statistical analysis
All data collected for flow cytometric analysis were determined by counting 20 000 RET or ~1.5x106 total red blood cells. Length of time for data collection varied between samples due to the differing number of RET (4–10 min).
Analysis of variance (ANOVA) was performed on the data. Dunnett's test, which compensates for multiple testing, was used as this allows several treatments to be compared with the control.
Defined data: %RET = (nRET/ntotal erythrocytes)x100%
%MNRET = (nMNRET/nRET)x100%
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Figure 3A
–D illustrates the resolution for the various erythrocyte populations obtained for a negative control mouse, a positive control mouse, a negative control rat and a positive control rat, respectively. The bivariate graphs demonstrate increases in DNA content on the x-axis (FL3) and anti-CD71–FITC binding on the y-axis (FL1). Clear differentiation between the different cell populations can be seen using anti-CD71–FITC and PI labelling for both mouse and rat peripheral blood.
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Mouse data
The results for the initial cyclophosphamide and MMS experiment analysed by Litron Laboratories are represented in Figure 4A and B
(%RET and %MNRET). Litron Laboratories demonstrated significant effects at 48 h with all doses of cyclophosphamide and with MMS at 100 mg/kg (Figure 4B).
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Figure 5A and B
shows %RET and %MNRET after oral administration of colchicine. The control MNRET frequencies (Figure 5B) in peripheral blood were 0.25 ± 0.10 (GlaxoWellcome) and 0.27 ± 0.04 (Litron). After oral administration, colchicine produced a marked increase in MNRET frequency at all sampling times at GlaxoWellcome and Litron Laboratories. Although, statistically significant effects were seen by GlaxoWellcome at 48 h post-treatment with colchicine at 1 and 2 mg/kg, statistically significant effects were only seen by Litron Laboratories at 48 h post-treatment with colchicine at 2 mg/kg. A marked reduction in RET frequency was also seen 48 h post-dosing with 2 mg/kg (Figure 5A). At 72 h excessive stem cell toxicity (>80% reduction in %RET) was observed and consequently at this sampling time the %MNRET frequency is not reported. These data are in agreement with previous work that has shown that colchicine induces MNRET dose dependently with a peak at 48 h after treatment in peripheral blood (Kondo et al., 1992; Cao et al., 1993). The magnitudes of the effects obtained within the two laboratories were very similar (Figure 5B).
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After i.p. administration of urethane a marked increase in MNRET frequency was seen at all sampling times at GlaxoWellcome and Litron Laboratories (Figure 6B
). Statistically significant effects were seen by GlaxoWellcome at 48 h post-treatment with urethane at 750 mg/kg and at 48 and 72 h post-treatment with 1000 mg/kg. Statistically significant effects were seen by Litron Laboratories at 48 h post-treatment with urethane at both 750 and 1000 mg/kg. The peak responses were seen at 48 h after i.p. administration of urethane at 750 and 1000 mg/kg (Figure 6B). Significant reductions in RET frequency were observed at 72 h post-treatment with 750 mg/kg and at 48 and 72 h post-treatment with 1000 mg/kg (Figure 6A). These results concur with published data and the magnitudes of the effects were similar for each laboratory, illustrating the reproducibility of this method.
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Rat data
Control frequencies of %MNRET in rat peripheral blood were 0.11 ± 0.03 (GlaxoWellcome) and 0.11 (Litron). Colchicine was negative for MN induction when administered by the oral and i.p. routes in both laboratories (Figure 7B
). Although an increase in MNRET frequency was seen at 72 h after oral dosing by both laboratories, at 48 h after i.p. dosing excessive stem cell toxicity was seen at GlaxoWellcome and Litron Laboratories and the %MNRET frequency is not reported. Unacceptable clinical signs prohibited analysis of animals at 72 h post-dosing.
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At GlaxoWellcome urethane gave statistically significant effects at 48 and 72 h post-dosing with 750 and 1000 mg/kg (Figure 8B
). Litron Laboratories obtained significant effects at 48 h at 750 mg/kg and at 48 and 72 h at 1000 mg/kg. The peak response was at 72 h post-dosing for these two doses, and slightly higher responses were obtained by GlaxoWellcome compared with Litron at both doses. Results were consistent with published data (Westmoreland and Gatehouse, 1991; Wakata et al., 1998).
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In the acetaldehyde experiment control MNRET frequencies in peripheral blood were 0.13 ± 0.06 (GlaxoWellcome, 20 animals analysed) and 0.14 (Litron, five animals analysed). Acetaldehyde gave statistically significant effects at 48 h at 125 mg/kg at GlaxoWellcome. In both laboratories significant effects were observed at the 24 and 48 h sample times after i.p. treatment with 250 mg/kg (Figure 9B
).
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Discussion |
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The main objective of this study was to compare the single laser flow cytometric micronucleus test methodology between two laboratories and to ascertain whether the analytical technique would give reproducible results.
In the initial experiment using cyclophosphamide and MMS doubling or greater effects were seen with cyclophosphamide at 10, 20 and 40 mg/kg at Litron Laboratories 48 h post-dose. Doubling or greater effects were also seen with MMS at 100 mg/kg. This preliminary study demonstrated that samples could be taken, frozen and dispatched to Litron without severely compromising the analysis outcome. Consequently, a more comprehensive evaluation of the method was initiated using mouse and rat peripheral blood.
In the mouse colchicine induced a marked increase in micronuclei, with an optimum at 48 h for both dose levels. At 1 mg/kg 1.07% (GlaxoWellcome)–1.6% (Litron Laboratories) RET contained micronuclei, compared with a published value of 1.8% after conventional manual slide analysis (Hayashi et al., 1989). After i.p. administration urethane induced statistically significant, dose-related increases in micronucleus frequency at both doses, which peaked at 48 h. At 1000 mg/kg 3.55–5.11% RET contained micronuclei, compared with a published value of 3.3% after conventional manual slide analysis (Wakata et al., 1998). These data illustrate the robustness of this analytical technique.
In the rat colchicine gave uniformly negative results after oral and i.p. dosing up to 6 mg/kg body wt. For both sets of data the results agree with published negative data in the rat after conventional manual slide analysis (Wakata et al., 1998). Consequently, it appears that it is not possible to detect the aneugenic effects of colchicine in rat peripheral blood after oral or i.p. dosing by increasing cell sample size and restricting analysis to immature reticulocytes.
After i.p. administration to rats urethane induced significant, dose-related increases in micronucleus frequency at 24, 48 and 72 h post-dosing. At 750 and 1000 mg/kg maximum increases were seen at 72 h. At 1000 mg/kg 1.03–1.69% RET contained micronuclei, compared with a published value of 1.6% at 72 h post-dosing (Wakata et al., 1998).
Acetaldehyde induced statistically significant effects in the rat after i.p. administration at 250 mg/kg in both laboratories. Maximum increases were seen at 48 h post-dose (0.33–0.39% RET), in agreement with published positive data obtained for rat peripheral blood by conventional manual slide analysis (0.33% RET) (Wakata et al., 1998).
The single laser FCM methodology gave consistent results across two laboratories when identical mouse and rat peripheral blood samples were analysed. The results obtained with urethane and colchicine (mouse and rat) and acetaldehyde (rat) are in good quantitative agreement with published data obtained using conventional manual slide analysis. The use of this methodology did not allow detection of the aneugenic activity of colchicine in rat peripheral blood after oral and i.p. administration.
In conclusion, use of the single laser flow cytometric procedure to quantify micronucleus frequency in both mouse and rat peripheral blood has been shown to give consistent data between two laboratories and compares favourably with previously published data. Automated analysis of micronucleus frequency in rodent peripheral blood using this technique increases the speed of analysis, reduces the resources required and may provide a useful way of incorporating such investigations into routine toxicological rodent safety studies. Further comparative studies are therefore warranted. Additional analysis of inter-sample variability and further inter-laboratory validation studies are being performed along with measurement of CD71–FITC antigen fluorescence to determine the optimum population for micronucleus measurement. Data collected to date suggest that with the use of malaria-infected blood as a standard, this automated scoring system could make a major contribution to the minimization of any inter-laboratory variation.
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Acknowledgments |
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The authors would like to thank Nikki Hall for her expert assistance throughout the experiments and Dr Stephen Dertinger for assistance with manuscript preparation.
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Notes |
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2 To whom correspondence should be addressed. Tel: +44 1920 882171; Fax: +44 1920 882679; Email: gmh0469{at}gsk.com
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Received on March 7, 2001; accepted on August 10, 2001.
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