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Mutagenesis Advance Access published online on June 4, 2007
Mutagenesis, doi:10.1093/mutage/gem015
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© The Author 2007. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Prior bleeding enhances the sensitivity of peripheral blood and bone marrow micronucleus tests in rats

Ajit Vikram, Poduri Ramarao and Gopabandhu Jena*

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab-160062, India

The rat peripheral blood micronucleus (MN) assay is not considered to be a sufficient biomarker of genotoxin exposure due to the selective elimination of micronucleated cells from peripheral circulation by the spleen. However, several recent reports suggest that peripheral blood reticulocytes of rats may represent a suitable cell population for use in the MN assay. The MN assay in rats with prior bleeding was thus conducted to determine the sensitivity of the bioassay. Hirai et al. reported that prior bleeding enhances the sensitivity of the in vivo MN test in mice. Based on these findings, the rat was used as a model to see the effect of prior bleeding on the sensitivity of the peripheral blood and bone marrow MN assays. In the present experiment, young male Sprague–Dawley (SD) rats ranging in age from 21 to 24 days were used. However, for the comparison of strain-specific induction of MN, Wistar rats were used. The kinetics of MN formation were investigated in adult, young bled and non-bled SD rats treated with cyclophosphamide (CP). For the MN kinetic study, CP was administered intraperitoneally 2 h after bleeding and sampling was done at intervals of 12, 24, 36, 48 and 96 h after chemical administration. Significant increases in MN induction activity in both bone marrow and peripheral blood were observed with prior bleeding. To further validate the influence of prior bleeding in the induction of MN frequency, two other known genotoxins (chlorambucil and mitomycin C) were used. It was concluded that prior bleeding can significantly increase the sensitivity of MN induction in both bone marrow and peripheral blood of rats compared with non-bled animals. Once validated, this model may be suitable for detecting different genotoxins, especially weak and marginally active clastogens.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The micronucleus (MN) test is widely used for screening of different agents for their genotoxic potential (1Go). It is a recommended bioassay in the test battery for genotoxicity evaluation and has become increasingly popular for regulatory acceptance. For the in vivo genotoxicity studies, the rat is less preferred than the mouse due to certain inherent shortcomings. Micronucleated cells formed in the bone marrow in the presence of clastogens can be detected easily in peripheral blood of mouse, but in case of adult rat, spleen selectively removes micronucleated RBC, thereby decreasing its sensitivity (2Go,3Go). The peripheral blood MN assay has several advantages over the bone marrow MN assay, e.g. (i) easy sample preparation with small quantity of blood, (ii) ease in counting, (iii) evaluation at various intervals using same animals, (iv) evaluation of cumulative damage and (v) rapid detection using high throughput screening (3Go,4Go). Many attempts in the past have been made to increase the suitability and sensitivity of rat peripheral blood MN assay for genotoxicity evaluation. Most of these remain unvalidated due to technical reasons, such as reproducibility in experiments and regulatory acceptance (5Go,6Go). Hamada et al. (7Go) evaluated the suitability of male rats of different age groups and found the sensitivity up to 13 weeks of age for the induction of MN employing bone marrow and peripheral blood erythrocytes. However, they reported that the sensitivity decreases with advancement of age. The splenectomized rat model was used to improve the sensitivity of peripheral blood MN assay, but this posed several technical problems (2Go,3Go). Greater sensitivity of MN assay is reported after administration of exogenous erythropoietin (8Go–10Go). Further, Hirai et al. (11Go) reported that prior removal of blood in mice can stimulate the erythropoesis, thereby increasing the sensitivity of bone marrow MN bioassay.

Recent reports suggest that young rat MN test sensitivity can be increased significantly by using anti-CD71 antibodies in flow cytometric analysis for the assessment of genotoxicity after acute and chronic exposures to chemicals, equivalent to human dosage (12Go). The International Workshop on Genotoxicity Testing discussed these new aspects of the in vivo MN test. It suggested suitability of rat peripheral blood reticulocytes (RETs) to serve as the target cell population for MN analysis. It also discussed the possibility of an in vivo MN assay other than the one using bone marrow. The biological relevance of the dose and time of sampling and above all the regulatory acceptance of data derived from automated scoring were also discussed. The working group reached a consensus that blood-derived RETs from rats as well as mice are acceptable when young RETs are analyzed under a proper assay protocol and with adequate sample size (13Go–16Go). In the present paper, we have made an attempt to see the effect of prior bleeding on the sensitivity of the rat peripheral blood and bone marrow MN assay using different known mutagens. Further, we evaluated the influence of prior bleeding on the maximum induction of MN in peripheral blood at different time points both in adult and in young rats. A strain-specific difference in the induction of MNs with prior bleeding was also considered for evaluation.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
All the animal experiments were approved by the Institutional Animal Ethics Committee. Experiments were performed on male Sprague–Dawley (SD) and Wistar rats which were procured from the Institute's Central Animal Facility and kept under controlled environmental conditions at room temperature (22 ± 2°C), humidity (50 ± 10%) and automatically controlled light and dark cycles (0600–1800 h). To analyze the ‘strain-specific difference’ on the induction of MN with prior bleeding, a study was conducted using Wistar rats. Standard laboratory animal feed was purchased from a commercial supplier and fed to the animals ad libitum. Animals were acclimatized to the experimental conditions prior to the start of dosing for a period of 2–3 days. Young SD and Wistar rats (21–24 days, 60 ± 5 g) were used for this experiment.

Chemicals
Cyclophosphamide (CP; CAS No. 50-18-0) and chlorambucil (CB, CAS No. 305-03-3) were procured from Sigma-Aldrich. Mitomycin C (MMC; CAS No. 50-07-7, MITOCIN-10) was obtained from Cadila Pharmaceuticals Pvt. Ltd, India. CP and MMC solutions were freshly prepared in distilled water. CB was first dissolved in minimal amount of absolute ethanol and later volume was adjusted with distilled water. The chemicals were injected intraperitoneally (i.p.) immediately after preparation. Control animals were injected with normal saline.

Experimental design
Blood removal results in high rate of blood-cell formation in order to compensate for the loss. This phenomenon reflected in terms of increased RETs-to-total erythrocyte ratio. RETs-to-total erythrocyte ratio was analyzed with both 0.5 ml (data not shown) as well as 1 ml blood removal from the rat. A significant (P < 0.001) increase was observed with 1 ml blood removal. Hence, all experiments were carried out with 1 ml prior blood removal. The experimental procedures for bleeding, administration of chemical and sampling are shown in Fig. 1. One milliliter of blood was removed from the orbital plexus 2 h before the genotoxin exposure. The sampling was done at 12, 24, 36, 48 and 96 h after the genotoxin exposure. The MN frequencies were evaluated at different time intervals to determine the time of maximum induction with CP (Fig. 2). In order to further validate the sensitivity of this model, two other potent alkylating agents, CB and MMC, were selected. The sampling for these two compounds was done only at the maximum time of induction as observed with CP. In addition to the peripheral blood MN assay, bone marrow sampling was also done at this time point to compare the effect of prior bleeding on the sensitivity of MN induction. Strain-specific induction of MN was assessed with CP by using SD and Wistar rats and sampling was done only at the time of maximum induction. Non-bled animals receiving the vehicle of the test compound served as controls for non-bled animals, and pre-bled animals receiving the vehicle of the test compound served as controls for pre-bled animals. RETs-to-total erythrocyte ratio was determined in order to calculate the effect of blood removal on the rate of erythropoesis in control as well as in pre-bled groups per se. This has further been calculated with all treatment groups.


Figure 1
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  		Fig. 1. Experimental design showing the time of blood removal, chemical administration and subsequent time of sampling. ‘B’ indicates the time for blood removal; ‘G’ indicates time for chemical administration. CP (100 mg/kg) -induced MN frequency was analyzed at different time points (12, 24, 36, 48 and 96 h) in peripheral blood of young bled, young non-bled and adult rats. The maximum induction of MN was observed at 36 h after CP administration in SD rats. This sampling time (36 h) was kept constant for the other two compounds (CB, MMC) after chemical administration.

 


Figure 2
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  		Fig. 2. Induction of MN by CP (100 mg/kg) in young bled, young non-bled and adult SD rats at different time points. The sensitivity of MN induction was further enhanced with prior bleeding in young rats. The maximum induction was obtained at 36 h after CP administration. All the values are shown as mean ± standard error of the mean (n = 5), **P < 0.01, ***P < 0.001, a versus adult and b versus young non-bled group.

 
Peripheral blood MN assay
Peripheral blood smear was prepared as described by Holden et al. (17Go) with some modifications (18Go). Blood was collected from tail tip, and smear was prepared on pre-cleaned slides. The smear was allowed to dry at room temperature and fixed in absolute methanol for 5 min. After fixation, slides were stained with acridine orange (AO) and washed twice with phosphate buffer (pH 6.8) as described by Hayashi et al. (19Go).

Bone marrow MN assay
Bone marrow slides were prepared as described by Jena and Bhunya (18Go,20Go) with some modifications. Bone marrow was isolated from the femur bone with the help of syringes and homogenized with fetal bovine serum (FBS). After centrifugation, the supernatant was discarded and the pellet was resuspended with the residual FBS. From this suspension, a smear was prepared on a clean grease-free slide and fixed in absolute methanol for 5 min (21Go). After fixation, slides were stained with AO and washed twice with phosphate buffer (pH 6.8) as described by Hayashi et al. (22Go).

Data scoring and photomicrographs
Slides were observed under oil immersion objective (x100) using an Olympus fluorescent microscope (Model BX 51) connected to digital photomicrograph software (OLYSIA BioReport). In total, 2000 cells were counted for each animal for each time point to calculate the MN frequencies. The RETs-to-total erythrocyte ratio was calculated by counting a total of 1000 erythrocytes in peripheral blood. Some representative photomicrographs were taken to elucidate the rare aberrations.

Statistical analysis
Results are shown as mean ± standard error of the mean for each group. Statistical analysis was performed using Jandel Sigma Stat (Version 2.03) software. The significance of difference between two groups was evaluated using Student's t-test. For multiple comparisons, one-way analysis of variance (ANOVA) was used. In case ANOVA shows significant differences, post hoc analysis was performed with Tukey's test. P <0.05 was considered to be statistically significant.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Sensitization of rat peripheral blood MN assay
The data indicate that CP (100 mg/kg, i.p.) induced more MN in the peripheral blood of young rats in comparison to adult rats. Further, prior bleeding significantly increased the MN frequencies in young rats over the non-bled young rats (Fig. 2). The increase in sensitivity of assay with prior bleeding in the induction of MN in peripheral blood RETs of young rats has been further confirmed with genotoxins like CB (30 mg/kg) and MMC (2 mg/kg) administered i.p. A significant increase in MN frequency was induced by CP (100 mg/kg, P < 0.001), CB (30 mg/kg, P < 0.05) and MMC (2 mg/kg, P < 0.05) in peripheral blood of rats over respective controls. For a strain-specific comparison, Wistar rats were used and a significant increase in MN frequency with CP (100 mg/kg) was obtained over the respective control (P < 0.001) (Fig. 3). Figure 4 shows fluorescent photomicrographs of MNs in young rat peripheral blood RET and bone marrow polychromatic erythrocytes stained with AO.


Figure 3
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  		Fig. 3. Effect of prior blood removal on the sensitivity of peripheral blood MN assay in young SD rats analyzed after 36 h of drug treatment. The sensitivity was compared between bled and non-bled SD rats with CP (100 mg/kg), CB (30 mg/kg) and MMC (2 mg/kg). All the values are shown as mean ± standard error of the mean (n = 5), the level of significance was determined at *P < 0.05, ***P < 0.001 with respective non-bled groups.

 


Figure 4
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  		Fig. 4. Photomicrographs showing (a) single MN in RET (orange red) of peripheral blood and (b) PCE with multiple MNs in bone marrow stained by AO. Bar length (20 µm) represents the magnification (x100).

 
Sensitization of rat bone marrow MN assay
Bone marrow is the site of rapid cell proliferation and hence it is the most preferred organ for the evaluation of genotoxicity of different chemicals. Prior removal of 1 ml of blood from young rats resulted in significant increases in MN frequency induced by CP (100 mg/kg, P < 0.01), CB (30 mg/kg, P < 0.01) and MMC (2 mg/kg, P < 0.05) in bone marrow of rats. In Wistar rats, removal of blood resulted in a significant increase in MN frequency with CP (100 mg/kg) over the respective control (P < 0.01) (Fig. 5).


Figure 5
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  		Fig. 5. Effect of prior blood removal on the sensitivity of bone marrow MN assay in young SD rats analyzed after 36 h of drug treatment. The sensitivity was compared between bled and non-bled SD rats with CP (100 mg/kg), CB (30 mg/kg) and MMC (2 mg/kg). All the values are shown as mean ± standard error of the mean (n = 5), the level of significance was determined at *P < 0.05, ***P < 0.001 with respective non-bled groups.

 
Sensitization of RETs/total erythrocytes ratio in peripheral blood
The RETs-to-total erythrocyte ratio was significantly increased in pre-bled animals as compared to non-bled animals. All the drug treatment (CP, CB and MMC) resulted in suppression of the RETs-to-total erythrocyte ratio in comparison to control animals. Even with chemical treatment, a significant increase in the ratio was observed with CP (P < 0.01) and MMC (P < 0.05) over the respective control groups, but not with CB (Fig. 6). Figure 7 shows fluorescent photomicrographs of RETs in bled and non-bled young rat peripheral blood stained with AO.


Figure 6
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  		Fig. 6. Effect of prior blood removal on RETs/total erythrocyte ratio in young SD rats. RETs/total erythrocyte ratio was analyzed after 36 h of genotoxin (CP 100 mg/kg, CB 30 mg/kg, MMC 2 mg/kg) exposure. All the values are shown as mean ± standard error of the mean (n = 5), the level of significance was determined at *P < 0.05, **P < 0.01, ***P < 0.001 with respective non-bled groups.

 


Figure 7
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  		Fig. 7. Photomicrographs showing the incidence of RETs (orange red) and normochromatic erythrocytes (green) in peripheral blood of (a) non-bled and (b) pre-bled SD rats.

 
Induction of multiple MNs
Multiple micronucleated cells were observed in both peripheral blood as well as bone marrow with different drugs (Table I). A higher number of multiple micronucleated cells were present in pre-bled animals. In one instance, a cell with four MNs was observed (Fig. 4).


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  		Table I. Influence of prior bleeding on the frequencies of multiple MNs with different genotoxic agents (CP 100 mg/kg, CB 30 mg/kg, MMC 2 mg/kg) in peripheral blood and bone marrow of SD rats

 
Strain-specific induction of MN
Prior blood removal significantly increases the MN frequencies in both peripheral and bone marrow erythrocytes of Wistar rats as compared with animals without bleeding. It has been further observed that CP induced more MN in SD rats as compared with Wistar rats (Fig. 8).


Figure 8
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  		Fig. 8. Effect of prior blood removal on the sensitivity of peripheral blood and bone marrow MN assay in SD and Wistar rats. MN frequency was analyzed after 36 h of CP (100 mg/kg) treatment. All the values are shown as mean ± standard error of the mean (n = 5), the level of significance was determined at *P < 0.05, ***P < 0.001 with respective non-bled groups.

 

   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The spleen of adult rats efficiently removes micronucleated RBC from circulation resulting in the loss of sensitivity of the bioassay (15Go,23Go). Removal of blood leads to increase in the rate of RBC formation in the bone marrow. Prior withdrawal of blood increases the blood-cell formation rate in bone marrow by stimulating the release of erythropoietin inside the body in a controlled and physiological way. This offers a better model for evaluating MNs in the peripheral blood of young rats. It has already been reported that erythropoietin increases the sensitivity of in vivo MN assay (9Go,10Go). Higher rate of cell proliferation is considered as a critical factor in both genotoxicity and carcinogenicity induction (24Go,25Go). Neonatal and young rats peripheral blood MN assay is used to evaluate the genotoxicity of compounds because up to 30 days of birth rat splenic red pulp (where micronucleated cells are captured and removed) is not able to remove micronucleated RETs efficiently from the peripheral blood circulation (5Go,23Go). Several scientists who have attempted to use splenectomized rat model to improve the sensitivity of the MN assay have confronted technical problems (2Go,26Go). Schlegal and MacGregor (3Go) have suggested that chemicals or agents that affect the normal splenic function (ability to remove spontaneously occurring micronucleated erythrocytes) might give false-positive responses. Concern for safe and effective use of drugs in pediatric populations increases the importance of conducting experiments in juvenile animals. Further, studies on juvenile animals can help to identify ‘unique’ toxicity not seen in adult animals (27Go). There is no major difference between cytochrome P-450 level in juvenile and adult rats. Most of the metabolic enzyme expression reaches a maximum at 3–4 weeks of age and thereafter remains constant (13Go,28Go–30Go).

Younger rats have shown significantly high numbers of micronucleated RETs as compared to adults. This has been further increased by prior blood removal. The maximum peak of MN induction in peripheral blood was observed at 36 h after chemical administration. This observation was in agreement with the findings of Sewerynek et al. (31Go) that the MN induction in peripheral blood was time dependent with lipopolysaccharide treatment and the peak values being reached at 36–48 h after the treatment. Increase in the sensitivity in young rats may be due to (i) inefficient removal of micronucleated cells from circulation, (ii) higher cell proliferation rate and (iii) a higher reserve of stem cells in the bone marrow. One of the interesting features noted was that animals with prior blood removal induced more than one MN in the bone marrow polychromatic erythrocytes as well as in peripheral blood RETs as compared with non-bled animals. In one circumstance, even a single bone marrow PCE contained four MNs. Prior blood removal increased the sensitivity of MN induction in Wistar rats. However, the induction was less than that in SD rats. Strain-specific differences in the induction of MN have already been reported with other chemicals in the rat model (32Go).

The peripheral blood model offers several potential advantages over the existing model to evaluate genotoxicity of different compounds, e.g. (i) ease of sample collection, (ii) multiple time-point evaluation, (iii) detection of weak genotoxic agents and (iv) minimized animal use in toxicological evaluation. Genotoxicity studies using the MN assay can be integrated with classical toxicological and toxicokinetic studies as rat is the most preferred species for long-term evaluation (33Go). As the sensitivity has been increased further with prior bleeding, this model may be used for the evaluation of MN in peripheral blood by using fluorescent activated cell sorter. However, more studies using genotoxins having different mechanisms of action are needed before the practical use of this model in a real genotoxic risk assessment process.


   Notes
 
* To whom correspondence should be addressed. NIPER Communication NO-395; Tel: +91 172 2214682 87; Email: gbjena{at}yahoo.com


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received on January 20, 2007; revised on March 9, 2007; accepted on March 13, 2007.


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