Erythropoiesis and the induction of micronuclei in mouse spleen determined by flow cytometry

Mutation Research 394 Ž1997. 17–28

Erythropoiesis and the induction of micronuclei in mouse spleen determined by flow cytometry Lilianne Abramsson-Zetterberg ) , Jan Grawe, ´ Gosta ¨ Zetterberg Department of Genetics, Uppsala UniÕersity, Box 7003, S-750 07 Uppsala, Sweden Received 25 February 1997; revised 13 June 1997; accepted 17 June 1997

Abstract Erythrocytes from the spleen of CBA mice have been prepared for analyses by flow cytometry. About 80% of the polychromatic erythrocytes ŽPCE. in the spleen originate from erythropoiesis in the spleen, while the remaining 20% come from the peripheral blood. Analyses of the RNA content of PCE revealed that splenic PCE do not mature into normochromatic erythrocytes ŽNCE. in the spleen but leave the organ at a more immature stage. A considerable part of the PCE from bone marrow also mature into NCE in the bone marrow. The rate of RNA breakdown in PCE follows an exponential function. Time-courses for the appearance of micronucleated PCE ŽMPCE. from spleen and from bone marrow were determined by analysis of samples taken with short intervals after an acute dose of 0.1 Gy X-rays. The time-courses were identical for MPCE from the spleen and the bone marrow: The frequency of MPCE ŽfMPCE. starts to increase at about 10 h after irradiation and reaches its maximum after about another 20 h, upon which fMPCE returns to control level. The first induced MPCE in peripheral blood appear at about 20 h after irradiation. The effects of the carcinogen DMBA, 9,10-dimethyl-1,2-benzanthracene, at low doses were determined in PCE from spleen and bone marrow. The sensitivity was found to be about the same for erythroblasts in the spleen and the bone marrow. Protracted exposure to g-irradiation at a very low dose rate Ž44 mGyrday. gave a similar increase of fMPCE in bone marrow and spleen. The suitability of using splenic erythrocytes in the micronucleus test is discussed. q 1997 Elsevier Science B.V. Keywords: Micronucleus test; Mouse; Erythrocyte; Spleen; Bone marrow; Erythropoiesis; Flow cytometry

1. Introduction The micronucleus test in bone marrow erythrocytes of mice is the most frequently used in vivo short-term assay of genotoxic chemicals. Being an in vivo test it is supposed to take into account important pharmacokinetic parameters like activation,

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detoxification and tissue distribution. However, concern has been expressed that some chemicals or their metabolites do not reach the bone marrow in sufficient concentrations, if at all ŽKirkland. w1x. Thus false negative results may be obtained in the assay. Ashby w2x reported that a number of hepatocarcinogens, active in the UDS-test with hepatocytes, were inactive in the bone marrow micronucleus test. Using young erythrocytes from fetal mouse liver, Cole et al. w3x showed that short-lived liver metabolites induced micronuclei. Nakamura et al. w4x found that

1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 1 9 - 8

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L. Abramsson-Zetterberg et al.r Mutation Research 394 (1997) 17–28

5-fluorouracil was active in fetal liver but inactive in the bone marrow of the pregnant mouse. Bone marrow is the principal, but not the only haem atopoietic organ in adult m am m als. Haematopoiesis occurs also in the spleen w5x, making it an organ from which erythrocytes for the micronucleus assay may be taken. Thus, the effects of chemicals or metabolites distributed to the spleen, but not to the bone marrow, may be detected in splenic erythrocytes. A number of investigations have been made to compare the sensitivity of the erythrocytes from bone marrow with that of erythrocytes derived from spleen in adult mice. Shindo et al. w6x found that the frequency of micronucleated polychromatic erythrocytes, fMPCE, induced by three chemicals was about the same in both organs. A more pronounced response to treatment with the carcinogen dimethylnitrosamine, DMN, was registered in spleen erythrocytes compared to that in bone marrow erythrocytes w7x, but for a number of other chemicals and X-rays the response was found to be similar in the two organs w8x. Martelli et al. w9x concluded that the spleen is not a useful alternative to the bone marrow in the micronucleus assay. They found a much higher toxic effect in spleen compared to in bone marrow after treatment with one direct acting carcinogen and two indirect acting ones. With the high doses applied, the reduction of polychromatic erythrocytes, PCE, was so strong in the spleen that the determination of fMPCE was difficult. In animals of a different species, in rats, dibenzw a,h xanthracene ŽDBA. increased fMPCE in erythrocytes from the spleen, but not from the bone marrow w10x. The mammalian spleen has a diversity of functions, which vary in expression depending on animal species and age. Virtually all blood is circulated through the spleen which filters and removes defective cells and old cells, including erythrocytes w5x. In the laboratory mouse Ž Mus domesticus. the ability of the spleen to eliminate erythrocytes containing micronuclei is very limited, as evidenced by the fact that protracted exposure to genotoxic agents results in accumulation of micronucleated erythrocytes in the peripheral blood w11–13x. However, in humans and rats the spleen very effectively removes micronucleated erythrocytes from peripheral blood. The spleen is also a reservoir for storage of blood cells, and in certain mammalian species this capacity

is significant. In response to stress or to hemorrhage, a concentrated mass of erythrocytes is forced into the circulation with a simultaneous increase in erythropoiesis w5x. Thus, when isolating the spleen for harvesting erythrocytes for the micronucleus assay, it may not be easy to determine whether these cells originate from erythropoiesis in spleen or in bone marrow, or whether they come from the peripheral circulation and are present in the spleen at the moment of its removal from the mouse. This will influence the conclusions about the sensitivity of splenic erythroblasts. In the present investigation we have used the flow cytometer-based micronucleus assay developed by us w14–17x to answer the following questions. Ža. Can erythrocytes from the spleen be isolated and prepared for analyses by the flow cytometer? Žb. Do the young erythrocytes, PCE, harvested in the spleen, originate from erythropoiesis in the spleen, or are they transported in the peripheral blood from bone marrow to the spleen for maturation? Žc. Do splenic erythrocytes, in comparison with bone marrow derived erythrocytes, respond with higher or lower fMPCE to low doses of the indirect-acting carcinogen 9,10-dimethyl-1,2-benzanthracene ŽDMBA.? Žd. Do the results suggest that DMBA-induced micronucleated erythrocytes become enriched in the spleen for removal? Že. Can the spleen be considered a useful alternative to bone marrow as a source of young erythrocytes for use in the micronucleus assay? The flow cytometric analyses provided data, not directly necessary for the micronucleus assay, but of great value in describing erythropoiesis in the spleen, which is a prerequisite for drawing conclusions about the suitability of splenic erythrocytes in the micronucleus test.

2. Materials and methods 2.1. Animals Male CBA mice aged 8–10 weeks, weighing about 25 g, were used in the experiments. The mice were bought from B and K Universal, Sollentuna, Sweden, and acclimatized for at least 1 week in an animal house at the Department of Genetics, Uppsala

L. Abramsson-Zetterberg et al.r Mutation Research 394 (1997) 17–28

University, where they were allowed free access to solid food and tap water. The experiment has been reviewed and approved by the Uppsala Ethical Committee on Animal Experiments, application C 285r94. 2.2. Chemical treatment DMBA, 9,10-dimethyl-1,2-benzanthracene, CAS 53-97-6 ŽSigma, Labkemi, Stockholm. was dissolved in corn oil to a concentration of 0.8 or 0.4 mgrml. DMBA was administered as a single dose, i.p., 100 mlr10 g, 72 h before the harvesting of the cells. 2.3. Irradiation In the time-course study the mice were exposed to X-irradiation at 0-time, using an X-ray tube ŽMuller ¨ MG300, CHF Muller, Hamburg, Germany.. The tube ¨ setting was 260 kV with 4 mm Al filtering and a current of 11 mA. Irradiation was given at a distance of 70 cm, giving a dose rate of 0.47 Gyrmin. The dose rate was measured continuously during exposure with a dosimeter ŽFarmer 2570, Nuclear Enterprises, Reading, UK.. At each of the times indicated in Fig. 4, one animal was killed, except for at 28, 32 and 36 h after irradiation, when three mice were used at each time. During the extended g-irradiation, the animals were exposed during 4 days to a 137Cs-source of 30 Ci. The dose rate was determined to be 44 mGyrday, using a RNI-10R dosimeter ŽRoland Nilsson Instrument, Goteborg, Sweden.. The mice were killed at ¨ the end of exposure. 2.4. Collection and purification of blood, bone marrow and spleen When sampling peripheral blood the animals were lightly anesthetized and blood, about 100 ml, was drawn from the orbital plexus into heparinized tubes using a Pasteur pipette. Each of triplicate samples of 5 ml blood were layered on 1 ml of a 65% Percoll ŽPharmacia Biosystems, Uppsala, Sweden. solution in phosphate-buffered saline ŽPBS. and centrifuged for 20 min at 600 = g. The supernatant, including platelets and the majority of nucleated cells, was

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carefully aspirated, leaving a pellet of erythrocytes and some nucleated cells. Immediately after the sampling of blood, the mice were killed by cervical dislocation. Both femurs were then excised and using a syringe, the bone marrow from the two femurs was flushed into a conical test tube Ž4.5 ml; 12r75 mm, Greiner, Kebo, Spanga, Sweden. with 1 ml of RPMI-1640 medium ˚ ŽGibco, UK. per femur. The samples were first mixed and then left for 1–2 min to allow large aggregates to sediment. The uppermost 1.9 ml in each tube was then transferred to a new test tube and centrifuged for 5 min at 600 = g. The remaining pellet was mixed with 40 ml PBS and, in another conical tube, layered onto a 1-ml 65% Percoll solution which was centrifuged for 20 min at 600 = g. Using a Pasteur pipette connected to water suction, the supernatant was carefully removed from the pellet. After addition of 40 ml PBS, the cells were carefully resuspended. The spleen was disintegrated using a wire-net of stainless steel immersed in a Petri dish containing 9 ml of a solution of RPMI, 60%, and fetal calf serum ŽLife Technologies, Taby, ¨ Sweden., 40%. The serum was added to eliminate the risk of coagulation of the cells. The backside of a spoon was used to scrape the spleen through the net. The splenic capsule was left on the net. The cells were dispersed by aspirating the mixture several times through a syringe Ž0.6 mm gauge.. The cell suspension was pipetted to a 15-ml plastic tube and left for about 5 min to allow large aggregates to settle. The cell suspension was then transferred to three conical test tubes, which were centrifuged for 5 min at 600 = g. The supernatant was discarded and the pellet was resuspended in 40 ml PBS. The cell suspension was layered onto 1 ml of 67% Percoll in a new conical tube and centrifuged for 20 min at 600 = g. In order to get rid of the majority of the nucleated cells and the platelets, the supernatant was carefully removed, as when harvesting the bone marrow cells. It was difficult to prepare samples from the spleen with equally good quality as samples from the bone marrow. For some unaccountable reason this problem is aggravated after i.p. injection with oil. When we sorted out the cells defined as micronucleated PCE from splenic samples, the percentage of true events was about 60. Thus, the fMPCE values found

L. Abramsson-Zetterberg et al.r Mutation Research 394 (1997) 17–28

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in samples from spleen have been reduced by 40% ŽTable 1.. 2.5. Erythrocyte fixation and staining The procedure for fixation has been described earlier w17x. The cells were suspended in 40 ml PBS and pipetted into a vigorously agitated tube containing 1.25 ml of fixative Žmainly according to a method by Hayashi et al. w18x., which was a 1% solution of glutaraldehyde made in Sørensens phosphate buffer, 1r20 M, pH 6.8, with 30 mgrml sodium dodecyl sulfate included. The samples were coded and stored overnight at 48C, allowing the cells to settle. The fixative was aspirated and 1-ml staining buffer was added upon which the samples were gently mixed. The staining buffer was prepared by adding 1 ml Hoechst 33342 stock ŽHO 342, Sigma; 500 mM in

distilled water. and 100 ml Thiazole orange stock ŽTO, 1 mgrml in methanol; Molecular probes, Eugene, OR, USA. to 100 ml PBS. The staining continued for 45 min at 378C. Samples were mixed every 10 min by inverting the tubes. We have tested the possibility of storing samples of peripheral blood from animals, which would be of practical importance. The storage of blood at room temperature in heparinized tubes for more than half a day gives a change in the light scattering of the cells. On the other hand, cells that are fixed can be stored for several weeks without any problem. 2.6. Flow cytometric analysis The samples were analysed on a dual laser FACStar Plus flow cytometer ŽBeckton Dickinson, Sunnyvale, CA, USA. equipped with standard opticals

Table 1 Results of the study with DMBA Treatment

No. of animals

PCE analysis fPCE Ž%.

MNCE analysis nNCE

nMNCE

MPCE analysis fMNCE" S.D. Ž‰.

fMNCE Ž‰. corrected for true events

nPCE

nMPCE

fMPCE" S.D. Ž‰.

fMPCE Ž‰. corrected for true events

Bone marrow

Control 4 mgrkg 8 mgrkg

5 5 5

59.5 49.3 50.6

400 911 504 701 490 401

431 741 886

1.09 " 0.12 1.47 " 0.18 b 1.82 " 0.41 b

95% true events 1.0 1.4 1.7

1.10 " 0.2 1.23 " 0.1 1.17 " 0.3

65% true events 0.71 0.80 0.76

0.85 " 0.08 0.80 " 0.04 0.94 " 0.20

95% true events 0.81 0.76 0.89

981 156 980 873 978 361

1039 1596 2647

1.06 " 0.2 1.62 " 0.2 2.70 " 0.7

c b

95% true events 1.0 1.5 2.6

Spleen

Control 4 mgrkg 8 mgrkg

5 5 5

5.9 3.3 3.4

1 015 711 1 062 943 1 043 154

1111 1311 1207

640 380 556 477 559 854

1603 2797 2592

2.60 " 0.5 4.99 " 0.5 4.94 " 1.6

c a

60% true events 1.6 3.0 3.0

Peripheral blood

Control 4 mgrkg 8 mgrkg

5 5 5

2.3 1.2 1.1

1 014 070 986 830 987 396

865 794 915

1 022 526 1327 884 299 1811 868 444 2587

1.27 " 0.2 2.05 " 0.3 3.15 " 0.8

b b

95% true events 1.2 1.9 3.0

The frequencies of micronucleated normochromatic erythrocytes ŽfMNCE. and polychromatic erythrocytes ŽfMPCE. are given both as direct flow data, and corrected for the proportion of true events as determined by sorting. Statistical analysis was done with two-tailed Student’s t-test with p-values adjusted for the reuse of control groups. a p - 0.05; b p - 0.01; c p - 0.001.

L. Abramsson-Zetterberg et al.r Mutation Research 394 (1997) 17–28

and with an argon ion laser operating at the 488 nm line, 200 mW as primary laser, and a second argon ion laser operating at the 351–364 nm multiple UV lines at 100 mW. Standard FACStar Plus filter sets were used, a 530r30 nm bandpass filter for TO fluorescence, and a 424r44 nm bandpass filter for HO fluorescence. A live gate was used in the FSC and SSC parameters to exclude residual nucleated cells ŽNC. and debris, thereby restricting data acquisition to an almost pure population of erythrocytes w15x. 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. Ideally, the number of erythrocytes analysed should be the same for each organ. As noted above, we prepare only one sample from the bone marrow, two from the peripheral blood and three from the spleen. The number of residual nucleated cells is higher in the spleen than in the bone marrow. Therefore the number of events analysed from each type of sample is different. From each peripheral blood sample 100 000 cells were collected, from each of the triplicate spleen samples 80 000 and from the bone marrow sample 200 000. This resulted in about 200 000 erythrocytes analysed in total per animal from each organ using the FSC threshold, and about 180 000 PCE using the TO threshold. CellQuest software was used for data acquisition and analysis.

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2.8. Calculation of PCE, MPCE and MNCE frequencies For each sample, dot plots representing DNA content vs. RNA content were displayed. Regions of interest were defined for NCE, MNCE, PCE and MPCE. The relative frequencies were then calculated. The calculations have been described previously w17x.

3. Results and discussion This investigation is divided into three different parts. Samples taken from bone marrow, spleen and peripheral blood have been analysed on a flow cytometer. In the first part, dot plots of erythrocytes from bone marrow, spleen and peripheral blood have been used to study the maturation of PCE in the different organs. In part two a time schedule is presented for the appearance of MPCE in the three organs after a low dose of 0.1 Gy X-rays. In the third part, the results are given from two different experiments where mice have been treated with DMBA or an extended low dose of g-irradiation in order to study the sensitivity of erythrocytes from the spleen. 3.1. The maturation of PCE in spleen, bone marrow and peripheral blood

2.7. Sorting In order to determine the reliability of the flow cytometric analyses of samples from the spleen, especially for the cells defined as micronucleated erythrocytes, these cells were sorted out on slides. In addition sorting was also made from samples of bone marrow and peripheral blood. To avoid salt buildup on the slides, the sheath fluid for sorting was diluted 1:500 with PBS. The slides were scored as wet mounts in a fluorescence microscope at a magnification of 630 = . In mice that were injected with corn oil, the proportion of true events was found to be about 60% and 65% for the cells from the spleen defined as MPCE and MNCE, respectively. Independent of the type of treatment, the proportion of true events for MNCE and MPCE was found to be more than 90% in the bone marrow and peripheral blood.

It is assumed that a PCE contains most RNA just after its birth, i.e. when it was formed by enucleation of the late erythroblast. During maturation to an NCE, the PCE may leave the site of erythropoiesis and eventually arrive in the peripheral blood. The distinction between PCE and NCE is based on the RNA content of the cells. When RNA reaches such a low value that the PCE can no longer be distinguished from the large population of NCE, it has become an NCE. The methods to define this border have included Wright’s staining, acridine orange staining, or flow cytometry using TO to stain RNA. With flow cytometry one can quantify the RNA content of a cell. The dot plots from the three different tissues, spleen, bone marrow and peripheral blood, are shown in Fig. 1a, b and c, respectively. They are all taken

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L. Abramsson-Zetterberg et al.r Mutation Research 394 (1997) 17–28

Fig. 1. Representative flow cytometry dot plots of erythrocytes from the spleen Ža., bone marrow Žb. and peripheral blood Žc. of one untreated mouse. NC, nucleated cells; NCE, normochromatic erythrocytes; PCE, polychromatic erythrocytes. The number of cells in each plot was adjusted to show about 1500 PCE. Note the position of the PCE population in the different samples. The highest Thiazole orange ŽTO. fluorescence Žindicating the lowest mean age of PCE. was found in the spleen, followed by the bone marrow and the peripheral blood PCE population.

from a single control mouse, but identical results have been obtained in all the analyses. To get a correct comparison between the PCE populations in Fig. 1a, b and c from the three tissues, we have analysed the same number of PCE from each organ. It is apparent that there is a difference in maturation status between the PCE populations. There is a higher proportion of young PCE in the spleen com-

pared to bone marrow and peripheral blood, as indicated by the TO fluorescence intensity. In Fig. 2a the distributions of PCE according to the RNA content are shown as a histogram. The population of cells below each of the three curves ŽFig. 2a. can be looked upon as a snapshot. If a second snapshot is taken some hours later, the position of each PCE has changed. Between the two

Fig. 2. Frequency distributions of the linear Thiazole orange ŽTO. fluorescence of the populations of polychromatic erythrocytes ŽPCE. in the spleen, bone marrow, and peripheral blood from one untreated mouse Ža.. Frequency distributions of the linear TO fluorescence of the PCE populations in the spleen and bone marrow after subtraction of the fraction originating from peripheral blood PCE. The calculation was based on the proportion of normochromatic erythrocytes ŽNCE. in each organ and a 2% proportion of PCE in the peripheral blood. Thus, the curves illustrate only newly produced PCE, with no contribution from peripheral blood Žb..

L. Abramsson-Zetterberg et al.r Mutation Research 394 (1997) 17–28

snapshots all PCE have become older and have a smaller RNA content, i.e. moved further to the left on the dot plot. Although all cells have changed their positions, the two snapshots look the same. The identity of the two snapshots depends on the balance between the inflow and the outflow of PCE. Comparing the two curves in Fig. 2a, representing the bone marrow and spleen, it can be seen that the width of the peaking PCE population in the spleen is narrower and has its maximum more to the right than the curve representing the bone marrow, implicating an earlier outflow of PCE from spleen to peripheral blood, provided that the progress of maturation is the same in the two organs. The latter seems very likely, since it has been observed that erythropoiesis is a programmed process of the cell, independent of site, and has been shown to continue with the same pattern, including enucleation and maturation, in bone marrow, spleen, peripheral blood, and also in vitro w19–22x. The fraction of the curves ŽFig. 2a. for spleen and bone marrow to the right of the maximum represents PCE originating from erythroblasts that have lost their nucleus quite recently and contain a considerable amount of RNA. The shape of the curves to the right of the peaks ŽFig. 2a. for bone marrow and spleen reveals that there are proportionally fewer cells with a high RNA content than such with a lower RNA content. The peaks of the curves represent cells with the commonest RNA content. This distribution may be explained in different ways, depending on which one of the following two assumptions is preferred: Ži. there is a variation of the RNA content among the newly formed PCE, and the rate of RNA breakdown is constant and independent of the RNA content, or Žii. the RNA content of the newborn PCE is almost constant, and the RNA breakdown is higher when the RNA content is high, and decreases with decreasing RNA content, leading to an increasing number of cells with lower RNA content. The second assumption Žii. is supported by the results in Fig. 3. We determined the RNA content of MPCE at different time after an acute X-ray dose of 1.0 Gy and found that the rate of RNA breakdown followed an exponential function. The shape of the curves for bone marrow and spleen to the left of their peaks is determined by the outflow of PCE to the peripheral blood. The PCE

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Fig. 3. The decrease with time Žabscissa. of the mean RNA content measured as Thiazole orange ŽTO. fluorescence Žordinate. of maturing micronucleated polychromatic erythrocytes ŽMPCE., induced by an acute X-ray dose, 1 Gy. The values of the TO fluorescence were calculated from flow cytometric analyses performed in a previous experiment w17x.

leave their organ of birth at a varying degree of maturation, containing different amounts of RNA. In comparison with the conditions in the bone marrow, there is a proportionally earlier outflow of PCE from the spleen. Because of a higher proportion of young PCE Žwith a higher RNA content. in the spleen than in the bone marrow, an initial decrease in the percentage of PCE can be expected to be more pronounced in the spleen when a toxic treatment impairs the cell proliferation. This was also found after the treatments with DMBA ŽTable 1.. Several authors w6–9x also found a similar pattern in the two organs concerning induced decrease of the percentage of PCE. The samples of erythrocytes taken from the bone marrow or from the spleen always contain a proportion of PCE originating from the peripheral blood flowing through the organs. With a mean value of about 2% PCE in peripheral blood, about 8% PCE in the spleen, and about 50% PCE in the bone marrow, the contribution from the PCE originating from peripheral blood can be calculated to be about 1r5 in spleen and less than 1r50 in bone marrow. Thus, to better determine the length of time spent by PCE in bone marrow and spleen, the proportion of PCE representing the peripheral blood should be subtracted from the curve presented in Fig. 2a. This was done in Fig. 2b, where the curves illustrate only

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L. Abramsson-Zetterberg et al.r Mutation Research 394 (1997) 17–28

newly produced PCE, with no contribution from peripheral blood. In the diagram ŽFig. 2b. it can be seen that no PCE are maturing to NCE in the spleen. The transition of the PCE to NCE must occur at another site, presumably in the peripheral blood. The situation in the bone marrow is somewhat different. Here a great proportion of the PCE, produced in the bone marrow, also reach the NCE stage in the same organ. This explains why the induced fMNCE in bone marrow can be higher than in spleen or peripheral blood, which has been found in this investigation ŽTable 1. and also by Martelli et al. w9x. Thus, since all erythrocytes produced in the spleen leave the spleen as PCE, the NCE and MNCE found in the spleen originate from peripheral blood. Accordingly, the fMNCE measured in the spleen should be about the same as the fMNCE value in the peripheral blood, which we also found after treatment with DMBA ŽTable 1.. It is known that the spleen removes micronucleated erythrocytes in other species, like man and rat w5x. If this occurs also in mouse, one would expect MNCE to be sequestered in the spleen, and fMNCE would thus be higher in splenic samples than in samples from bone marrow or peripheral blood. However, the results in Tables 1 and 2 show that the spontaneous and induced fMNCE was not higher in the spleen. In Fig. 2a it can also be seen that in peripheral blood there is a continuous increase of the proportion of PCE with decreasing RNA content until the NCE stage is reached. This circumstance implicates that

the PCE in the peripheral blood remain there until they reach the NCE stage. It has been suggested that PCE produced in bone marrow are transported to the spleen for maturation to NCE w5x. This does not seem to be the case in the present investigation. 3.2. The time-course of micronucleated polychromatic erythrocytes in bone marrow, spleen and peripheral blood It has long been known that the spleen is a site for erythropoiesis in adult mice Žsee w5x for references.. This can be shown in several ways. A higher frequency of PCE in the spleen than in the peripheral blood is an indication of that erythropoiesis occurs in the spleen. But the higher percentage of PCE is, however, not necessarily due to erythropoiesis, but could instead depend on the fact that the spleen is the site for the maturation of PCE w5x. However, the dot plots and curves shown in Fig. 1a–c and Fig. 2a,b clearly indicate that the main part of the PCE population isolated from the spleen is produced in the same organ. Fig. 4 shows the time-course of MPCE induced by a low X-ray dose, 0.1 Gy. The result is unequivocal; the maximum for fMPCE in the spleen does not occur after the maximum in the bone marrow, implicating an induction of MPCE in the spleen, independent of the induction in the bone marrow. The values for the maximum fMPCE, obtained after a dose of 0.1 Gy, were almost the same in the two organs,

Table 2 Frequencies of polychromatic erythrocytes ŽfPCE. and micronucleated polychromatic erythrocytes ŽfMPCE. in the spleen, bone marrow, and peripheral blood of mice killed after 4 days of continuous low-level exposure to g-irradiation Ž44 mGyrday. Treatment

No. of animals

PCE analysis

MPCE analysis

fPCE Ž% " SD.

nPCE

nMPCE

fMPCE Ž‰ " SD.

Bone marrow Control Cs-exposed

7 5

64.9 " 4.5 56.8 " 4.8

1 198 211 451 234

1386 1673

1.16 " 0.15 3.70 " 0.55

Spleen Control Cs-exposed

7 5

11.8 " 2.2 9.1 " 5.2

980 887 745 058

1382 2084

1.46 " 0.22 2.80 " 0.22

Peripheral blood Control Cs-exposed

7 5

2.6 " 0.3 2.3 " 0.3

1 381 110 907 019

1599 2526

1.16 " 0.18 2.78 " 0.20

L. Abramsson-Zetterberg et al.r Mutation Research 394 (1997) 17–28

Fig. 4. The frequency of micronucleated polychromatic erythrocytes ŽfMPCE. in spleen ŽSP., bone marrow ŽBM. and peripheral blood ŽPB. as a function of time after an acute dose of X-rays, 0.1 Gy. At each sampling time Žexcept at 0 h when two non-irradiated mice were used. peripheral blood, bone marrow and spleen were taken from one irradiated mouse of the inbred strain CBA-CA. At each sampling time the values were based on the analysis of about 180 000 PCE from the peripheral blood, 190 000 from the bone marrow, and 150 000 PCE from the spleen of each animal, except at 36 h when only 67 000 splenic PCE were analysed. The positions of the peaks of the curves were verified in an additional experiment where samples were taken at 28, 32 and 36 h after irradiation, from three animals at each time.

about 7‰. Fig. 4 also indicates that, when studying the effects of a chemical mutagen, the maximum fMPCE should occur at the same time in spleen and bone marrow, if the chemical has an equal distribution and metabolism in both organs. As stated above ŽSection 3.1., a comparison of the fMNCE values found in bone marrow, spleen and peripheral blood did not support that the spleen is a site for sequestering of micronucleated erythrocytes in the mouse w5x. Also, if such a deposition of MPCE is of importance the curve in Fig. 4 representing the fMPCE in spleen should have had a different shape, i.e. the decline of fMPCE should have been less steep Žbecause of the input of fMPCE from the peripheral blood flowing through.. The values obtained for bone marrow and peripheral blood in this investigation verify our results in

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an earlier report w17x where we in similar experiments studied the time-course of MPCE in bone marrow and peripheral blood, partly in order to describe the progression of erythrocyte maturation in mice. We applied an acute X-ray dose to induce micronuclei that were used as a tag on young erythrocytes which could be followed during maturation. Because of the low dose of X-rays, not interfering with cell proliferation, the time-course of MPCE can be considered to represent the time-course for all PCE for the period after the last mitosis in the succession of erythroblasts. Thus the time from the last mitosis to the extrusion of the main nucleus can be seen as the period following the time of irradiation until the first appearance of MPCE in the bone marrow or the spleen. As seen in Fig. 4, the length of this period was about the same for MPCE in the bone marrow and the spleen, about 10 h, which we also have found for bone marrow in a previous investigation w17x. Another 10 h Žtransition time. is needed until the MPCE from the bone marrow and the spleen enter the peripheral blood. In a similar experiment Žresults not shown., the mice were given an acute dose of 0.5 Gy. In order to obtain a time-course of fMPCE, repeated samples were taken from blood, bone marrow and spleen. It was found that 0.5 Gy X-rays induced a decrease of the percentage of PCE, and that this was more pronounced in the spleen than in the bone marrow. A dose-dependent increase of fMPCE occurred. The maximum of fMPCE in the spleen was about 16‰. 3.3. Treatments with DMBA and g-irradiation In a preliminary experiment with the carcinogen DMBA, 9,10-dimethyl-1,2-benzanthracene, dissolved in corn oil Žresults not shown., the mice were given a dose of 25 mgrkg. The samples from bone marrow, spleen and peripheral blood were harvested at different time, 30, 42, 54 and 66 h after treatment. The dose of 25 mg DMBArkg caused a strong depression of cell proliferation resulting in a low percentage of PCE. At 66 h the fMPCE values and also the depression of cell proliferation were the highest. In another experiment Žresults not shown. we treated the mice with 2, 4, and 10 mg DMBArkg. The results indicated that maximum fMPCE occurred after 67 h also at these doses. The results

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from these introductory studies were the background for choosing a post exposure period of 72 h in a third experiment. Here, a total of 15 mice were used; a control group of 5 mice, 5 mice given a dose of 4 mg DMBArkg, and 5 mice given a dose of 8 mgrkg. The mice were given a single i.p. injection. The animals in the control group were injected with the same volume of corn oil. At 72 h after the injection, samples were taken from the three organs; spleen, bone marrow and peripheral blood. Table 1 shows the results from the experiment. In all three organs, a significant increase of fMPCE was induced by DMBA. In the bone marrow and the peripheral blood, the increase was dose-dependent. In the spleen, the fMPCE values were almost the same for the two different doses. Because of the low doses used, the decrease of PCE frequency was slight; in spleen and peripheral blood about 40% and in the bone marrow about 15%. There was no significant increase of fMNCE in peripheral blood or spleen. In bone marrow a dose-dependent increase in fMNCE was apparent. The results given here are in accordance with the conclusions given above about the production of PCE and their maturation. A significant increase of fMPCE occurred at dose levels considerably lower than in earlier reports on the effects of DMBA in the micronucleus assay with mice w2,9,23–27x. In our study the two sites for erythropoiesis, bone marrow and spleen, did not exhibit any significant differences in sensitivity, presumably due to DMBA having a similar pattern of distribution and metabolism in the two organs. The absence of a dose-dependent response in the spleen cannot be given a reasonable interpretation. In the preliminary experiment, we registered a dose-related increase of fMPCE from mice given doses of 2, 4 and 10 mg DMBArkg. Martelli et al. w9x demonstrated a partly different response between the two organs, bone marrow and spleen. The doses applied were much higher, 50 and 100 mgrkg, and a great depression in the proliferation occurred, making it difficult to score a sufficient number of cells for determination of fMPCE. We found that the values of fMNCE were very similar in the spleen and the peripheral blood. This can be expected, since we have shown above that there is no transition from PCE to NCE in the spleen, so the NCE found in samples from the spleen are there

because peripheral blood circulates in the spleen. The higher value of fMNCE that we found in bone marrow can be explained by the fact that among the PCE produced in bone marrow, a portion matures to NCE in the same organ. Previously we have shown that the flow cytometer-based micronucleus assay can detect an increase of fMPCE and fMNCE in bone marrow and peripheral blood by g-irradiation at a low dose rate w12,13x. In order to compare the response of splenic erythroblasts with those of bone marrow origin, we g-irradiated 5 mice during 4 days at a very low dose rate of 44 mGyrday. Seven mice constituted the control group and were kept outside the exposure room. Samples were taken from bone marrow, spleen and peripheral blood. The fMPCE after irradiation were 3.7‰, 2.8‰, and 2.8‰ in bone marrow, spleen and peripheral blood, respectively ŽTable 2.. It is concluded that samples from the spleen can be used to detect the chromosome damaging effect of low doses of low-LET ionizing radiation, and that the sensitivity is about the same as it is in bone marrow. When analysing the concentration of a compound in the target organ, the spleen is easier to handle than the bone marrow. For example, we attempted to determine the concentration of radon daughters in the femoral bone marrow of mice exposed to radon by inhalation, and found it difficult to quantify the mass of the bone marrow samples Žweighing less than 10 mg.. In comparison, the spleen is much easier to isolate and has a greater mass, which should give more reproducible results. Using the flow cytometer, the time needed for analysis is very much reduced in comparison with microscope analysis. Thus, extra samples, like those from the spleen, can easily be managed. We recommend that samples from the spleen are routinely included in testing to increase the information from the test and to make better use of the test animals. Further refinement of the isolation of splenic erythrocytes should give samples as good as those from the bone marrow and peripheral blood. 4. Conclusions Using flow cytometry, one can follow changes of the quantity of RNA in PCE, and thus obtain information about how maturation of erythrocytes pro-

L. Abramsson-Zetterberg et al.r Mutation Research 394 (1997) 17–28

ceeds in different organs. The comparison of the distribution of the RNA content of PCE populations in spleen, bone marrow and peripheral blood clearly showed that erythropoiesis occurs in the spleen. This was also supported by the time-courses determined for X-ray-induced MPCE in spleen, bone marrow and peripheral blood. The outflow to peripheral blood of PCE produced in the spleen occurs at an earlier stage than it does in bone marrow. Erythrocytes produced in the spleen enter the peripheral blood while they still are in the PCE stage. In bone marrow a great part of PCE finish their maturation into NCE while they remain in the same organ. We got no indications that PCE from the bone marrow are transported to the spleen for maturation, nor that MPCE or MNCE are sequestered in the spleen for elimination. The effects of low doses of the carcinogen DMBA could be determined in erythrocytes from both spleen and bone marrow, and the response was the same for cells from the two organs. This was also the case after g-irradiation for 4 days at the very low dose rate of 44 mGy per day. We conclude that erythrocytes from mouse spleen can be used in the flow cytometer-based micronucleus test for detection of the effects of low doses of genotoxic agents, and that this may be of special advantage in cases where the chemical under test, or its metabolites, are specifically distributed to the spleen. Acknowledgements The investigation was supported by the Swedish National Board for Laboratory Animals and by the Nilsson-Ehle Foundation. We thank Kerstin Santesson for skilful technical assistance, and the Department of Radioecology, Swedish University of Agricultural Sciences, for the use of the g-source. The flow cytometer was funded by the Knut and Alice Wallenberg Foundation. References w1x D.J. Kirkland, Genetic toxicology testing requirements. Official and unofficial views from Europe, Environ. Mol. Mutagen. 21 Ž1993. 8–14.

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