Comparison of three-colour flow cytometry and slide-based microscopy for the scoring of micronucleated reticulocytes in rat bone-marrow and peripheral blood

Mutation Research 758 (2013) 12–17

Contents lists available at ScienceDirect

Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres

Comparison of three-colour flow cytometry and slide-based microscopy for the scoring of micronucleated reticulocytes in rat bone-marrow and peripheral blood Changhui Zhou a , Qingli Wang b , Zheng Wang a , Yan Chang a,∗ a b

National Shanghai Center for New Drug Safety Evaluation & Research, China State Institute of Pharmaceutical Industry, Shanghai 201203, China Center for Drug Evaluation, State Food and Drug Administration, Beijing 100038, China

a r t i c l e

i n f o

Article history: Received 22 February 2013 Received in revised form 11 May 2013 Accepted 8 July 2013 Available online 23 September 2013 Keywords: Micronucleus Genotoxicity Polychromatic erythrocytes Cyclophosphamide Clastogen Aneugen

a b s t r a c t The aim of this study was to perform the first transferability assessment in China of the micronucleus (MN) scoring method based on three-colour flow cytometry (FCM). This was accomplished for both rat bone-marrow and peripheral blood specimens following exposure to a variety of genotoxic and nongenotoxic chemicals, whereby micronucleus induction was measured both with FCM and with traditional microscopy. In an initial study, rats were treated with vehicle or cyclophosphamide (CP) for 2 consecutive days by oral gavage, and blood and bone marrow were sampled at 24 h after the second treatment. Staining with acridine orange (AO) of methanol-fixed slides was used for microscopical analysis and 2000 reticulocytes (RET) or polychromatic erythrocytes (PCE) were scored for MN frequency. The erythrocytes in the remaining bone-marrow cell suspensions were eluted on cellulose columns. The eluted bone marrow as well as the peripheral blood cells was fixed, incubated and analyzed by FCM. In another experiment, the performance of the FCM-MN method was further evaluated with five clastogens (urethane, 5-fluorouracil, mitomycin C, methylmethane sulfonate and 6-thioguanine), two aneugens (vincristine sulfate and colchicine) and two non-genotoxic new drugs (compounds A and B), whose results were negative in the routine mouse-micronucleus test (MNT). The MN frequencies in rat peripheral blood induced by the positive chemicals were found to be lower than the frequencies in rat bone-marrow by both scoring methods. However, a high level of agreement for the MN frequencies in both compartments was obtained. Good correspondence between the two analysis methods was also achieved. These data provide support that the three-colour FCM method is more rapid and objective than manual microscopy, while yielding comparable data. It further supports the premise that rat peripheral blood may be an alternative to rat bone marrow in the MNT. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The micronucleus (MN) resulting from a chromosome fragment or a lagging whole chromosome is a well-established biomarker of cytogenetic damage [1]. The erythrocyte-based in vivo micronucleus test (MNT), which was established in the 1970s [2], is a key part of the standard battery of genetic toxicology tests required for the registration of new pharmaceuticals [3–5], with the mouse being the most widely used rodent species. However, the rat may be the preferred animal model for pharmaceutical and/or chemical industry toxicology safety assessment. Use of rats allows for an expanded collection of data including information on

∗ Corresponding author at: National Shanghai Center for New Drug Safety Evaluation & Research, 199 Guoshoujing Road, Zhangjiang Hi-tech Park, Pudong, Shanghai 201203, China. Tel.: +86 21 50800333x208. E-mail address: [email protected] (Y. Chang). 1383-5718/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2013.07.010

pharmacokinetics, drug metabolism, distribution, and excretion that would not be possible in the mouse due to limited sample availability. Therefore, integration of the MNT into routine toxicological studies has become an acceptable and sometimes preferred option that effectively reduces the number of animals required in preclinical safety studies [6,7]. Peripheral blood erythrocytes have been accepted as an appropriate target for MN assessment [4,5]. Until relatively recently, there had been a historical bias against using rat peripheral blood erythrocytes because of splenic elimination of micronucleated erythrocytes [8,9]. However, it was the consensus of the 2005 International Workshop on Genotoxicity Testing (IWGT) group that accumulated data show that peripheral blood reticulocytes (RET) are an acceptable cell population for evaluating micronuclei in rats as well as mice [10]. Data from the Collaborative Study Group for the micronucleus test [11] suggest that rat peripheral blood may be appropriate for the enumeration of MN, if scoring is limited to the youngest fraction of polychromatic erythrocytes (PCE) (types I and

C. Zhou et al. / Mutation Research 758 (2013) 12–17

13

II) as classified by Vander et al. [12]. By restricting the analysis to the youngest PCE (RET), immature erythrocytes can be scored prior to being captured by the spleen. The manual microscopical scoring of MN can be subjective, time-consuming, labour-intensive, and dependent upon staining quality. More importantly, a relatively small number of PCE or RET (typically 2000 cells per animal) are evaluated in slide preparations, which can lead to considerable scoring variability due to the rarity of MN. However, the three-colour flow cytometric method is an automated scoring platform that has been employed successfully by numerous groups. Briefly, this methodology involves adding fixed whole blood (or column-fractionated bone marrow) to a labelling solution that simultaneously exposes cells to anti-CD71-FITC, a platelet-specific antibody (anti-CD61-PE) and RNase. After an adequate incubation period, the cells are re-suspended in a propidium iodide (PI)containing buffer and are thereafter ready for analysis by flow cytometry [13]. This technique can restrict the analysis to the youngest PCE (RET) by selectively choosing CD71-high positive PCE (RET) in bone marrow (peripheral blood) of rats, and within several minutes 10- to 100-fold more cells can be scored compared with what can be achieved with microscopy. Furthermore, the reproducibility of flow cytometry-generated data is much greater than microscopical scoring when a biological standard is used to calibrate the FCM system [14]. Therefore, analysis by flow cytometry increases the capacity and the sensitivity of the in vivo MNT. The goal of the current studies was to validate the threecolour flow method at our laboratory in China. Our intention was to replace microscopy-based scoring with the automated analysis platform in studies run under Good Laboratory Practice (GLP) requirements, so long as the resulting data supported the change. The work presented from the initial study with CP established the three-colour (MicroFlow® ) FCM analysis method, originally developed by Litron Laboratories, in both rat bone-marrow and peripheral blood in our laboratory. Method validation proceeded with known aneugenic, clastogenic, and non-genotoxic agents, and involved parallel collection of FCM- and microscopy-based data. The results from the validation studies and the comparison between blood and bone marrow are discussed.

heat-inactivated fetal bovine serum (FBS) (Gibco, Invitrogen) was added to each remaining blood sample. Ten microliters of each FBS-diluted blood suspension was used to prepare slides for microscopy-based scoring. These slides were allowed to air-dry and then fixed with absolute methanol for 10 min prior to conventional acridine-orange staining (MeOH-AO) [15]. Following euthanasia of the animal, both femurs were removed, minimally trimmed and bone marrow was gently flushed and repeatedly aspirated with approximately 3 ml of FBS. Following centrifugation (100 × g for 5 min), the cells were re-suspended in approximately 500 ␮l FBS. Duplicate bone-marrow smears were prepared, allowed to air-dry, and then fixed with absolute methanol for 10 min for AO staining. Replicate bone-marrow pellets were re-suspended in approximately 2 ml of MicroFlow buffer solution and added drop-wise to the center of previously prepared cellulose columns. The columns were prepared by fitting a circular disc cut from an 8-␮m polycarbonate membrane (GE Whatman, Cat. No. 110414) into the bottom of a 20-cc syringe (this differed from the Fisher lens paper recommended in the manual) and then adding 1.2 g of a cellulose powder mixture [16]. The erythrocytes were eluted from the columns by adding 13 ml of MicroFlow buffer solution to the center of the cellulose matrix (drop-wise at first so the column matrix was not disturbed and the addition rate gradually increased after a meniscus formed above the matrix). The erythrocyte eluate was centrifuged at 250 × g for 10 min, and the supernatant was aspirated, leaving 200–300 ␮l in which the cell pellet was resuspended. Approximately 180 ␮l of each erythrocyte fraction sample was fixed and stored in ultra-cold methanol for subsequent analysis by flow cytometry according to procedures described in the Rat Bone Marrow MicroFlowPLUS manual (v090203).

2. Materials and methods

2.6. Statistical methods

2.1. Animals and treatment

Statistical analyses were performed with SAS v9.1 Software. For each treatment group, the mean and standard deviation for the percentage RET and the percentage MN-RET was calculated. One-way ANOVA with Dunnett’s test and Fisher’s exact test were used, respectively, to assess whether there were treatment-related changes in the percentages of RET and MN-RET (significance indicated by P < 0.05). Spearman correlation coefficients were calculated for the percentage MN-RET obtained with the two scoring methods and from two compartments.

Male Sprague-Dawley (SD) rats, approximately 7–8 weeks old, were purchased from Sino-British Sippr/BK Lab. Animal Ltd., Co. (Shanghai, PRC). Animals were randomly assigned to treatment groups and group-housed (in treatment groups) with five animals per cage in a room with a relative humidity of 40–70%, temperature of 20–26 ◦ C and a 12-h light/dark cycle. The animals were acclimated for approximately 1 week before the experiment was initiated. Food and water were available ad libitum throughout the acclimation and experimentation periods. Animal studies were approved by the appropriate Animal Care and Use Committee of National Shanghai Center for New Drug Safety Evaluation & Research. Control and the animals that received the test chemical were treated once daily (for route, see Table 1) for two consecutive days; animals were euthanized approximately 24 h following the last treatment. 2.2. Chemicals and other reagents Details regarding test compounds, route of administration and vehicle can be found in Table 1. Dose levels were based on literature review or internal study reference. Flow-cytometry reagents, including fixed malaria-infected rat blood (malaria bio-standard) were from Rat MicroFlowPLUS Kits (available from Litron Laboratories, Rochester, NY and BD Biosciences Pharmingen, San Diego, CA). 2.3. Blood and bone-marrow sample collection and storage Approximately 24 h after the last administration, low-volume blood samples (approximately 100 ␮l) were collected by retro-orbital bleeding using a capillary into tubes containing 350 ␮l Anticoagulant/Diluent solution. Of each diluted blood sample, 180 ␮l were fixed with ultra-cold methanol for FCM analysis according to procedures described in the Rat Peripheral Blood MicroFlowPLUS manual (v090203). Fixed samples were stored at −75 to −85 ◦ C until analysis. An equal volume of

2.4. Standard acridine-orange staining and slide scoring Slides were stained with acridine orange (Sigma–Aldrich), a fluorescent DNAand RNA-specific stain [15]. For each animal, 2000 uniformly stained bone marrow and blood RET were scored to calculate the percentage MN-RET. The percentage RET was determined by inspecting 500 or 1000 total erythrocytes per bone marrow or blood sample, respectively. 2.5. Flow cytometry-based scoring Peripheral blood or bone-marrow samples were washed to remove fixative and labelled for analysis by flow cytometry. Data acquisition was performed with a 488-nm capable instrument (FACSCalibur, Becton Dickinson, San Jose, CA). AntiCD71-FITC, anti-CD61-PE, and propidium-iodide fluorescence signals were detected in the FL1, FL2, and FL3 channels, respectively. For analysis of peripheral blood samples, the instrument setup with the Plasmodium berghei-infected rat blood (malaria bio-standards) followed details as described in the MicroFlow manual. Each cell population of interest was counted by use of CellQuest 3.3 software and acquisition was terminated when a total of 20,000 CD71 high-positive RET were acquired for most samples. In some cases, high toxicity limited the total number of RET that could be counted, but at least 5000 RET per rat were acquired.

3. Results 3.1. Cyclophosphamide (CP) CP, a classical clastogen, was administered by oral gavage at different doses (5, 10, 20 mg/kg bw) to establish the three-colour flow-cytometry system in our laboratory. Analysis of both bone marrow and blood slides stained with AO showed dose-related increases in MN-RET in rats treated with CP. FCM measurements of MN-RET in bone marrow and peripheral blood samples from these same animals also showed dose-related positive responses (Fig. 1). The treatment concentrations tested ranged from relatively non-cytotoxic to cytotoxic levels, as was clear from a dose-related reduction in the percentage of RET in both bone marrow and peripheral blood (Fig. 1). The analysis of peripheral blood produced slightly smaller increases in the frequency of micronuclei when compared with the results in bone marrow by the two scoring methodologies. However, the correspondence of MN-RET values between the scoring

14

C. Zhou et al. / Mutation Research 758 (2013) 12–17

Table 1 Chemical and dosing information.a Chemical (abbreviation)

CAS No.

Supplier

Doseb

Route

Solvent

Cyclophosphamide (CP) Urethane (Ure) 5-fluorouracil (5-FU) Mitomycin C (MMC) Methylmethane sulfonate (MMS) Thioguanine (6-TG) Vincristine sulfate (Vinc) Colchicine (Col) Compound A (CA) Compound B (CB)

6055-19-2 51-79-6 51-21-8 50-07-7 66-27-3 154-42-7 2068-78-2 64-86-8 NA NA

Sigma Sigma Sigma Roche Sigma Sigma Sigma Sigma Company A Company B

5, 10, 20 750 20 1.6 50 4 0.1 8 2000 2000

p.o. i.p. i.p. i.p. i.p. p.o. i.p. p.o. p.o. p.o.

0.9% NS 0.9% NS 0.9% NS 0.9% NS 0.9% NS 0.9% NS 0.9% NS 0.9% NS 0.5% CMC-Na 0.5% MC

a b

NS: normal saline; CMC-Na: sodium carboxymethylcellulose; MC: methylcellulose; NA: not applicable. Dose in mg/kg bw.

methods was high (r = 0.962). Furthermore, the good agreement between bone marrow and peripheral blood data is reflected by a correlation coefficient of 0.955 (Fig. 2). 3.2. Validation of the study results Each of the seven reference genotoxicants induced significantly (P < 0.01) elevated frequencies of MN in both the bone marrow and the peripheral blood compartment of rats (Table 2). No statistically significant increases in %MN-RET were observed in either compartment for compounds A and B (Table 2). %MN-PCE and %MN-RET values in the two compartments were highly correlated, r = 0.803. Scoring methodologies were found to be in good agreement as evidenced by an r-value of 0.940 (Fig. 3). Statistical analysis of micronucleus data for peripheral blood and bone marrow produced equivalent results, providing further support that analysis of peripheral blood is as sensitive as that of bone marrow in detecting micronucleus induction. In general, both scoring methods showed remarkably similar results for percentage PCE (RET) observed in bone marrow and blood, with slightly lower frequencies observed with FCM. The reason for this is likely related to the fact that the FCM method focuses on the very youngest of the erythrocyte population, i.e., those RETs identified as having the highest levels of CD71-bound transferrin [17] (Table 3).

over manual, slide-based scoring methods. Unlike microscopybased methodologies, the FCM method scores cells in suspension. Individual cells flow one-by-one past a focused laser beam, and light scatter and fluorescence signals are detected by photomultiplier tubes (PMT) and captured by an in-line computer [18]. The flow cytometry-based micronucleus detection method has many advantages. That is, automation provides much large numbers of micro-nucleated cells to be detected/scored, and this improves assay power; also the higher throughput capacity saves resources, as labour- and time-intensive manual scoring is eliminated; and bio-standard samples allow for easy calibration of the flow cytometer and for transferability of the method. The three-colour labelling method (MicroFlow technology) has recently emerged as a powerful tool for assaying MN in peripheral blood erythrocytes of several species including human [19–28]. The mouse model has been extensively validated in international collaborative studies [29,30]. In the case of species other than mice (e.g., rats, dogs, non-human primates as well as humans),

4. Discussion The development of automated FCM-based methods to measure MN frequencies in erythrocytes represents a technological advance

Fig. 1. Comparison of the mean percentages of RET and MN-RET obtained by flow cytometric and fluorescent microscopy analysis for the induction of micronuclei in the bone marrow (BM) and in the peripheral blood (PB) of rats treated with 0, 5, 10, 20 mg/kg CP (¯x ± s, n = 5). **P < 0.01, when compared with the respective negative control.

Fig. 2. Linear regression analysis of micronucleus frequencies collected from 20 rats treated with 0.9% normal saline or cyclophosphamide, illustrating the correlation between manual microscopical and flow cytometric analysis (A) and correlation between peripheral blood (PB) and bone marrow (BM) by flow cytometry or manual microscopical analysis (B).

C. Zhou et al. / Mutation Research 758 (2013) 12–17

15

Table 2 Frequencies of MN-PCE in rat bone-marrow and frequencies of MN-RET in rat peripheral blood treated with seven known genotoxicants and two known non-genotoxicants obtained by FCM and fluorescence microscopy (¯x ± s, n = 5). Treatment

Dose (mg/kg bw)

BM microscopy

NS Ure 5-FU MMC MMS 6-TG Vinc Col CMC-Na CA MC CBa

0 750 20 1.2 50 4 0.1 8 0 2000 0 500

0.10 1.27 1.21 1.83 1.01 1.09 4.00 0.67 0.12 0.13 0.13 0.12

± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.19** 0.10** 0.44** 0.25** 0.13** 0.70** 0.22** 0.06 0.08 0.04 0.03

BM FCM 0.17 1.06 1.26 2.08 0.85 1.12 6.92 0.54 0.19 0.16 0.21 0.21

± ± ± ± ± ± ± ± ± ± ± ±

0.13 0.21** 0.40** 0.18** 0.27** 0.28** 0.61** 0.12** 0.03 0.03 0.06 0.05

PB microscopy

PB FCM

0.07 ± 0.03 0.53 ± 0.12** 0.81 ± 0.13** 0.85 ± 0.11** 0.60 ± 0.18** 0.70 ± 0.20** NA 0.41 ± 0.26** 0.11 ± 0.07 0.08 ± 0.03 0.08 ± 0.04 0.12 ± 0.03

0.12 0.58 1.15 0.99 0.56 0.67 1.66 0.38 0.16 0.19 0.12 0.14

± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.24** 0.44** 0.32** 0.12** 0.11** 0.77** 0.29** 0.04 0.06 0.05 0.09

PB: peripheral blood; BM: bone marrow; NA: not available because of the high RET toxicity. a Two rats died and only three rats were left for statistics analysis. ** P < 0.01, statistically significantly increased frequencies of MN-RET or MN-PCE over the respective negative control.

the possible confounding effect of splenic activity needs to be considered. The main objective of this study was to provide data supporting the use of three-colour labelling FCM for the detection of micronuclei in both bone narrow and peripheral blood of rats exposed to a variety of genotoxic and non-genotoxic agents. CP was chosen as a model genotoxic compound [23,24], because it is a classical clastogen used in our laboratory as well as other laboratories around the world as the positive control in the MNT according to GLP standards. In addition, a significant amount of historical data for CP has been accumulated in our laboratory. CP produced statistically significant dose-related increases in MN in both bone marrow and peripheral blood compared with the concurrent vehicle control. This indicated that the three-colour labelling FCM method to assess MN formation in rats was reliably established in our laboratory. The validation studies were conducted with seven genotoxic agents (five clastogens and two aneugens) with different mechanisms of action and two non-genotoxic new drugs (compounds A and B – whose results were negative in the routine MNT). All genotoxic compounds evaluated here induced statistically significant increases in MN frequency in bone marrow by both FCM and microscopy. Similar effects were seen when peripheral blood was evaluated (Table 2). Urethane (a common anaesthetic) induced significantly increased %MN-RET in rat peripheral blood at 24 h after the second i.p. administration at 750 mg/kg. This is in agreement with a published value of ∼0.6% at 72 h after giving a single i.p. injection

[31]. At 1.2 mg/kg bw, MMC (a DNA cross-linking agent) induced a somewhat higher %MN-RET in rat bone-marrow by both FCM and manual microscopy than the published data of 1.25 mg/kg bw MMC [32], however, the %MN-RET in rat peripheral blood compared favourably with previous data [33]. MMS (a typical DNA-alkylating agent) induced statistically significant effects after i.p. administration at 50 mg/kg bw in both rat bone-marrow and peripheral blood (Table 2), with the %MN-RET in peripheral blood in close agreement with published data [22]. The nucleoside analogue 6-TG was shown to be a potent inducer of MN by both analysis methods and in both compartments at the 4 mg/kg bw dose level where a marked reduction in PCE (or RET) was observed (Table 2). The induced MN-RET frequencies observed in our study were somewhat lower than those previously published [22]. The reason for this discrepancy is not known at this time. The standard deviations of the mean values of %MN-RET and %RET from bone marrow and blood induced by vincristine sulfate and colchicine were larger compared with those of the other test compounds (Tables 2 and 3). This may be related to the aneugenic mechanism of action of these compounds. It is well known that aneugens have a very narrow dose-window for MN induction, which is typically close to cytotoxic levels [34]. Furthermore, in order to compare the results across compartments most effectively, it is necessary to achieve a steady-state level of MN induction, and this is unlikely to have occurred with the two dosings used in these studies.

Table 3 Frequencies of PCE in rat bone-marrow and frequencies of RET in rat peripheral blood treated with seven known genotoxicants and two known non-genotoxicants obtained by FCM and fluorescence microscopy (¯x ± s, n = 5). Treatment

Dose (mg/kg bw)

BM microscopy

NS Ure 5-FU MMC MMS 6-TG Vinc Col CMC-Na CA MC CBa

0 750 20 1.2 50 4 0.1 8 0 2000 0 500

49.48 49.82 34.57 43.99 40.57 36.06 29.07 36.80 53.20 48.83 42.73 56.46

± ± ± ± ± ± ± ± ± ± ± ±

2.90 7.80 5.27# 3.80 4.57 7.49# 1.73## 1.59# 4.29 2.15 0.74 1.55&&

BM FCM 41.03 36.47 16.02 35.19 40.77 23.74 6.35 37.99 37.39 41.73 40.36 62.05

± ± ± ± ± ± ± ± ± ± ± ±

0.50 3.30 4.84## 1.45 6.64 4.75## 4.25## 4.59 0.32 3.79 4.92 2.74&&

BM: bone marrow; PB: peripheral blood; NA: not available because of the high RET toxicity. a Two rats died and only three rats were left for statistics analysis. # P < 0.05, statistically significantly decreased frequencies of PCE and RET over the respective negative control. ## P < 0.01, statistically significantly decreased frequencies of PCE and RET over the respective negative control. && P < 0.01, statistically significantly increased frequencies of PCE and RET over the respective negative control.

PB microscopy

PB FCM

3.72 ± 1.65 1.36 ± 0.33## 1.05 ± 0.26## 1.12 ± 0.24## 1.98 ± 0.05## 1.23 ± 0.24## NA 1.69 ± 0.60## 2.20 ± 0.64 1.76 ± 0.29 2.10 ± 0.36 4.68 ± 0.52&&

1.31 0.68 0.20 0.49 1.04 0.24 0.07 0.55 1.12 0.64 1.44 2.43

± ± ± ± ± ± ± ± ± ± ± ±

0.28 0.19## 0.07## 0.11## 0.21 0.09## 0.03## 0.27## 0.09 0.24## 0.25 0.17&&

16

C. Zhou et al. / Mutation Research 758 (2013) 12–17

Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by a grant from the State Project for Essential Drug Research and Development (No. 2012ZX09302002). The authors would like to acknowledge the encouragement and expert advice of Dorothea K. Torous and Stephen D. Dertinger from Litron Laboratories, a company that owns patents of In Vivo MicroFlow® kits. References

Fig. 3. Linear regression analysis of micronucleus frequencies measured in all animals in the validation study, illustrating the correlation between manual microscopical analysis and measurement by flow cytometry (A) and between analysis by flow cytometry in peripheral blood, and in bone marrow (B).

Data in this study revealed generally higher frequencies of MN-RET in bone-marrow slide preparations than by FCM analysis of peripheral blood (Fig. 1 and Table 2). This observation is consistent with very efficient splenic selection, even though FCM analysis was restricted to the most highly expressing CD71 subpopulation of erythrocytes (i.e., the youngest and most immature reticulocytes). Nonetheless, for all compounds tested, a consistently high level of agreement was obtained between microscopy and FCM (Figs. 2 and 3). In addition, for the genotoxic and non-genotoxic compounds tested in both blood and bone marrow, no major differences in MN response were observed. The use of cellulose columns to obtain relatively pure erythrocyte fractions was essential in utilizing the MicroFlow technology for rat bone marrow. Although the additional fractionation step results in longer sample processing time prior to fixation, the data acquisition time (usually within a minute) on the flow cytometer for rat bone-marrow cells is considerably shorter than for peripheral blood, because of the higher percent of PCE. In conclusion, the three-colour FCM method described and validated in this study offers a rapid, objective, reproducible procedure for micronucleus evaluation in both rat bone-marrow and peripheral blood. Following validation, this method has been routinely used in our laboratory for drug-safety evaluation. It has also been successfully implemented in multiple other laboratories, demonstrating its robustness. Cumulatively, these data indicate that analysis of peripheral blood is as effective as that of bone marrow in detecting micronucleus responses. This provides a useful and effective way of performing acute MNT, with further efficiencies possible through integration of the assay into routine rodent toxicological safety studies.

[1] M. Hayashi, J.T. MacGregor, D.G. Gatehouse, I.-D. Adler, D.H. Blakey, S.D. Dertinger, G. Krishna, T. Morita, A. Russo, S. Sutou, In vivo rodent erythrocyte micronucleus assay: aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring – a report from the International Workshop on Genotoxicity Test Procedures (IWGTP), Environ. Mol. Mutagen. 35 (2000) 234–252. [2] J.A. Heddle, A rapid in vivo test for chromosome damage, Mutat. Res. 18 (1973) 187–190. [3] International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Topic S2B, Genotoxicity: a standard for genotoxicity testing of pharmaceuticals. ICH, Step 4 Guideline, July 16, 1997. [4] International Conference on Harmonization, Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2 (R1), 2011, http://www.ich.org/fileadmin/Public Web Site/ICH Products/ Guidelines/Safety/S2R1/Step4/S2R1 Step4.pdf (dated 30.11.11). [5] OECD, Guideline for the testing of chemicals. Mammalian Erythrocyte Micronucleus Test, Guideline 474, July, 1997, http://www.oecd.org/ dataoecd/18/34/1948442.pdf [6] G. Krishna, G. Urda, J. Theiss, Principles and practices of integrating genotoxicity evaluation into routine toxicology studies: a pharmaceutical industry perspective, Environ. Mol. Mutagen. 32 (1998) 115–120. [7] S. Hamada, S. Sutou, T. Morita, A. Wakata, S. Asanami, S. Hosoya, S. Ozawa, K. Kondo, M. Nakajima, H. Shimada, K. Osawa, Y. Kondo, N. Asano, S.-I. Sato, H. Tamura, N. Yajima, R. Marshall, C. Moore, D.H. Blakey, L.M. Schechtman, J.L. Weaver, D.K. Torous, R. Proudlock, S. Ito, C. Namiki, M. Hayashi, 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), Environ. Mol. Mutagen. 37 (2001) 93–110. [8] R. Schlegel, J.T. MacGregor, The persistence of micronuclei erythrocytes in the peripheral circulation of normal and splenectomized Fisher 344 rats: implications for cytogenetic screening, Mutat. Res. 127 (1984) 169–174. [9] M. Hayashi, Y. Kodama, T. Awogi, T. Suzuki, A.O. Asita, T. Sofuni, The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyclophosphamide-treated rats, Mutat. Res. 278 (1992) 209–213. [10] M. Hayashi, J.T. MacGregor, D.G. Gatehouse, D.H. Blakey, S.D. Dertinger, L. Abramsson-Zetterberg, G. Krishna, T. Morita, A. Russo, N. Asano, H. Suzuki, W. Ohyama, D. Gibson, 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, Mutat. Res. 627 (2007) 10–30. [11] A. Wakata, Y. Miyamae, S. Sato, T. Suzuki, T. Morita, N. Asano, T. Awogi, K. Kondo, M. Hayashi, 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. [12] J.B. Vander, S.R. Ellis, C.A. Harris, Reticulocyte counts by means of fluorescence microscopy, J. Lab. Clin. Med. 62 (1963) 132–140. [13] S.D. Dertinger, K. Camphausen, J.T. MacGregor, M.E. Bishop, D.K. Torous, S. Avlasevich, et al., Three-color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood, Environ. Mol. Mutagen. 44 (2004) 427–435. [14] S.D. Dertinger, D.K. Torous, N.E. Hall, C.R. Tometsko, T.A. Gasiewicz, Malariainfected erythrocytes serve as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes by flow cytometry, Mutat. Res. 464 (2000) 195–200. [15] M. Hayashi, T. Sofuni, M. Ishidate Jr., An application of acridine orange fluorescent staining to the micronucleus test, Mutat. Res. 120 (1983) 241–247. [16] G. Krishna, Comparative micronucleus quantitation in pre- and post-column fractionated mouse bone marrow by manual and flow analysis, Mutat. Res. 302 (1993) 119–127. [17] M.J. LeBaron, M.R. Schisler, D.K. Torous, S.D. Dertinger, B.B. Gollapudi, Influence of counting methodology on erythrocyte ratios in the mouse micronucleus test, Environ. Mol. Mutagen. 54 (2013) 222–228. [18] M. Hayashi, J.T. MacGregor, D.G. Gatehouse, I. Adler, D.H. Blakey, S.D. Dertinger, G. Krishna, T. Morita, A. Russo, S. Sutou, In vivo rodent erythrocyte micronucleus

C. Zhou et al. / Mutation Research 758 (2013) 12–17

[19]

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

assay: Part II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring, Environ. Mol. Mutagen. 35 (2000) 234–252. S. Dertinger, D. Torous, N. Hall, C. Tometsko, Measurement of cytogenetic damage in rodent blood with a single-laser flow cytometer, Curr. Protoc. Cytom. (2003) 21, Unit 7.21. M. De Boeck, B. van der Leede, F. Van Goethem, A. De Smedt, M. Steemans, A. Lampo, P. Vanparys, Flow cytometric analysis of micronucleated reticulocytes: time- and dose-dependent response of known mutagens in mice, using multiple blood sampling, Environ. Mol. Mutagen. 46 (2005) 30–42. K.L. Witt, E. Livanos, G.E. Kissling, D.K. Torous, W. Caspary, R.R. Tice, L. Recio, Comparison of flow cytometry- and microscopy-based methods for measuring micronucleated reticulocyte frequencies in rodents treated with nongenotoxic and genotoxic chemicals, Mutat. Res. 649 (2008) 101–113. D.K. Torous, N.E. Hall, F.G. Murante, S.E. Gleason, C.R. Tometsko, S.D. Dertinger, Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy, Toxicol. Sci. 74 (2003) 309–314. S.D. Dertinger, M.E. Bishop, J.P. McNamee, M. Hayashi, T. Suzuki, N. Asano, M. Nakajima, J. Saito, M. Moore, D.K. Torous, J.T. MacGregor, Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: I. Intra- and interlaboratory comparison with microscopic scoring, Toxicol. Sci. 94 (2006) 83–91. J.T. MacGregor, M.E. Bishop, J. McNamee, M. Hayashi, N. Asano, A. Wakata, M. Nakajima, A. Aidoo, M.M. Moore, S.D. Dertinger, Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal damage in the rat, Toxicol. Sci. 94 (2006) 92–107. S.D. Dertinger, R.K. Miller, K. Brewer, T. Smudzin, D.K. Torous, D.J. Roberts, S.L. Avlasevich, S.M. Bryce, S. Sugunan, Y. Chen, Automated human blood micronucleated reticulocyte measurements for rapid assessment of chromosomal damage, Mutat. Res. 626 (2007) 111–119. S.B. Harper, S.D. Dertinger, M.E. Bishop, A.M. Lynch, M. Lorenzo, M. Saylor, J.T. MacGregor, Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: III. An efficient method of monitoring chromosomal damage in the beagle dog, Toxicol. Sci. 100 (2007) 406–414. C.E. Hotchkiss, M.E. Bishop, S.D. Dertinger, W. Slikker Jr., M.M. Moore, J.T. MacGregor, Flow cytometric analysis of micronuclei in peripheral blood

[28]

[29]

[30]

[31]

[32]

[33]

[34]

17

reticulocytes: IV. An index of chromosomal damage in the rhesus monkey (Macaca mulatta), Toxicol. Sci. 102 (2008) 352–358. S.D. Dertinger, D.K. Torous, M. Hayashi, J.T. MacGregor, Flow cytometric scoring of micronucleated erythrocytes: an efficient platform for assessing in vivo cytogenetic damage, Mutagenesis 26 (2011) 139–145. D.K. Torous, N.E. Hall, S.D. Dertinger, M.S. Diehl, A.H. IlliLove, K. Cederbrant, K. Sandelin, G. Bolcsfoldi, L.R. Ferguson, A. Pearson, J.B. Majeska, J.P. Tarca, D.R. Hewish, L. Doughty, M. Fenech, J.L. Weaver, D.D. Broud, D.G. Gatehouse, G.M. Hynes, P. Kwanyuen, J. McLean, J.P. McNamee, M. Parenteau, V. Van Hoof, P. Vanparys, M. Lenarczyk, J. Siennicka, B. Litwinska, M.G. Slowikowska, P.R. Harbach, C.W. Johnson, S. Zhao, C.S. Aaron, A.M. Lynch, I.C. Marshall, B. Rodgers, C.R. Tometsko, Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories, Environ. Mol. Mutagen. 38 (2001) 59–68. D.K. Torous, N.E. Hall, A.H. Illi-Love, M.S. Diehl, K. Cederbrant, K. Sandelin, I. Pontén, G. Bolcsfoldi, L.R. Ferguson, A. Pearson, J.B. Majeska, J.P. Tarca, G.M. Hynes, A.M. Lynch, J.P. McNamee, P.V. Bellier, M. Parenteau, D. Blakey, J. Bayley, B.J. van der Leede, P. Vanparys, P.R. Harbach, S. Zhao, A.L. Filipunas, C.W. Johnson, C.R. Tometsko, S.D. Dertinger, Interlaboratory validation of a CD71-based flow cytometric method (MicroFlow® ) for the scoring of micronucleated reticulocytes in mouse peripheral blood, Environ. Mol. Mutagen. 45 (2005) 44–55. G.M. Hynes, D.K. Torous, C.R. Tometsko, 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, Mutagenesis 17 (2002) 15–23. R.D. Fiedlera, S.K. Weinerb, M. Schuler, Evaluation of a modified CD71 MicroFlow® method for the flow cytometric analysis of micronuclei in rat bone marrow erythrocytes, Mutat. Res. 703 (2010) 122–129. Z. Cammerer, A. Elhajouji, M. Kirsch-Volders, W. Suter, Comparison of the peripheral blood micronucleus test using flow cytometry in rat and mouse exposed to aneugens after single-dose applications, Mutagenesis 22 (2007) 129–134. M. Kirsch-Volders, M. Aardema, A. Elhajouji, Concepts of thresholds in mutagenesis and carcinogenesis, Mutat. Res. 464 (2000) 3–11.