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The time-course of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood
Fundamental and Molecular Mechanisms of Mutagenesis
ELSEVIER
Mutation Research 350 (1996) 349-358
The time-course of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood Lilianne Abramsson-Zetterberg Department
Received
oj’Genetics.
Uppsala
*, Gijsta Zetterberg, Jan Graw6
Unirersi&.
1 1 July 1995; revised 26 October
Bm
7003.
S-750
07 Uppsala.
1995; accepted 27 October
.SNX&VI
1995
Abstract The time-course of micronucleated polychromatic erythrocytes (MPCE) in mouse bone marrow and peripheral blood, induced by an acute 0.1 Gy dose of X-rays, was determined using flow cytometric analysis, which made frequent sampling possible and allowed use of a dose low enough not to affect erythroid cell proliferation. The frequency of MPCE (NPCE) began to increase in the bone marrow at IO h after irradiation and reached a maximum at 28 h after irradiation. In the peripheral Hood fMPCE began to increase at 20 h after irradiation and peaked at about 40 h after irradiation. The time-course found is discussed on the basis of data on the differentiation of erythroid cells. The results indicate that the micronuclei registered in polychromatic erythrocytes may originate from lesions induced not only during the last cell cycle but also during earlier ones. After an acute dose of 1.0 Gy of X-rays the maximum fMPCE was delayed both in bone marrow and peripheral blood reflecting an effect on the cell cycle progression of erythroblasts. k?~ords:
Micronucleus
test; Mouse; Time-course;
Erythropoiesis;
X-ray
1. Introduction The in vivo micronucleus test using erythrocytes in bone marrow or peripheral blood from mice has extensive application in testing programs where results from mammalian in vivo exposure are required (Auletta et al., 1993; Kirkland, 1993). Recommendations and guidelines for test performance have been published (Heddle et al., 1983; Mavoumin et al., 1990). One problem has been discussed and investi-
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gated repeatedly, namely how long after exposure should samples be taken for a maximum yield of micronucleated erythrocytes to be registered (Salamone and Heddle, 1983; Sofuni, 1992). Proper timing of the sampling in a testing situation is influenced by the ability of the chemical to interfere with the proliferation of erythroblasts and by pharmacokinetic parameters, and in many cases it may be necessary to determine the schedule for sampling when the influence on erythropoiesis by the specific test chemical is unknown. The basis of all sampling schedules, however, is the kinetics of erythrocyte development in the bone marrow and peripheral blood. Only a few reports have been published on
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this subject. Using radiolabeling Tarbutt and Blackett (19681, Lala (19721, Jenssen and Ramel (1978) and Mary et al. ( 1980) have determined important parameters in the erythropoiesis of rodents. Cole et al. (198 1) described a model for micronucleus induction and development in the bone marrow of mice after exposure to single doses of methyl methanesulphonate or gamma-radiation. and Hayashi et al. (1984) reported a simulation analysis of the kinetics of micronucleus formation in mouse bone marrow polychromatic erythrocytes after exposure to three different chemicals using frequent sampling. Ludwikow and Ludwikow (1993). using the data from Hayashi et al. (I 9841, reported a mathematical model to explain the effects of the chemicals in terms of the damage leading to micronucleus induction in assuming a two-compartment distribution represented by the polychromatic and normochromatic erythrocytes in bone marrow. Chaubey et al. (1993) extended the observation to micronucleated erythrocytes in peripheral blood, using essentially the same experimental approach as in the work reported here. However, the flow cytometric analysis developed by Grawt et al. (1992, 1993a, b) and used here, has made it possible to determine these parameters with much higher accuracy than is possible with the microscopy-based analysis, especially after a dose that does not interfere with cell proliferation and after which the yield of micronuclei is low. We present results on the time-course of the appearance of micronucleated polychromatic erythrocytes (MPCE) in the bone marrow and also in the peripheral blood of mice exposed to an acute 0.1 Gy dose of X-rays. It has been found in earlier experiments (GrawC et al., 1992) and shown also here that the dose applied does not interfere with cell proliferation. Thus, the behaviour of the X-ray-induced MPCE can be considered to represent the behaviour of the total population of the polychromatic erythrocytes (PCE), an approach that has been used in earlier studies (Jenssen and Ramel, 1978: Cole et al., 1981; Hart and Hartley-Asp. 1983; Hayashi et al., 1984; Heddle, 1990; Chaubey et al.. 1993; McFee et al., 19941. Treatments that prolong the cell cycles of the erythroblasts have been found to delay the peak of MPCE frequency in bone marrow and peripheral blood (Jenssen and Ramel, 1978; Cole et al., 1981,
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Hart and Hartley-Asp, 1983; McFee et al., 1994). Below we report the results of an acute dose of 1.0 Gy of X-rays.
2. Materials
and methods
2. I. Animals Two strains of mice were used. Male CBA-S mice aged 7-8 weeks and weighing approx. 25 g were obtained from the breeding stock of the Department of Pathology, Swedish University of Agricultural Sciences, and used for the experiment the results of which are shown in Fig. 2. For the other experiments we used male mice of the same age and weight from another inbred CBA-strain, CBA-CA, which were bought from B&K Universal AB, Sollentuna, Sweden. The animals were housed and fed in accordance with good laboratory practice. The experiment has been reviewed and approved by the Uppsala Ethical Committee on Animal Experiments (application C 182/931. 2.2. Irradiation
and sampling
The X-ray tube (Mi.iller MG300. CHF Miiller, Hamburg, Germany) setting was 260 kV with 4 mm Al filtering. The tube current was 11 mA at a distance of 70 cm. giving a dose rate of 0.47 Gy/min. The dose rate was measured continuously during exposure with a dosimeter (Farmer 2570, Nuclear Enterprises, Reading, UK). After irradiation with a dose of 0.1 Gy at 0 h blood and bone marrow samples were obtained at 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36, 48, 60 and 72 h after treatment. Samples from four unirradiated mice were collected. two together with the animals killed at 4 h after irradiation and two at the end of the sampling period. In the second experiment with a dose of 0.1 Gy sampling times were the same as in the first experiment. except for an extra sample at 42 h after treatment and omission of the sampling at 4 and 6 h after treatment. Also unirradiated animals were analysed, one in the beginning and two at the end of the sampling period. When a dose of 1.O Gy was used, sampling times were adjusted to cover the expected delay of the
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maximum fMPCE both in bone marrow and peripheral blood. 2.3. Blood collection
and pur$cation
Animals were lightly anaesthetized and blood, about 200 ~1, was drawn from the orbital vein into heparinized tubes using a Pasteur pipette. Each of triplicate samples of 5 ~1 blood were layered on 1 ml of a 65% Percoll (Pharmacia Biosystems, Uppsala, Sweden) solution in phosphate-buffered saline (PBS) and centrifuged for 20 min at 600 X g. The supernatant, including platelets and the majority of nucleated cells, was carefully aspirated, leaving a pellet of erythrocytes and some nucleated cells. Immediately after the sampling of blood from the orbital vein the mice were killed by cervical dislocation. Both femurs were then excised and using a syringe the bone marrow was flushed into a test tube (Falcon 2052, Beckton-Dickinson Labware, NJ) with 1 ml of RPMI-1640 medium (Gibco. UK) per femur. The samples were first mixed and then left for l-2 min to allow large aggregates to sediment. The uppermost 1.9 ml in each tube was then transferred to a new tube and centrifuged for 5 min at 600 X g. The supernatant was discarded and the pellet was resuspended in 25 ~1 PBS. This cell suspension was layered onto 2 ml of 65% Percoll in a new tube and centrifuged for 20 min at 600 X g. 2.4. Elythrocyte
,fiuation and staining
The cells in a pellet obtained as above were diluted in 25 ~1 PBS and during vigorous agitation pipetted into a tube containing 1.25 ml of fixative (modified from the method of Hayashi et al., 1992), which was a 1% solution of glutaraldehyde (70% double-distilled EM grade, TAAB Laboratories, Reading, UK) made in PBS with 30 @g/ml sodium dodecyl sulphate (SDS; Sigma, St. Louis, MO, USA) included. Compared to the technique reported earlier (Grawe et al., 1992, 1993a, b; Zetterberg and GrawC. 1993) this modification proved to give more reproducible results. The samples were coded and stored overnight at 4°C allowing the cells to settle. The fixative was aspirated, staining buffer added and the samples were gently mixed. The staining buffer was
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prepared by adding 1 ml Hoechst 33342 stock (H0342, Sigma; 500 FM in distilled water) and 50 ~1 thiazole orange stock (TO, 1 mg/ml in methanol; Molecular probes, Eugene, OR. USA) to 100 ml PBS. The staining continued for 45 min at 37°C. Samples were mixed every 10 min by inverting the tubes.
2.5. Flow cytometric
analysis
The samples were analysed on a FACStar Plus flow cytometer (Beckton-Dickinson, Sunnyvale, CA, USA) equipped with an argon ion laser (S-P 2016, Spectra-Physics, Mountain View, CA, USA) operating at the 488 nm line, 200 mW as primary laser and a second argon ion laser operating at the 35 I-364 nm multiple UV lines at 100 mW (S-P 2025, Spectra-physics). The analysis rate was 1000 erythrocytes/s with a threshold in forward scatter (FSC) set to include all intact cells. Peak values for FSC, side scatter (SSC). TO fluorescence and HO342 fluorescence signals were collected in list mode on a Hewlett-Packard 300 computer (Hewlett-Packard, Fort Collins, CO, USA) running FACStar Plus software. Standard FACStar Plus filter sets were used. The FSC signals were acquired using a linear scale. while the SSC, TO, and HO342 signals were acquired using a 4 decade log scale. A live gate was used in the FSC and SSC parameters to exclude residual nucleated cells (NC) and debris. thereby restricting data acquisition to an almost pure population of erythrocytes (Grawe et al., 1993a). 80000 events were collected from each sample. For analysis of polychromatic erythrocytes (PCE) only a threshold level was established for TO fluorescence which excluded virtually all normochromatic erythrocytes (NCE) (Grawt et al., 1993a). Use of the TO fluorescence threshold increased the analysis rate to 500-1000 PCE/s. In this way files containing data from about 75000 PCE were generated from each sample. Each sample was analysed twice. first using the FSC threshold set to include all intact erythrocytes, then with the TO threshold for analysis of PCE only (Fig. 1). From the peripheral blood, triplicate samples were analysed, while from the bone marrow only one sample per animal was analysed.
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Blood
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All erythrocytes . . . . . . .. . . . . . .
.
PC&; control
PCES 12h after exposure
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3. Calculation
rt al. /Mutation
of PCE and MPCE frequencies
For each sample dot plots representing DNA content vs. RNA content were displayed. Regions of interest were defined for NCE and PCE using files containing data from the entire erythrocyte population (Fig. 1a.b) and MPCE using files that were acquired using a TO fluorescence threshold to eliminate NCE (Fig. 1c-j). The absolute number of events in the regions of interest for NCE, PCE and MPCE. respectively (,NCE. ,,PCE, “MPCE) were summed for the triplicate samples from peripheral blood. In some cases less than three parallel samples were analysed. The relative frequencies were then calculated as follows: fPCE = ( ilPCE +,,MPCE)/(
,,NCE +,,PCE
+ (??MPCE) fMPCE ==,IMPCE/(
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individual PCE or MPCE decreases with time as the cell loses RNA. At 12 h after exposure a clear influx of MPCE is evident in the bone marrow. The MPCE population shows a high TO fluorescence. Very little is seen in the peripheral blood at 12 h. At 20 h a large increase is seen in the bone marrow. and by now also in the peripheral blood. The TO fluorescence of the lMPCE population is still high, indicating a continuing influx of nascent MPCE to the bone marrow. At 36 h after exposure the frequency of MPCE is high in both bone marrow and peripheral blood. The TO fluorescence has decreased. indicating an ageing of the MPCE population. Compared to 20 h, few MPCE are seen with the highest level of TO fluorescence (about 100 units on the TO axis). This indicates that the influx of new PCE containing treatment-induced micronuclei into the bone marrow is ceasing.
,,MPCE + !,PCE)
Fig. 1 illustrates the appearance of the flow cytometric data and also shows how regions of interest for the NCE. PCE and MPCE populations were determined. One can also see some nucleated cells present in the HO-TO fluorescence dot plots of the bone marrow samples.
4. Results and discussion -1.I. Dot plots generated by jlow cytometry of samples taken at d@erent times after irradiation (I.0 GJ of X-rays) gice CI qualitative picture of the timecourse of MPCE Fig. I shows how the population of PCE containing micronuclei induced by the treatment (1 .O Gy of X-rays) appear in bone marrow and peripheral blood and subsequently mature. The TO fluorescence of an
Fig. I Dot plots of HO 342 vs. TO tluorescence while plots e-j are from animals exposed to I.0 and MNCE used for the analysis of the relative gated on light scatter as described in the text. In fluorescence units.
4.2. Quantitative assessment by ,flo~, cytometry generates data ,for jMPCE in bone marrow arld peripheral blood as a ,fitnction of time qfrer an acute exposure to 0. I Gy of X-ray
The time-course of MPCE in bone marrow and peripheral blood after the acute dose of X-rays is shown in Fig. 2. From each animal four samples were taken, one from the bone marrow and three samples from the peripheral blood. In Fig. 2 each value for bone marrow represents about 75 000 analysed PCE. Each value from peripheral blood is based on the analysis of about 185000 PCE. Such a large number of analysed cells increases substantially the reliability for each point. In earlier experiments (Abramsson-Zetterberg et al., 1995) using five CBA-S animals for each fMPCE value we conducted a statistical analysis and found that the differences between parallel samples
of erythrocytes in peripheral blood and bone marrow. Plots a-d are from control animals. Gy of X-rays and killed at the times indicated. Regions of interest for PCE, NCE, MPCE frequencies of the cell populations are shown. All plots are derived from files that were plots a and b 10000 events are shown, for the rest 80000 events. Scale units are arbitrary
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a
X
0
10
20
X
30
40
50
60
70
80
Time after irradiation (h) Fig. 2. of time on the analysis
The frequency of micronucleated polychromatic erythrocytes (fMPCE) in bone marrow (X) and peripheral blood ( n ) as a function after an acute dose of X-rays. 0.1 Gy. Animals from an in-house bred strain, CBA-S. were used. Each bone marrow value is based analysis of about 75000 PCE taken from one animal, and each fMPCE value representing the peripheral blood is based on the of about 185 000 PCE taken from one animal.
from one mouse were greater than the differences between the five animals given the same dose of ionizing radiation. In an identical experiment we used CBA-CA mice and obtained essentially the same results (not shown) confirming the time-course shown in Fig. 2 conceming the time for the start of an increase in fh4PCE both in bone marrow and peripheral blood. Also the peaks for fMPCE in bone marrow and peripheral blood occurred at the same times after irradiation seen in Fig. 2.. i.e., at about 28 and 40 h, respectively. The high reliability of the flow-cytometrically determined fMPCE for each experimental point is reflected in Fig. 2. Using flow-cytometric analysis we were able to obtain a sufficient number of values to describe the fMPCE trajectory after irradiation so that a distinct curve is apparent. Since all values lie consistently on the curve its shape and properties strongly suggest a high reliability for each point. The frequency of PCE was determined in all samples from bone marrow and peripheral blood of mice exposed to an acute dose of 0.1 Gy of X-rays. The variation between animals is of the same order as has been observed in earlier experiments (GrawC et al., 1992), and the conclusion is drawn that the
dose applied did not cause any notable depression of the proliferation of erythroblasts. It is possible to follow (i) the time schedule for erythrocyte maturation, i.e., the length of the period between the last mitosis until erythroblasts extrude their main nucleus and become PCE; (ii) the passage from bone marrow to peripheral blood of PCE; and iii) the duration as PCE in peripheral blood before the cell is classified as a normochromatic erythrocyte (NCE). 4.3. Evthrocyte
maturation
time is about IO h
From the results obtained in the two experiments using a dose of 0.1 Gy it is easy to postulate the length of the period between the last mitosis until erythroblasts extrude their main nucleus. This period represents the interval from the time of irradiation until the fMPCE starts to increase over the spontaneous level in the bone marrow. The results in both experiments indicate that about 10 h are required after the time of irradiation before the increase of MPCE can be observed in the bone marrow. This is in agreement with the results of Cole et al. (1981). Heddle (1990), Jenssen and Ramel (1978) and Chaubey et al. (1993) although in all these reports
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the doses applied were considerably higher and the intervals between sampling were longer. Hart and Hartley-Asp (1983) reported a different result. They noted an increase in the fMPCE in bone marrow as early as 6 h after an acute dose of ionizing radiation (0.45 Gy). Hayashi et al. (1984) found that MPCE appeared in the bone marrow at 6-8 h after the start of treatment with three different chemicals. 4.4. PCE hulje a transition about IO h
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In the peripheral blood system the fMPCE started to increase over the level of the control group after about 20 h and peaked 16-20 h later, i.e.. at 36-40 h after irradiation. Thereafter the frequency of MPCE in the peripheral blood system started to decrease gradually and at 72 h the level was still somewhat higher compared to the control group, although the difference was small. Thus, between the first appearance of MPCE in the bone marrow until they reach the peripheral blood system it takes about 10 h, giving a transition time of about 10 h of PCE in the bone marrow. Some estimates have been published for this transition time, based on a comparison of the bone marrow time schedule and the peripheral blood schedule. Chaubey et al. (19931, Hayashi et al. (1990) and Iwakura et al. (1992) presented MPCE values after different kinds of treatment in both bone marrow and peripheral blood, but a delay of the cell cycle oc-
time in bone marrow of
The frequency of MPCE in the bone marrow increased during a period of about 18 h and reached the maximum at 28 h after irradiation. From 28 h after the treatment the fMPCE in the bone marrow decreased, first rapidly and later on more slowly, and approached the control level after about 72 h after irradiation.
x
x x
x
x
. .
x
n
X
time after irradiation
(h)
Fig. 3. The frequency of micronucleated polychromatic erythrocytes (fMPCE) in bone marrow (XI and peripheral blood (W 1 as a function of time after an acute dose of X-rays, 1.0 Gy. Animals from an inbred strain of mice, CBA-CA, were used. Each bone marrow value is based on the analysis of about 75 000 PCE taken from one animal, and each fMPCE value representing the peripheral blood is based on the analysis of about 225 000 PCE taken from one animal.
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curred and samples were not frequently taken so a more precise transition time was not easily determined. However, Hayashi et al. (1984) reported a transition time of 18-22 h based on treatment with three different chemicals and using frequent sampling. Cole et al. (198 1) postulated a transition time of 10 h, Jenssen and Ramel (1978). and Lala et al. (1966) reported a value of about 24 and 12 h, respectively. In another two experiments using frequent sampling after an acute X-ray dose of 1.O Gy, we followed the time-course of MPCE in bone marrow and peripheral blood (Fig. 3). We found a delay of the peaks for MPCE frequency both in bone marrow and peripheral blood. However, the difference between the time of initial appearance of MPCE in bone marrow and in peripheral blood was the same. about 10 h, indicating that the transition time of PCE in bone marrow is not influenced by the magnitude of the dose. The time of maximum frequency in both bone marrow and peripheral blood. on the other hand, should be influenced by doses that interfere with the length of the cell cycles of erythroblasts. This was found after a dose of 1.O Gy of X-rays. The results are shown in Fig. 3. The effect on cell proliferation also gave lower proportion of PCE among the total number of erythrocytes both in bone marrow and peripheral blood. In bone marrow, where the normal proportion of PCE is about 50%. a dose of 1.0 Gy caused a decrease to between 15 and 25%~ PCE, the minimum occurring at 50-60 h after irradiation. Also in peripheral blood, where the proportion of PCE shows a higher variation between animals, the proportion of PCE reached a minimum between 50 and 60 h after irradiation. It can be observed that the difference in time between the peaks of fMPCE in bone marrow and peripheral blood was less pronounced than after 0.1 Gy (Fig. 2).
4.5. The duration time as PCE in peripheral about 1.5 h
blood is
A maximal duration time of the PCE in the peripheral blood system can be determined from the graph obtained after a dose of 0.1 Gy of X-rays (Fig. 2). After the peak, about 36 to 40 h after irradiation the frequency of MPCE declined again. At the time
Reseurch 350 (19961 349-358
when the frequency of MPCE decreased the maturation from PCE to NCE withdraws MPCE at a higher rate than the rate at which MPCE enter from the bone marrow into the peripheral blood system. From our graph (Fig. 2) it can be seen that the time from entrance of MPCE into peripheral blood till a maximum is obtained is 16 to 20 h, and accordingly the duration time of PCE in peripheral blood must be shorter than 16-20 h. Another way of measuring the duration time is to multiply the proportion of PCE in the peripheral blood system with the life length of erythrocytes in the blood. The data on erythrocyte life span in mice are at variance (Bannerman, 1983) probably due to methodological differences. However. the time required for the frequency of induced micronuclei in normochromatic erythrocytes to return to control values indicates the length of the period required for replacement of the total population of red cells and seems most relevant in the context here. Schlegel and MacGregor (1982) and GrawC et al. (1993b) found this period to be approx. 30 days. The mean proportion of PCE in peripheral blood in the present investigation was found to be 2%. This gives a PCE duration time in peripheral blood of about 15 h.
4.6. The bone marrow curue indicates that lesions induced in earlier cell cycles can gice rise to micronuclei There are very few reports about the cell cycle length of erythroblasts in mice. A detailed study of the erythropoiesis in adult rodents was made by Tarbutt and Blackett (1968) Tarbutt (1969) and Mary et al. (1980). The results were that the cell cycles are about 10 h with some variation between different cell cycles during the erythropoiesis. Considering these results and relating them to our results it seems clear that lesions induced not only during the last cell cycle contribute to the yield of micronuclei. If the bone marrow is looked upon as a window with a width of 10 h, representing the transition time of PCE, then the irradiated PCE from the last cell cycle. finished 10 h earlier (the maturation period), constitute a population of cells gradually appearing in the window: the first members to appear in the window were in late G, at the time of
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irradiation and the last cells to enter the window are those that were in early G, at the time of exposure. Thus the entire population of cells from the last cell cycle would occupy the window at 20 h after irradiation, i.e.. when the peak of MPCE would be reached. During the following 10 h, the bone marrow window would be emptied from MPCE and the curve representing fMPCE would thus reach control level at 30 h after irradiation. Even if a cell cycle time of 15 h is assumed the maximum should have been reached much earlier than at 28 h, the time determined here. Thus. the results obtained here do not support the assumption that MPCE originate from lesions induced only during the last cell cycle of erythroblasts. The maximum of fMPCE occurred at 28 h and the control level was not reached until about 70 h after X-ray exposure. Also the shape determined for the bone marrow curve is not compatible with an assumption that lesions giving rise to double-strand breaks in DNA only during the last cell cycle are responsible for the MPCE observed in the bone marrow. Since the yield of DNA double-strand breaks should be proportional to the amount of DNA in the cells during their last cell cycle the highest yield would be obtained from cells in G,. This would generate a convex curve before the-maximum. This is contradicted by our results. Even at the fairly low dose of 0.1 Gy X-rays ionizations should occur in every cell. It seems unlikely that the cells that develop micronuclei due to damage in their last cell cycle would constitute a specific population of cells with a prolonged cell cycle and/or maturation period compared to the main population of cells in which the DNA damage has not given rise to micronuclei. The lowest dose of X-rays used, 0.1 Gy, was shown not to influence the proportion PCE: NCE in the bone marrow, indicating that for the main population of cells no delay in the cell cycle progression occurred. Furthermore, a dose of 1.0 Gy substantially influenced the PCE: NCE ratio, and as seen in Fig. 3, also the peak of MPCE frequency in bone marrow occurred 16 h later (at 44 hl than after 0.1 Gy (when fMPCE peaked at 28 h). Thus it seems likely that micronucleated cells were affected to a similar degree by X-rays as the main population of cells without micronuclei, and that the substantial proportion of
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MPCE appearing during the interval 30-60 h after irradiation was due to damage induced in earlier cell cycles. Further information concerning the induction and repair of DNA lesions that are responsible for in vivo chromosomal damage giving rise to micronuclei in erythrocytes may be obtained through investigations using a similar approach as described above combined with studies on erythroblasts in vitro.
Acknowledgements We acknowledge the financial support from the Borgstrom Foundation. We thank Dr. Anders Johannisson for generous help with the flow cytometer and Britt-Marie Svedenst%l for advice and help with the X-irradiation. We are indebted to the Department of Pathology, Swedish University of Agricultural Sciences for housing the mice. One of us (L. A-Z) was financially supported by a fellowship from the Sven and Lilly Lawski Foundation. The flow cytometer has been funded by the Knut and Alice Wallenberg Foundation.
References Abramsson-Zetterberg, L., J. GrawC and G. Zetterberg (1995) Flow cytometric analysis of micro-nucleus induction in mice by internal exposure to ‘I’ Cs at very low dose rates. Int. J. Radiat. Biol., 67. 29-36. Auletta, A.E., K.L. Dearfield and M.C. Cimino (I9931 Mutagenicity test schemes and Guidelines: U.S. EPA Office of Pollution Prevention and Toxics and Office of Pesticide Programs. Env. Mol. Mutagen. 21. 38-45. Bannerman, R.M. (1983) Hematology, In: H.L. Foster. J.D. Small and J.G. Fox tEds.1. The Mouse in Biomedical Research. Academic Press. New York. Vol. III. pp. 293-3 12. Chaubey, R.C., H.N. Bhilwade. B.N. Joshi and P.S. Chauhan (19931 Studies on the migration of micronucleated erythrocytes from bone marrow to the peripheral blood in irradiated Swiss mice. Int. J. Radiat. Biol., 63, 239-235. Cole, R.J.. N. Taylor. J. Cole and C.F. Arlett (1981) Short-term tests for transplacentally active carcinogens. I. Micronucleus formation in fetal and maternal mouse erythroblasts. Mutation Res.. 80. 111-157. Craw& J.. Cl. Zetterberg and H. AmnCus (1992) Flow-cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood. Cytometry. 13. 750-758. Grawe, J.. G. Zetterberg and H. Am&s (lYY3a) DNA content determination of micronucleated polychromatic erythrocytes
358
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induced by clastogens and spindle poisons in mouse bone marrow and peripheral blood, Mutagenesis, 8, 249-255. GrawC. J., G. Zetterberg and H. Amneus (1993b3 Effects of extended low dose-rate exposure to “‘Cs detected by flow-cytometric enumeration of micronucleated erythrocytes in mouse peripheral blood, Int. J. Radiat. Biol., 63, 339-347. Hart, J.W. and B. Hartley-Asp (19831 Induction of micronuclei in the mouse, Revised timing of the final stage of erythropoiesis, Mutation Res.. 120, 127-132. Hayashi, M., T. Sofuni and M. Ishidate Jr. (19841 Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow, Mutation Res., 127, 129-137. Hayashi, M., T. Morita. Y. Kodama, T. Sofuni and M. Ishidate Jr. (1990) The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides, Mutation Res.. 245, 245-249. Hayashi. M., H. Norppa, T. Sofuni and M. Ishidate Jr. (1992) Flow cytometric micronucleus test with mouse peripheral erythrocytes, Mutagenesis, 7, 257-264. Heddle, J.A. (19901 Micronuclei in viva. In: M.L. Mendelsohn and R.J. Albertini (Eds.), Mutation and Environment, Part B, Wiley-Liss, New York, pp. 185-194. Heddle, J.A.. M. Hite, B. Kirkhart, K. Mavoumin, J.T. MacGregor. G.W. Newell and M.F. Salamone (19831 The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 133, 61-118. Iwakura, K., H. Tamura, A. Matsumoto. S. Ajimi, S. Ogura, K. Kakimoto, T. Matsumoto and M. Hayashi (1992) The micronucleus assay with peripheral blood reticulocytes by acridine orange supravital staining with I-P-parabinofuranosylcytosine, Mutation Res., 278. 13 1- 137. Jenssen. D. and C. Ramel (1978) Factors affecting the induction of micronuclei at low doses of X-rays. MMS and dimethylnitrosamine in mouse erythroblasts, Mutation Res., 58, 51-65. Kirkland, D.J. (19931 Genetic toxicology testing requirements. Official and unofficial views from Europe, Env. Mol. Mutagen., 21, 8-14. Lala, P.K. (1972) DNA synthesis time of bone marrow cells in healthy and ascites-tomor-bearing mice, Cell Tissue Kinet., 5, 79-85.
Reseurch 350 (1996134-358 Lala, P.K., H.M. Patt and M.A. Maloney (1966) An evaluation of erythropoiesis in canine marrow, Acta Haematol., 35,3 1 l-3 18. Ludwikow, F. and G. Ludwikow (1993) A two-compartment model of micronucleus formation in erythrocytes and its application to mouse bone marrow. I. Rectangulary transient action of clastogen in stationary conditions, J. Theor. Biol.. 165, 417-428. Mary, J.Y., A.J. Valleron, H. Croizat and E. Frindel (1980) Mathematical analysis of bone marrow erythropoiesis: Application to C,H mouse data, Blood Cells, 6, 241-254. Mavournin, K.H., D.H. Blakey, M.C. Cimino. M.F. Salamone and J.A. Heddle (1990) The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res., 239, 29-80. McFee, A.F., S.B. Cook and M.G. Abbott (19941 Negative doseresponse relationship for radiation-induced micronuclei in polychromatic erythrocytes of mice, Env. Mol. Mutagen.. 23. 128-131. Salamone, M.F. and J.A. Heddle (1983) In: F.J. deSerres and A. Hollaender (Eds.1, Chemical Mutagens, Principles and Methods for Their Detection. Vol. 8. pp. 11 l-149, Plenum, New York. Schlegel, R. and J.T. MacGregor (19821 The persistence of micronuclei in peripheral blood erythrocytes: Detection of chronic chromosome breakage in mice, Mutation Res., 104, 367-369. Sofuni, T. (19921 Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary of a report of the 5th collaborative study by SCGMT/JEMS-MMS, The collaborative study group for the micronucleus test, Mutation Res.. 278, 83-98. Tarbutt, R.G. (1969) Erythroid cell proliferation in hypertransfused rats, Br. J. Haematol., 17, 191-198. Tarbutt, R.G. and N.M. Blackett (1968) Cell population kinetics of the recognizable erythroid cells in the rat, Cell Tissue Kinet., 1, 65-80. Zetterberg, G. and J. Grawe (1993) Flow cytometric analysis of micronucleus induction in mouse erythrocytes by gammairradiation at very low dose rates, Int. J. Radiat. Biol., 64, 555-564.
ELSEVIER
Mutation Research 350 (1996) 349-358
The time-course of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood Lilianne Abramsson-Zetterberg Department
Received
oj’Genetics.
Uppsala
*, Gijsta Zetterberg, Jan Graw6
Unirersi&.
1 1 July 1995; revised 26 October
Bm
7003.
S-750
07 Uppsala.
1995; accepted 27 October
.SNX&VI
1995
Abstract The time-course of micronucleated polychromatic erythrocytes (MPCE) in mouse bone marrow and peripheral blood, induced by an acute 0.1 Gy dose of X-rays, was determined using flow cytometric analysis, which made frequent sampling possible and allowed use of a dose low enough not to affect erythroid cell proliferation. The frequency of MPCE (NPCE) began to increase in the bone marrow at IO h after irradiation and reached a maximum at 28 h after irradiation. In the peripheral Hood fMPCE began to increase at 20 h after irradiation and peaked at about 40 h after irradiation. The time-course found is discussed on the basis of data on the differentiation of erythroid cells. The results indicate that the micronuclei registered in polychromatic erythrocytes may originate from lesions induced not only during the last cell cycle but also during earlier ones. After an acute dose of 1.0 Gy of X-rays the maximum fMPCE was delayed both in bone marrow and peripheral blood reflecting an effect on the cell cycle progression of erythroblasts. k?~ords:
Micronucleus
test; Mouse; Time-course;
Erythropoiesis;
X-ray
1. Introduction The in vivo micronucleus test using erythrocytes in bone marrow or peripheral blood from mice has extensive application in testing programs where results from mammalian in vivo exposure are required (Auletta et al., 1993; Kirkland, 1993). Recommendations and guidelines for test performance have been published (Heddle et al., 1983; Mavoumin et al., 1990). One problem has been discussed and investi-
_ Corresponding 67 27 05.
author. Tel.: +46-18
67 13 01: Fax: +46-18
0027-5107/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0027.5 107(95)00208-1
gated repeatedly, namely how long after exposure should samples be taken for a maximum yield of micronucleated erythrocytes to be registered (Salamone and Heddle, 1983; Sofuni, 1992). Proper timing of the sampling in a testing situation is influenced by the ability of the chemical to interfere with the proliferation of erythroblasts and by pharmacokinetic parameters, and in many cases it may be necessary to determine the schedule for sampling when the influence on erythropoiesis by the specific test chemical is unknown. The basis of all sampling schedules, however, is the kinetics of erythrocyte development in the bone marrow and peripheral blood. Only a few reports have been published on
350
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this subject. Using radiolabeling Tarbutt and Blackett (19681, Lala (19721, Jenssen and Ramel (1978) and Mary et al. ( 1980) have determined important parameters in the erythropoiesis of rodents. Cole et al. (198 1) described a model for micronucleus induction and development in the bone marrow of mice after exposure to single doses of methyl methanesulphonate or gamma-radiation. and Hayashi et al. (1984) reported a simulation analysis of the kinetics of micronucleus formation in mouse bone marrow polychromatic erythrocytes after exposure to three different chemicals using frequent sampling. Ludwikow and Ludwikow (1993). using the data from Hayashi et al. (I 9841, reported a mathematical model to explain the effects of the chemicals in terms of the damage leading to micronucleus induction in assuming a two-compartment distribution represented by the polychromatic and normochromatic erythrocytes in bone marrow. Chaubey et al. (1993) extended the observation to micronucleated erythrocytes in peripheral blood, using essentially the same experimental approach as in the work reported here. However, the flow cytometric analysis developed by Grawt et al. (1992, 1993a, b) and used here, has made it possible to determine these parameters with much higher accuracy than is possible with the microscopy-based analysis, especially after a dose that does not interfere with cell proliferation and after which the yield of micronuclei is low. We present results on the time-course of the appearance of micronucleated polychromatic erythrocytes (MPCE) in the bone marrow and also in the peripheral blood of mice exposed to an acute 0.1 Gy dose of X-rays. It has been found in earlier experiments (GrawC et al., 1992) and shown also here that the dose applied does not interfere with cell proliferation. Thus, the behaviour of the X-ray-induced MPCE can be considered to represent the behaviour of the total population of the polychromatic erythrocytes (PCE), an approach that has been used in earlier studies (Jenssen and Ramel, 1978: Cole et al., 1981; Hart and Hartley-Asp. 1983; Hayashi et al., 1984; Heddle, 1990; Chaubey et al.. 1993; McFee et al., 19941. Treatments that prolong the cell cycles of the erythroblasts have been found to delay the peak of MPCE frequency in bone marrow and peripheral blood (Jenssen and Ramel, 1978; Cole et al., 1981,
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Hart and Hartley-Asp, 1983; McFee et al., 1994). Below we report the results of an acute dose of 1.0 Gy of X-rays.
2. Materials
and methods
2. I. Animals Two strains of mice were used. Male CBA-S mice aged 7-8 weeks and weighing approx. 25 g were obtained from the breeding stock of the Department of Pathology, Swedish University of Agricultural Sciences, and used for the experiment the results of which are shown in Fig. 2. For the other experiments we used male mice of the same age and weight from another inbred CBA-strain, CBA-CA, which were bought from B&K Universal AB, Sollentuna, Sweden. The animals were housed and fed in accordance with good laboratory practice. The experiment has been reviewed and approved by the Uppsala Ethical Committee on Animal Experiments (application C 182/931. 2.2. Irradiation
and sampling
The X-ray tube (Mi.iller MG300. CHF Miiller, Hamburg, Germany) setting was 260 kV with 4 mm Al filtering. The tube current was 11 mA at a distance of 70 cm. giving a dose rate of 0.47 Gy/min. The dose rate was measured continuously during exposure with a dosimeter (Farmer 2570, Nuclear Enterprises, Reading, UK). After irradiation with a dose of 0.1 Gy at 0 h blood and bone marrow samples were obtained at 4, 6, 8, 10, 12, 16, 20, 24, 28, 32, 36, 48, 60 and 72 h after treatment. Samples from four unirradiated mice were collected. two together with the animals killed at 4 h after irradiation and two at the end of the sampling period. In the second experiment with a dose of 0.1 Gy sampling times were the same as in the first experiment. except for an extra sample at 42 h after treatment and omission of the sampling at 4 and 6 h after treatment. Also unirradiated animals were analysed, one in the beginning and two at the end of the sampling period. When a dose of 1.O Gy was used, sampling times were adjusted to cover the expected delay of the
L. Ahra,nssorz-Zetterberg
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maximum fMPCE both in bone marrow and peripheral blood. 2.3. Blood collection
and pur$cation
Animals were lightly anaesthetized and blood, about 200 ~1, was drawn from the orbital vein into heparinized tubes using a Pasteur pipette. Each of triplicate samples of 5 ~1 blood were layered on 1 ml of a 65% Percoll (Pharmacia Biosystems, Uppsala, Sweden) solution in phosphate-buffered saline (PBS) and centrifuged for 20 min at 600 X g. The supernatant, including platelets and the majority of nucleated cells, was carefully aspirated, leaving a pellet of erythrocytes and some nucleated cells. Immediately after the sampling of blood from the orbital vein the mice were killed by cervical dislocation. Both femurs were then excised and using a syringe the bone marrow was flushed into a test tube (Falcon 2052, Beckton-Dickinson Labware, NJ) with 1 ml of RPMI-1640 medium (Gibco. UK) per femur. The samples were first mixed and then left for l-2 min to allow large aggregates to sediment. The uppermost 1.9 ml in each tube was then transferred to a new tube and centrifuged for 5 min at 600 X g. The supernatant was discarded and the pellet was resuspended in 25 ~1 PBS. This cell suspension was layered onto 2 ml of 65% Percoll in a new tube and centrifuged for 20 min at 600 X g. 2.4. Elythrocyte
,fiuation and staining
The cells in a pellet obtained as above were diluted in 25 ~1 PBS and during vigorous agitation pipetted into a tube containing 1.25 ml of fixative (modified from the method of Hayashi et al., 1992), which was a 1% solution of glutaraldehyde (70% double-distilled EM grade, TAAB Laboratories, Reading, UK) made in PBS with 30 @g/ml sodium dodecyl sulphate (SDS; Sigma, St. Louis, MO, USA) included. Compared to the technique reported earlier (Grawe et al., 1992, 1993a, b; Zetterberg and GrawC. 1993) this modification proved to give more reproducible results. The samples were coded and stored overnight at 4°C allowing the cells to settle. The fixative was aspirated, staining buffer added and the samples were gently mixed. The staining buffer was
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prepared by adding 1 ml Hoechst 33342 stock (H0342, Sigma; 500 FM in distilled water) and 50 ~1 thiazole orange stock (TO, 1 mg/ml in methanol; Molecular probes, Eugene, OR. USA) to 100 ml PBS. The staining continued for 45 min at 37°C. Samples were mixed every 10 min by inverting the tubes.
2.5. Flow cytometric
analysis
The samples were analysed on a FACStar Plus flow cytometer (Beckton-Dickinson, Sunnyvale, CA, USA) equipped with an argon ion laser (S-P 2016, Spectra-Physics, Mountain View, CA, USA) operating at the 488 nm line, 200 mW as primary laser and a second argon ion laser operating at the 35 I-364 nm multiple UV lines at 100 mW (S-P 2025, Spectra-physics). The analysis rate was 1000 erythrocytes/s with a threshold in forward scatter (FSC) set to include all intact cells. Peak values for FSC, side scatter (SSC). TO fluorescence and HO342 fluorescence signals were collected in list mode on a Hewlett-Packard 300 computer (Hewlett-Packard, Fort Collins, CO, USA) running FACStar Plus software. Standard FACStar Plus filter sets were used. The FSC signals were acquired using a linear scale. while the SSC, TO, and HO342 signals were acquired using a 4 decade log scale. A live gate was used in the FSC and SSC parameters to exclude residual nucleated cells (NC) and debris. thereby restricting data acquisition to an almost pure population of erythrocytes (Grawe et al., 1993a). 80000 events were collected from each sample. For analysis of polychromatic erythrocytes (PCE) only a threshold level was established for TO fluorescence which excluded virtually all normochromatic erythrocytes (NCE) (Grawt et al., 1993a). Use of the TO fluorescence threshold increased the analysis rate to 500-1000 PCE/s. In this way files containing data from about 75000 PCE were generated from each sample. Each sample was analysed twice. first using the FSC threshold set to include all intact erythrocytes, then with the TO threshold for analysis of PCE only (Fig. 1). From the peripheral blood, triplicate samples were analysed, while from the bone marrow only one sample per animal was analysed.
L. Abram.~s~n-Zetterber~
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Peripheral
et al. /Mutation
Blood
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C19%)34%
35x
Sone Marrow
All erythrocytes . . . . . . .. . . . . . .
.
PC&; control
PCES 12h after exposure
U
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3. Calculation
rt al. /Mutation
of PCE and MPCE frequencies
For each sample dot plots representing DNA content vs. RNA content were displayed. Regions of interest were defined for NCE and PCE using files containing data from the entire erythrocyte population (Fig. 1a.b) and MPCE using files that were acquired using a TO fluorescence threshold to eliminate NCE (Fig. 1c-j). The absolute number of events in the regions of interest for NCE, PCE and MPCE. respectively (,NCE. ,,PCE, “MPCE) were summed for the triplicate samples from peripheral blood. In some cases less than three parallel samples were analysed. The relative frequencies were then calculated as follows: fPCE = ( ilPCE +,,MPCE)/(
,,NCE +,,PCE
+ (??MPCE) fMPCE ==,IMPCE/(
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individual PCE or MPCE decreases with time as the cell loses RNA. At 12 h after exposure a clear influx of MPCE is evident in the bone marrow. The MPCE population shows a high TO fluorescence. Very little is seen in the peripheral blood at 12 h. At 20 h a large increase is seen in the bone marrow. and by now also in the peripheral blood. The TO fluorescence of the lMPCE population is still high, indicating a continuing influx of nascent MPCE to the bone marrow. At 36 h after exposure the frequency of MPCE is high in both bone marrow and peripheral blood. The TO fluorescence has decreased. indicating an ageing of the MPCE population. Compared to 20 h, few MPCE are seen with the highest level of TO fluorescence (about 100 units on the TO axis). This indicates that the influx of new PCE containing treatment-induced micronuclei into the bone marrow is ceasing.
,,MPCE + !,PCE)
Fig. 1 illustrates the appearance of the flow cytometric data and also shows how regions of interest for the NCE. PCE and MPCE populations were determined. One can also see some nucleated cells present in the HO-TO fluorescence dot plots of the bone marrow samples.
4. Results and discussion -1.I. Dot plots generated by jlow cytometry of samples taken at d@erent times after irradiation (I.0 GJ of X-rays) gice CI qualitative picture of the timecourse of MPCE Fig. I shows how the population of PCE containing micronuclei induced by the treatment (1 .O Gy of X-rays) appear in bone marrow and peripheral blood and subsequently mature. The TO fluorescence of an
Fig. I Dot plots of HO 342 vs. TO tluorescence while plots e-j are from animals exposed to I.0 and MNCE used for the analysis of the relative gated on light scatter as described in the text. In fluorescence units.
4.2. Quantitative assessment by ,flo~, cytometry generates data ,for jMPCE in bone marrow arld peripheral blood as a ,fitnction of time qfrer an acute exposure to 0. I Gy of X-ray
The time-course of MPCE in bone marrow and peripheral blood after the acute dose of X-rays is shown in Fig. 2. From each animal four samples were taken, one from the bone marrow and three samples from the peripheral blood. In Fig. 2 each value for bone marrow represents about 75 000 analysed PCE. Each value from peripheral blood is based on the analysis of about 185000 PCE. Such a large number of analysed cells increases substantially the reliability for each point. In earlier experiments (Abramsson-Zetterberg et al., 1995) using five CBA-S animals for each fMPCE value we conducted a statistical analysis and found that the differences between parallel samples
of erythrocytes in peripheral blood and bone marrow. Plots a-d are from control animals. Gy of X-rays and killed at the times indicated. Regions of interest for PCE, NCE, MPCE frequencies of the cell populations are shown. All plots are derived from files that were plots a and b 10000 events are shown, for the rest 80000 events. Scale units are arbitrary
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a
X
0
10
20
X
30
40
50
60
70
80
Time after irradiation (h) Fig. 2. of time on the analysis
The frequency of micronucleated polychromatic erythrocytes (fMPCE) in bone marrow (X) and peripheral blood ( n ) as a function after an acute dose of X-rays. 0.1 Gy. Animals from an in-house bred strain, CBA-S. were used. Each bone marrow value is based analysis of about 75000 PCE taken from one animal, and each fMPCE value representing the peripheral blood is based on the of about 185 000 PCE taken from one animal.
from one mouse were greater than the differences between the five animals given the same dose of ionizing radiation. In an identical experiment we used CBA-CA mice and obtained essentially the same results (not shown) confirming the time-course shown in Fig. 2 conceming the time for the start of an increase in fh4PCE both in bone marrow and peripheral blood. Also the peaks for fMPCE in bone marrow and peripheral blood occurred at the same times after irradiation seen in Fig. 2.. i.e., at about 28 and 40 h, respectively. The high reliability of the flow-cytometrically determined fMPCE for each experimental point is reflected in Fig. 2. Using flow-cytometric analysis we were able to obtain a sufficient number of values to describe the fMPCE trajectory after irradiation so that a distinct curve is apparent. Since all values lie consistently on the curve its shape and properties strongly suggest a high reliability for each point. The frequency of PCE was determined in all samples from bone marrow and peripheral blood of mice exposed to an acute dose of 0.1 Gy of X-rays. The variation between animals is of the same order as has been observed in earlier experiments (GrawC et al., 1992), and the conclusion is drawn that the
dose applied did not cause any notable depression of the proliferation of erythroblasts. It is possible to follow (i) the time schedule for erythrocyte maturation, i.e., the length of the period between the last mitosis until erythroblasts extrude their main nucleus and become PCE; (ii) the passage from bone marrow to peripheral blood of PCE; and iii) the duration as PCE in peripheral blood before the cell is classified as a normochromatic erythrocyte (NCE). 4.3. Evthrocyte
maturation
time is about IO h
From the results obtained in the two experiments using a dose of 0.1 Gy it is easy to postulate the length of the period between the last mitosis until erythroblasts extrude their main nucleus. This period represents the interval from the time of irradiation until the fMPCE starts to increase over the spontaneous level in the bone marrow. The results in both experiments indicate that about 10 h are required after the time of irradiation before the increase of MPCE can be observed in the bone marrow. This is in agreement with the results of Cole et al. (1981). Heddle (1990), Jenssen and Ramel (1978) and Chaubey et al. (1993) although in all these reports
L. Abramsson-Zetterberg
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the doses applied were considerably higher and the intervals between sampling were longer. Hart and Hartley-Asp (1983) reported a different result. They noted an increase in the fMPCE in bone marrow as early as 6 h after an acute dose of ionizing radiation (0.45 Gy). Hayashi et al. (1984) found that MPCE appeared in the bone marrow at 6-8 h after the start of treatment with three different chemicals. 4.4. PCE hulje a transition about IO h
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In the peripheral blood system the fMPCE started to increase over the level of the control group after about 20 h and peaked 16-20 h later, i.e.. at 36-40 h after irradiation. Thereafter the frequency of MPCE in the peripheral blood system started to decrease gradually and at 72 h the level was still somewhat higher compared to the control group, although the difference was small. Thus, between the first appearance of MPCE in the bone marrow until they reach the peripheral blood system it takes about 10 h, giving a transition time of about 10 h of PCE in the bone marrow. Some estimates have been published for this transition time, based on a comparison of the bone marrow time schedule and the peripheral blood schedule. Chaubey et al. (19931, Hayashi et al. (1990) and Iwakura et al. (1992) presented MPCE values after different kinds of treatment in both bone marrow and peripheral blood, but a delay of the cell cycle oc-
time in bone marrow of
The frequency of MPCE in the bone marrow increased during a period of about 18 h and reached the maximum at 28 h after irradiation. From 28 h after the treatment the fMPCE in the bone marrow decreased, first rapidly and later on more slowly, and approached the control level after about 72 h after irradiation.
x
x x
x
x
. .
x
n
X
time after irradiation
(h)
Fig. 3. The frequency of micronucleated polychromatic erythrocytes (fMPCE) in bone marrow (XI and peripheral blood (W 1 as a function of time after an acute dose of X-rays, 1.0 Gy. Animals from an inbred strain of mice, CBA-CA, were used. Each bone marrow value is based on the analysis of about 75 000 PCE taken from one animal, and each fMPCE value representing the peripheral blood is based on the analysis of about 225 000 PCE taken from one animal.
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curred and samples were not frequently taken so a more precise transition time was not easily determined. However, Hayashi et al. (1984) reported a transition time of 18-22 h based on treatment with three different chemicals and using frequent sampling. Cole et al. (198 1) postulated a transition time of 10 h, Jenssen and Ramel (1978). and Lala et al. (1966) reported a value of about 24 and 12 h, respectively. In another two experiments using frequent sampling after an acute X-ray dose of 1.O Gy, we followed the time-course of MPCE in bone marrow and peripheral blood (Fig. 3). We found a delay of the peaks for MPCE frequency both in bone marrow and peripheral blood. However, the difference between the time of initial appearance of MPCE in bone marrow and in peripheral blood was the same. about 10 h, indicating that the transition time of PCE in bone marrow is not influenced by the magnitude of the dose. The time of maximum frequency in both bone marrow and peripheral blood. on the other hand, should be influenced by doses that interfere with the length of the cell cycles of erythroblasts. This was found after a dose of 1.O Gy of X-rays. The results are shown in Fig. 3. The effect on cell proliferation also gave lower proportion of PCE among the total number of erythrocytes both in bone marrow and peripheral blood. In bone marrow, where the normal proportion of PCE is about 50%. a dose of 1.0 Gy caused a decrease to between 15 and 25%~ PCE, the minimum occurring at 50-60 h after irradiation. Also in peripheral blood, where the proportion of PCE shows a higher variation between animals, the proportion of PCE reached a minimum between 50 and 60 h after irradiation. It can be observed that the difference in time between the peaks of fMPCE in bone marrow and peripheral blood was less pronounced than after 0.1 Gy (Fig. 2).
4.5. The duration time as PCE in peripheral about 1.5 h
blood is
A maximal duration time of the PCE in the peripheral blood system can be determined from the graph obtained after a dose of 0.1 Gy of X-rays (Fig. 2). After the peak, about 36 to 40 h after irradiation the frequency of MPCE declined again. At the time
Reseurch 350 (19961 349-358
when the frequency of MPCE decreased the maturation from PCE to NCE withdraws MPCE at a higher rate than the rate at which MPCE enter from the bone marrow into the peripheral blood system. From our graph (Fig. 2) it can be seen that the time from entrance of MPCE into peripheral blood till a maximum is obtained is 16 to 20 h, and accordingly the duration time of PCE in peripheral blood must be shorter than 16-20 h. Another way of measuring the duration time is to multiply the proportion of PCE in the peripheral blood system with the life length of erythrocytes in the blood. The data on erythrocyte life span in mice are at variance (Bannerman, 1983) probably due to methodological differences. However. the time required for the frequency of induced micronuclei in normochromatic erythrocytes to return to control values indicates the length of the period required for replacement of the total population of red cells and seems most relevant in the context here. Schlegel and MacGregor (1982) and GrawC et al. (1993b) found this period to be approx. 30 days. The mean proportion of PCE in peripheral blood in the present investigation was found to be 2%. This gives a PCE duration time in peripheral blood of about 15 h.
4.6. The bone marrow curue indicates that lesions induced in earlier cell cycles can gice rise to micronuclei There are very few reports about the cell cycle length of erythroblasts in mice. A detailed study of the erythropoiesis in adult rodents was made by Tarbutt and Blackett (1968) Tarbutt (1969) and Mary et al. (1980). The results were that the cell cycles are about 10 h with some variation between different cell cycles during the erythropoiesis. Considering these results and relating them to our results it seems clear that lesions induced not only during the last cell cycle contribute to the yield of micronuclei. If the bone marrow is looked upon as a window with a width of 10 h, representing the transition time of PCE, then the irradiated PCE from the last cell cycle. finished 10 h earlier (the maturation period), constitute a population of cells gradually appearing in the window: the first members to appear in the window were in late G, at the time of
L. Abruitlsson-Zerrerber~
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irradiation and the last cells to enter the window are those that were in early G, at the time of exposure. Thus the entire population of cells from the last cell cycle would occupy the window at 20 h after irradiation, i.e.. when the peak of MPCE would be reached. During the following 10 h, the bone marrow window would be emptied from MPCE and the curve representing fMPCE would thus reach control level at 30 h after irradiation. Even if a cell cycle time of 15 h is assumed the maximum should have been reached much earlier than at 28 h, the time determined here. Thus. the results obtained here do not support the assumption that MPCE originate from lesions induced only during the last cell cycle of erythroblasts. The maximum of fMPCE occurred at 28 h and the control level was not reached until about 70 h after X-ray exposure. Also the shape determined for the bone marrow curve is not compatible with an assumption that lesions giving rise to double-strand breaks in DNA only during the last cell cycle are responsible for the MPCE observed in the bone marrow. Since the yield of DNA double-strand breaks should be proportional to the amount of DNA in the cells during their last cell cycle the highest yield would be obtained from cells in G,. This would generate a convex curve before the-maximum. This is contradicted by our results. Even at the fairly low dose of 0.1 Gy X-rays ionizations should occur in every cell. It seems unlikely that the cells that develop micronuclei due to damage in their last cell cycle would constitute a specific population of cells with a prolonged cell cycle and/or maturation period compared to the main population of cells in which the DNA damage has not given rise to micronuclei. The lowest dose of X-rays used, 0.1 Gy, was shown not to influence the proportion PCE: NCE in the bone marrow, indicating that for the main population of cells no delay in the cell cycle progression occurred. Furthermore, a dose of 1.0 Gy substantially influenced the PCE: NCE ratio, and as seen in Fig. 3, also the peak of MPCE frequency in bone marrow occurred 16 h later (at 44 hl than after 0.1 Gy (when fMPCE peaked at 28 h). Thus it seems likely that micronucleated cells were affected to a similar degree by X-rays as the main population of cells without micronuclei, and that the substantial proportion of
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MPCE appearing during the interval 30-60 h after irradiation was due to damage induced in earlier cell cycles. Further information concerning the induction and repair of DNA lesions that are responsible for in vivo chromosomal damage giving rise to micronuclei in erythrocytes may be obtained through investigations using a similar approach as described above combined with studies on erythroblasts in vitro.
Acknowledgements We acknowledge the financial support from the Borgstrom Foundation. We thank Dr. Anders Johannisson for generous help with the flow cytometer and Britt-Marie Svedenst%l for advice and help with the X-irradiation. We are indebted to the Department of Pathology, Swedish University of Agricultural Sciences for housing the mice. One of us (L. A-Z) was financially supported by a fellowship from the Sven and Lilly Lawski Foundation. The flow cytometer has been funded by the Knut and Alice Wallenberg Foundation.
References Abramsson-Zetterberg, L., J. GrawC and G. Zetterberg (1995) Flow cytometric analysis of micro-nucleus induction in mice by internal exposure to ‘I’ Cs at very low dose rates. Int. J. Radiat. Biol., 67. 29-36. Auletta, A.E., K.L. Dearfield and M.C. Cimino (I9931 Mutagenicity test schemes and Guidelines: U.S. EPA Office of Pollution Prevention and Toxics and Office of Pesticide Programs. Env. Mol. Mutagen. 21. 38-45. Bannerman, R.M. (1983) Hematology, In: H.L. Foster. J.D. Small and J.G. Fox tEds.1. The Mouse in Biomedical Research. Academic Press. New York. Vol. III. pp. 293-3 12. Chaubey, R.C., H.N. Bhilwade. B.N. Joshi and P.S. Chauhan (19931 Studies on the migration of micronucleated erythrocytes from bone marrow to the peripheral blood in irradiated Swiss mice. Int. J. Radiat. Biol., 63, 239-235. Cole, R.J.. N. Taylor. J. Cole and C.F. Arlett (1981) Short-term tests for transplacentally active carcinogens. I. Micronucleus formation in fetal and maternal mouse erythroblasts. Mutation Res.. 80. 111-157. Craw& J.. Cl. Zetterberg and H. AmnCus (1992) Flow-cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood. Cytometry. 13. 750-758. Grawe, J.. G. Zetterberg and H. Am&s (lYY3a) DNA content determination of micronucleated polychromatic erythrocytes
358
L. Abralnsson-Zetterbergg
et al. /Mutation
induced by clastogens and spindle poisons in mouse bone marrow and peripheral blood, Mutagenesis, 8, 249-255. GrawC. J., G. Zetterberg and H. Amneus (1993b3 Effects of extended low dose-rate exposure to “‘Cs detected by flow-cytometric enumeration of micronucleated erythrocytes in mouse peripheral blood, Int. J. Radiat. Biol., 63, 339-347. Hart, J.W. and B. Hartley-Asp (19831 Induction of micronuclei in the mouse, Revised timing of the final stage of erythropoiesis, Mutation Res.. 120, 127-132. Hayashi, M., T. Sofuni and M. Ishidate Jr. (19841 Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow, Mutation Res., 127, 129-137. Hayashi, M., T. Morita. Y. Kodama, T. Sofuni and M. Ishidate Jr. (1990) The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides, Mutation Res.. 245, 245-249. Hayashi. M., H. Norppa, T. Sofuni and M. Ishidate Jr. (1992) Flow cytometric micronucleus test with mouse peripheral erythrocytes, Mutagenesis, 7, 257-264. Heddle, J.A. (19901 Micronuclei in viva. In: M.L. Mendelsohn and R.J. Albertini (Eds.), Mutation and Environment, Part B, Wiley-Liss, New York, pp. 185-194. Heddle, J.A.. M. Hite, B. Kirkhart, K. Mavoumin, J.T. MacGregor. G.W. Newell and M.F. Salamone (19831 The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 133, 61-118. Iwakura, K., H. Tamura, A. Matsumoto. S. Ajimi, S. Ogura, K. Kakimoto, T. Matsumoto and M. Hayashi (1992) The micronucleus assay with peripheral blood reticulocytes by acridine orange supravital staining with I-P-parabinofuranosylcytosine, Mutation Res., 278. 13 1- 137. Jenssen. D. and C. Ramel (1978) Factors affecting the induction of micronuclei at low doses of X-rays. MMS and dimethylnitrosamine in mouse erythroblasts, Mutation Res., 58, 51-65. Kirkland, D.J. (19931 Genetic toxicology testing requirements. Official and unofficial views from Europe, Env. Mol. Mutagen., 21, 8-14. Lala, P.K. (1972) DNA synthesis time of bone marrow cells in healthy and ascites-tomor-bearing mice, Cell Tissue Kinet., 5, 79-85.
Reseurch 350 (1996134-358 Lala, P.K., H.M. Patt and M.A. Maloney (1966) An evaluation of erythropoiesis in canine marrow, Acta Haematol., 35,3 1 l-3 18. Ludwikow, F. and G. Ludwikow (1993) A two-compartment model of micronucleus formation in erythrocytes and its application to mouse bone marrow. I. Rectangulary transient action of clastogen in stationary conditions, J. Theor. Biol.. 165, 417-428. Mary, J.Y., A.J. Valleron, H. Croizat and E. Frindel (1980) Mathematical analysis of bone marrow erythropoiesis: Application to C,H mouse data, Blood Cells, 6, 241-254. Mavournin, K.H., D.H. Blakey, M.C. Cimino. M.F. Salamone and J.A. Heddle (1990) The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res., 239, 29-80. McFee, A.F., S.B. Cook and M.G. Abbott (19941 Negative doseresponse relationship for radiation-induced micronuclei in polychromatic erythrocytes of mice, Env. Mol. Mutagen.. 23. 128-131. Salamone, M.F. and J.A. Heddle (1983) In: F.J. deSerres and A. Hollaender (Eds.1, Chemical Mutagens, Principles and Methods for Their Detection. Vol. 8. pp. 11 l-149, Plenum, New York. Schlegel, R. and J.T. MacGregor (19821 The persistence of micronuclei in peripheral blood erythrocytes: Detection of chronic chromosome breakage in mice, Mutation Res., 104, 367-369. Sofuni, T. (19921 Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary of a report of the 5th collaborative study by SCGMT/JEMS-MMS, The collaborative study group for the micronucleus test, Mutation Res.. 278, 83-98. Tarbutt, R.G. (1969) Erythroid cell proliferation in hypertransfused rats, Br. J. Haematol., 17, 191-198. Tarbutt, R.G. and N.M. Blackett (1968) Cell population kinetics of the recognizable erythroid cells in the rat, Cell Tissue Kinet., 1, 65-80. Zetterberg, G. and J. Grawe (1993) Flow cytometric analysis of micronucleus induction in mouse erythrocytes by gammairradiation at very low dose rates, Int. J. Radiat. Biol., 64, 555-564.