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Comparison of micronucleus formation in mouse bone marrow and spleen
Mutation Research, 292 (1993) 63-67
63
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1161/93/$06.00
MUTENV 08876
Comparison of micronucleus formation in mouse bone marrow and spleen A. Martelli, G. Brambilla Campart, F. Benvenuto, A.M. Fresu and G. Brambilla Institute of Pharmacology, University of Genoa, 1-16132 Genoa, Italy
(Received 2 December 1992) (Revision received2 March 1993) (Accepted 2 March 1993)
Keywords: Micronucleatedpolychromaticerythrocytes;Spleen; Bone marrow
Summary The frequencies of micronucleated erythrocytes were compared in bone marrow and spleen of mice killed 24 and 48 h after a single i.p. dose of one directly acting carcinogen, N-nitroso-Noethylurea (NEU, 100 mg/kg), and two indirectly acting ones, N-nitrosodimethylamine (NDMA, 50 m g / k g ) and 7,12-dimethylbenz[a]anthracene (7,12-DMBA, 50 mg/kg). The t r e a t e d / c o n t r o l ratio of the incidence of micronucleated polychromatic erythrocytes (MnPCEs) was similar in the two tissues for N D M A at 24 h (sampling at 48 h was precluded by toxicity) and for 7,12-DMBA at 48 h, while it was higher in the bone marrow than in the spleen for N E U at both 24 and 48 h and for 7,12-DMBA at 24 h. Concerning micronucleated normochromatic erythrocytes (MnNCEs), their frequency in both tissues was always lower than that of MnPCEs; however, while in bone marrow a marked increase in their incidence was induced by N E U and 7,12-DMBA, any response was absent in spleen, thus suggesting that this organ does not sequester micronucleated erythrocytes. These results already indicate that the spleen is not a useful alternative to the bone marrow in the mieronucleus assay. Moreover, counting of MnPCEs in the spleen is made more difficult and prone to error by the low frequency of PCEs, and by their greater toxicity-induced reduction. This last effect was found to be enhanced by the use of old mice.
The micronucleus assay on mouse bone marrow polychromatic erythrocytes (PCEs), originally developed by Schmid (1975), is probably the most frequently used in vivo short-term genotoxicity test. Published data on chemicals tested by this assay have been reviewed and summarized by Mavournin et al. (1990). Correspondence: Dr. Antonietta Martelli, Istituto di Farmacologia dell'UniversitY,Viale Benedetto XV 2, 1-16132Genoa, Italy. Tel. 39 10 3538801, 39 10 3538849.
Krishna et al. (1990) recently proposed the spleen as a source of PCEs, and found that in CD-1 mice treated with N-nitrosodimethylamine (NDMA) the spleen uniformly yielded a higher proportion of micronucleated PCEs (MnPCEs) compared to bone marrow. Since the spleen is easier to remove than bone marrow and mice can survive after splenectomy, we deemed it of interest to verify if the micronucleus assay on spleen PCEs could represent an advantageous and valid alternative to the assay on bone marrow PCEs. In this paper we report the results obtained from a
64
comparative evaluation of the frequencies of MnPCEs in spleen and bone marrow of mice treated with a directly acting agent, N-nitroso-N-ethylurea (NEU), and two indirectly acting agents, 7,12-dimethylbenz[a]anthracene (7,12-DMBA) and N-nitrosodimethylamine (NDMA). NEU was tested not only in young but also in old mice, in order to evaluate the influence of age on the erythropoietic activity of the spleen. 4-Acetylaminofluorene (4-AAF), a weak/non-carcinogenic isomer of 2-acetylaminofluorene which has been found to behave as a bacterial mutagen in vitro and in contrast as a non-genotoxic agent in vivo (Ashby, 1983; Ashby et al., 1986), has also been tested to examine whether the end-point considered may identify 'false-positive' predictions made by in vitro assays.
Materials and methods
Chemicals NDMA was purchased from E. Merck (Darmstadt, Germany); NEU from Serva Feinbiochemica (Heidelberg, Germany); 7,12-DMBA from Fluka (Buchs, Switzerland); 4-AAF from MTM Research Chemicals (Eastgate, Morecambe, UK); fetal bovine serum from Boehringer (Mannheim, Germany). All the other chemicals, reagent grade, were obtained from E. Merck. Animals Male Swiss albino mice, 5-6 and 20-22 weeks old, were purchased from Nossan s.r.1. (Correzzana, Italy). During the study mice had free access to tap water and rodent chow S. Morini (S. Polo d'Enza, Italy).
TABLE 1 FREQUENCIES OF MICRONUCLEI IN PCEs AND NCEs OF SPLEEN AND BONE MARROW OF MICE TREATED WITH 7,12-DMBA, NEU, NMDA AND 4-AA_F Each dose group consisted of four mice; the values are the means of individual mouse data. Treatment
Time of death (h)
Number of PCEs observed
Frequency of PCEs (% + SD)
Control
24 48
4093 4182
19.4+ 6.4 17.1_+ 9.0
7,12-DMBA 50mg/kgi.p. NEU 100mg/kgi.p. NDMA 50 mg/kg i.p. 4-AAF 1000 mg/kg p.o.
24 48 24 48 24 48 24 48
4192 47 4342 2117 4112
24 48 24 48 24 48 24 48 24 48
4129 4571 4372 4224 4282 4197 4285
Frequency of MnPCEs (%,~ _+SD)
MnPCEs treated/control
Number of NCEs observed
Frequency of MnNCEs (%o + SD)
19327 24273
0.54_+0.62 0.10-+0.12
Spleen
a
4155 4104
1.22+ 0.94 0.75_+ 1.49
17.7-+ 5.5 1.1-+ 0.4 12.9+ 3.8 8.8_+ 9.9 29.8+_ 6.5 toxic 31.7_+ 9.4 42.8_+15.2
7.21-+ 3 . 6 5 36.46-+70.94 32.67+12.15 1.97_+ 2 . 6 7 7.60+_ 6 . 9 2
5.91_+ 2.99 47.62+95.23 26.77-+ 9.96 2.29_+ 3.77 6.23+ 5.67
20916 4164 31010 18213 10107
0.33+_0.45 0 0.47-+0.39 0.30_+0.41 0.16+0.31
0.47_+ 0 . 5 5 0.24_+ 0.49
0.39+_ 0.45 0.33_+ 0.65
10099 6537
0.30-+0.35 0
47.6-+23.5 48.0_+11.5 54.7_+ 8.1 15.7_+ 6.6 37.4_+ 6.0 20.8+ 9.6 62.9-+18.6 toxic 53.6_+19.1 60.9_+ 4.6
0.96_+ 1.11 0.59-t- 0.73 9.40-+ 9.30 27.47-+19.74 53.32_+34.47 9.66-+ 8.60 5.93-+ 3 . 9 4
10.67-+ 9.35 46.56-+33.46 55.54-+35.90 16.37_+14.57 6.17+_ 4.10
6325 5347 3754 26102 7399 20407 3046
0 0.42_+0.84 0.24_+0.49 2.71_+3.14 2.48_+ 1.66 2.81_+2.22 0.88+1.31
1.96_+ 3 . 2 9 0.49-+ 0 . 9 9
2.04_+ 3.43 0.84_+ 1.68
4683 2750
0 0.43_+0.86
Bone marrow
Control 7,12-DMBA 50 mg/kg i.p. NEU 100mg/kgi.p. NDMA 50 mg/kg i.p. 4-AAF 1000 mg/kg p.o.
4200 4243
a Total number of PCEs encountered in 16 slides.
65
Treatments NDMA (50 mg/kg) and NEU (100 mg/kg), dissolved in physiological saline, and 7,12-DMBA (50 mg/kg) dissolved in corn oil, were injected i.p.; 4-AAF was suspended in 1% carboxymethylcellulose in water and administered by oral gavage at the dose of 1000 mg/kg. The doses were administered in a volume of 0.01 ml/g. Controls received i.p. the same amount of corn oil. Isolation of PCEs from bone marrow and spleen Mice were killed by cervical dislocation 24 and 48 h after treatment as suggested by Mavournin et al. (1990). Bone marrow cells were isolated from both femurs by flushing the shafts with fetal bovine serum, and spleen cells were obtained by shaking the spleen after incision of the capsule in sterile Ca 2÷, Mg2+-free Hank's solution. Slides were prepared by centrifuging aliquots of a 1:5 diluted cell suspension at 500 rpm for 10 min in a Cytospin 2 (Shandon) centrifuge. The smears were air-dried, stained according to Cole et al. (1979), and examined by light microscopy to determine
both the incidence of MnPCEs per 1000 PCEs per animal, and the frequency of PCEs. Taking into account the hemocatheretic activity of the spleen, the same parameters were evaluated in normochromatic erythrocytes (NCEs). Results
Table 1 lists the frequencies of MnPCEs and micronucleated NCE (MnNCEs) observed in spleen and bone marrow of mice treated with a single dose of 7,12-DMBA, NEU, NDMA, and 4-AAF. The dose levels employed of 7,12-DMBA (Mizuhashi et al., 1989) and NEU (Sutou et al., 1989) have been previously found to be subtoxic but capable of inducing a positive response in the micronucleus assay; the one of NDMA was the same as used by Krishna et al. (1990); that of 4-AAF was derived from Ashby et al. (1986). A comparison of data shows that the treated/control ratio of MnPCEs was similar in the two tissues for NDMA and the 48-h sampling time of 7,12-DMBA, while for NEU and the 24-h sam-
TABLE 2 COMPARISON IN YOUNG AND OLD MICE OF THE FREQUENCIES OF MICRONUCLEI INDUCED BY NEU IN PCEs AND NCEs OF SPLEEN AND BONE MARROW Each dose group consisted of four mice; the values are the means of individual mouse data. Treatment
Time of death (h)
Age of mice
Number of PCEs observed
Frequency of PCEs (% -I-SD)
Frequency of MnPCEs (%o + SD)
Number of NCEs observed
Frequency of MnNCEs (%o + SD)
24 48 24 48 24 48 24 48
young
4093 4182 4172 4034 4342 2117 4069 219
19.4+ 17.1_+ 18.0_+ 16.5_+ 12.9_+ 8.8+ 8.4_+ 1.0_+
1.22+ 0.94 0.75_+ 1.49 1.42_+ 0.95 0.75_+ 0.96 32.67_+12.15 1.97_+ 2.67 31.13_+15.21 0
19327 24273 21578 21066 31010 18213 47106 13037
0.54_+0.62 0.10_+0.12 0.12_+0.17 0.14_+0.16 0.47_+0.39 0.30_+0.41 0.18_+0.17 0
24 48 24 48 24 48 24 48
young
4129 4571 4145 4239 4282 4197 4251 4038
47.6+23.5 48.0+ 11.5 40.7+18.1 39.1+ 4.2 37.4+ 6.0 20.8+ 9.6 38.6+ 2.5 10.8+ 5.1
0.96+ 1.11 0.59+ 0.73 1.15+ 0.88 0.74+ 0.94 53.32+34.47 9.66+ 8.60 45.83+47.04 3.20+ 3.95
6325 5347 7971 6739 7399 20407 6848 39459
0 0.42+0.84 0.14-1-0.27 0.14-1-0.28 2.48+1.66 2.81+2.22 2.56+4.27 0.94+0.83
Spleen
Control
NEU 100mg/kgi.p.
old young old
6.4 9.0 6.9 3.3 3.8 9.9 2.0 1.6
Bone marrow
Control
NEU 100 mg/kg i.p.
old young old
66 pling time of 7,12-DMBA the same ratio was higher in the bone marrow than in the spleen. Concerning NDMA, toxicity-induced lethality precluded any evaluation at 48 h. Negative results were obtained with 4-AAF in both tissues. The frequency of PCEs was constantly lower in the spleen than in the bone marrow. Moreover, at 48 h the toxicity-induced reduction of PCE frequencies for both 7,12-DMBA and NEU was greater in the spleen than in the bone marrow. 4-AAF produced, presumably because of its mitogenic activity, an increase in PCE frequency in both organs. The incidences of MnNCEs were markedly lower than those of MnPCEs in both tissues. However, while any increase over controls was absent in the spleen, a definite increment of MnNCEs was observed in the bone marrow at both sampling times with N E U and at 48 h with 7,12-DMBA. In order to examine the influence of age on both the erythropoietic activity of the spleen and the frequency of micronucleated cells, the clastogenic effect of N E U was evaluated in young and old mice. The results shown in Table 2 indicate that no meaningful differences exist between young and old animals in the absence of treatment, while the old mice exhibited after exposure to NEU a greater sensitivity to the toxic effect. A reduction of PCE frequencies was observed in both tissues being particularly evident at 48 h, but, due to the lower number of PCEs in the spleen, this toxic effect was more evident in this organ. Discussion The present study was undertaken to verify whether the spleen may represent, as suggested by Krishna et al. (1990), an additional or alternative source of PCEs in the micronucleus test, usually carried out on bone marrow cells. In fact in the adult mouse both spleen and bone marrow are hemopoietic organs; moreover, Krishna et al. (1990) formulated the hypothesis that, the spleen being the main site of erythrocyte destruction, it is possible that MnPCEs, which arise from the bone marrow, may be sequestered in the spleen. Our results demonstrate that counting of Mn-
PCEs in the spleen of Swiss albino mice is not a useful alternative to the bone marrow because of the following reasons. First of all in our experiments the frequency of MnPCEs, depending on the carcinogen, was equal to or lower than that observed in the bone marrow, thus excluding that the spleen sequesters micronucleated erythrocytes. Moreover, considering that the spleen is the main site of erythrocyte destruction, it could be expected that an increase of MnNCEs occurred due to the fact that PCEs released from bone marrow mature in the peripheral blood into NCEs (Mavournin et al., 1990). In contrast, the frequency of MnNCEs in the spleen was consistently lower than in the bone marrow. A double interpretation of this phenomenon could be advanced: (1) MnPCEs produced in the bone marrow are presumably destroyed in the same organ with no releasing of damaged cells in the general circulation; (2) the spleen sequesters and destroys these cells at an extremely fast rate. In any case the clastogenic effect of the tested compound cannot be monitored by the increase of the frequency of MnNCEs. In the second place, the number of PCEs per 1000 erythrocytes is always markedly lower in the spleen than in the bone marrow; in our experiment the ratio bone marrow P C E s / s p l e e n PCEs was about 2.5. Moreover, the spleen seems to be more sensitive to toxic effects than bone marrow; this implies a further reduction of PCEs. For instance, in mice treated with 7,12-DMBA we found at 48 h only 47 spleen PCEs in 16 slides from four mice. We have provocatively reported this finding in order to underline that in similar cases the significance of MnPCE frequency is questionable; the mean value of 36.5%c. MnPCEs reported in Table 1 comes from two MnPCEs found in one slide of a single mouse. This unfavorable event may be amplified by the use of old animals. Coming specifically to a comparison of our results with those published by Krishna et al. (1990), the main discrepancy is that in the spleen of mice treated with 50 m g / k g N D M A we did not observe a frequency of MnPCEs higher than in the bone marrow, but of the same order. At 24 h this frequency was lower in both tissues than that reported by Krishna et al. (1990); the evalua-
67
tion at subsequent times was precluded by the death of the majority of mice. At any rate we think that it is unwise to assume, as Krishna et al. (1990) did, that the spleen may be a useful additional source of PCEs on the basis of effects produced by N D M A dosages 2.5-5-fold higher than its LDs0, particularly if it is taken into account that present guidelines indicate the use in this test of a maximal dose between LDs0 and LD 100Taking into account that mice of a Swiss albino strain were used in this study whereas Krishna et al. (1990) employed mice of the CD-1 strain, our findings should be conservatively interpreted as indicating that the spleen of the former has a lower capability of sequestering micronucleated erythrocytes than that of the latter. Consequently the only reasonable conclusion which can at present be drawn is that micronucleus formation in mouse spleen cannot be recommended as a routine alternative procedure to the bone marrow micronucleus assay.
Acknowledgement This work was supported by funds from M.U.R.S.T. (quota 40%), Italy.
References Ashby, J. (1983) The unique role of rodents in the detection of possible human carcinogens and mutagens, Mutation Res., 115, 177-213. Ashby, J., P.A. Lefevre, B. Burlinson and D. Beije (1986) Potent mitogenic activity of 4-acetylaminofluorene to the rat liver, Mutation Res., 172, 271-279. Cole, R.J., N.A. Taylor, J. Cole and C.F. Arlett (1979) Transplacental effects of chemical mutagens detected by the micronucleus test, Nature, 277, 317-318. Krishna, G., M.L. Kropko and J.C. Theiss (1990) Dimethylnitrosamine-induced micronucleus formation in mouse bone marrow and spleen, Mutation Res., 242, 345-351. 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. Mizuhashi, F., K. Murata, T. Kitagaki, T. Nishitomi, A. Hashimoto and A. Kido (1989) Administration-route-related difference in the micronucleus test with 7,12-dimethylbenz[a]anthracene, Mutation Res., 223, 357-360. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Sutou, S., S. Sato, S. Hitotsumachi and Y. Kimura (1989) Administration-route-related difference in the micronucleus test with N-ethyl-N-nitrosourea, Mutation Res., 223, 377-381.
63
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1161/93/$06.00
MUTENV 08876
Comparison of micronucleus formation in mouse bone marrow and spleen A. Martelli, G. Brambilla Campart, F. Benvenuto, A.M. Fresu and G. Brambilla Institute of Pharmacology, University of Genoa, 1-16132 Genoa, Italy
(Received 2 December 1992) (Revision received2 March 1993) (Accepted 2 March 1993)
Keywords: Micronucleatedpolychromaticerythrocytes;Spleen; Bone marrow
Summary The frequencies of micronucleated erythrocytes were compared in bone marrow and spleen of mice killed 24 and 48 h after a single i.p. dose of one directly acting carcinogen, N-nitroso-Noethylurea (NEU, 100 mg/kg), and two indirectly acting ones, N-nitrosodimethylamine (NDMA, 50 m g / k g ) and 7,12-dimethylbenz[a]anthracene (7,12-DMBA, 50 mg/kg). The t r e a t e d / c o n t r o l ratio of the incidence of micronucleated polychromatic erythrocytes (MnPCEs) was similar in the two tissues for N D M A at 24 h (sampling at 48 h was precluded by toxicity) and for 7,12-DMBA at 48 h, while it was higher in the bone marrow than in the spleen for N E U at both 24 and 48 h and for 7,12-DMBA at 24 h. Concerning micronucleated normochromatic erythrocytes (MnNCEs), their frequency in both tissues was always lower than that of MnPCEs; however, while in bone marrow a marked increase in their incidence was induced by N E U and 7,12-DMBA, any response was absent in spleen, thus suggesting that this organ does not sequester micronucleated erythrocytes. These results already indicate that the spleen is not a useful alternative to the bone marrow in the mieronucleus assay. Moreover, counting of MnPCEs in the spleen is made more difficult and prone to error by the low frequency of PCEs, and by their greater toxicity-induced reduction. This last effect was found to be enhanced by the use of old mice.
The micronucleus assay on mouse bone marrow polychromatic erythrocytes (PCEs), originally developed by Schmid (1975), is probably the most frequently used in vivo short-term genotoxicity test. Published data on chemicals tested by this assay have been reviewed and summarized by Mavournin et al. (1990). Correspondence: Dr. Antonietta Martelli, Istituto di Farmacologia dell'UniversitY,Viale Benedetto XV 2, 1-16132Genoa, Italy. Tel. 39 10 3538801, 39 10 3538849.
Krishna et al. (1990) recently proposed the spleen as a source of PCEs, and found that in CD-1 mice treated with N-nitrosodimethylamine (NDMA) the spleen uniformly yielded a higher proportion of micronucleated PCEs (MnPCEs) compared to bone marrow. Since the spleen is easier to remove than bone marrow and mice can survive after splenectomy, we deemed it of interest to verify if the micronucleus assay on spleen PCEs could represent an advantageous and valid alternative to the assay on bone marrow PCEs. In this paper we report the results obtained from a
64
comparative evaluation of the frequencies of MnPCEs in spleen and bone marrow of mice treated with a directly acting agent, N-nitroso-N-ethylurea (NEU), and two indirectly acting agents, 7,12-dimethylbenz[a]anthracene (7,12-DMBA) and N-nitrosodimethylamine (NDMA). NEU was tested not only in young but also in old mice, in order to evaluate the influence of age on the erythropoietic activity of the spleen. 4-Acetylaminofluorene (4-AAF), a weak/non-carcinogenic isomer of 2-acetylaminofluorene which has been found to behave as a bacterial mutagen in vitro and in contrast as a non-genotoxic agent in vivo (Ashby, 1983; Ashby et al., 1986), has also been tested to examine whether the end-point considered may identify 'false-positive' predictions made by in vitro assays.
Materials and methods
Chemicals NDMA was purchased from E. Merck (Darmstadt, Germany); NEU from Serva Feinbiochemica (Heidelberg, Germany); 7,12-DMBA from Fluka (Buchs, Switzerland); 4-AAF from MTM Research Chemicals (Eastgate, Morecambe, UK); fetal bovine serum from Boehringer (Mannheim, Germany). All the other chemicals, reagent grade, were obtained from E. Merck. Animals Male Swiss albino mice, 5-6 and 20-22 weeks old, were purchased from Nossan s.r.1. (Correzzana, Italy). During the study mice had free access to tap water and rodent chow S. Morini (S. Polo d'Enza, Italy).
TABLE 1 FREQUENCIES OF MICRONUCLEI IN PCEs AND NCEs OF SPLEEN AND BONE MARROW OF MICE TREATED WITH 7,12-DMBA, NEU, NMDA AND 4-AA_F Each dose group consisted of four mice; the values are the means of individual mouse data. Treatment
Time of death (h)
Number of PCEs observed
Frequency of PCEs (% + SD)
Control
24 48
4093 4182
19.4+ 6.4 17.1_+ 9.0
7,12-DMBA 50mg/kgi.p. NEU 100mg/kgi.p. NDMA 50 mg/kg i.p. 4-AAF 1000 mg/kg p.o.
24 48 24 48 24 48 24 48
4192 47 4342 2117 4112
24 48 24 48 24 48 24 48 24 48
4129 4571 4372 4224 4282 4197 4285
Frequency of MnPCEs (%,~ _+SD)
MnPCEs treated/control
Number of NCEs observed
Frequency of MnNCEs (%o + SD)
19327 24273
0.54_+0.62 0.10-+0.12
Spleen
a
4155 4104
1.22+ 0.94 0.75_+ 1.49
17.7-+ 5.5 1.1-+ 0.4 12.9+ 3.8 8.8_+ 9.9 29.8+_ 6.5 toxic 31.7_+ 9.4 42.8_+15.2
7.21-+ 3 . 6 5 36.46-+70.94 32.67+12.15 1.97_+ 2 . 6 7 7.60+_ 6 . 9 2
5.91_+ 2.99 47.62+95.23 26.77-+ 9.96 2.29_+ 3.77 6.23+ 5.67
20916 4164 31010 18213 10107
0.33+_0.45 0 0.47-+0.39 0.30_+0.41 0.16+0.31
0.47_+ 0 . 5 5 0.24_+ 0.49
0.39+_ 0.45 0.33_+ 0.65
10099 6537
0.30-+0.35 0
47.6-+23.5 48.0_+11.5 54.7_+ 8.1 15.7_+ 6.6 37.4_+ 6.0 20.8+ 9.6 62.9-+18.6 toxic 53.6_+19.1 60.9_+ 4.6
0.96_+ 1.11 0.59-t- 0.73 9.40-+ 9.30 27.47-+19.74 53.32_+34.47 9.66-+ 8.60 5.93-+ 3 . 9 4
10.67-+ 9.35 46.56-+33.46 55.54-+35.90 16.37_+14.57 6.17+_ 4.10
6325 5347 3754 26102 7399 20407 3046
0 0.42_+0.84 0.24_+0.49 2.71_+3.14 2.48_+ 1.66 2.81_+2.22 0.88+1.31
1.96_+ 3 . 2 9 0.49-+ 0 . 9 9
2.04_+ 3.43 0.84_+ 1.68
4683 2750
0 0.43_+0.86
Bone marrow
Control 7,12-DMBA 50 mg/kg i.p. NEU 100mg/kgi.p. NDMA 50 mg/kg i.p. 4-AAF 1000 mg/kg p.o.
4200 4243
a Total number of PCEs encountered in 16 slides.
65
Treatments NDMA (50 mg/kg) and NEU (100 mg/kg), dissolved in physiological saline, and 7,12-DMBA (50 mg/kg) dissolved in corn oil, were injected i.p.; 4-AAF was suspended in 1% carboxymethylcellulose in water and administered by oral gavage at the dose of 1000 mg/kg. The doses were administered in a volume of 0.01 ml/g. Controls received i.p. the same amount of corn oil. Isolation of PCEs from bone marrow and spleen Mice were killed by cervical dislocation 24 and 48 h after treatment as suggested by Mavournin et al. (1990). Bone marrow cells were isolated from both femurs by flushing the shafts with fetal bovine serum, and spleen cells were obtained by shaking the spleen after incision of the capsule in sterile Ca 2÷, Mg2+-free Hank's solution. Slides were prepared by centrifuging aliquots of a 1:5 diluted cell suspension at 500 rpm for 10 min in a Cytospin 2 (Shandon) centrifuge. The smears were air-dried, stained according to Cole et al. (1979), and examined by light microscopy to determine
both the incidence of MnPCEs per 1000 PCEs per animal, and the frequency of PCEs. Taking into account the hemocatheretic activity of the spleen, the same parameters were evaluated in normochromatic erythrocytes (NCEs). Results
Table 1 lists the frequencies of MnPCEs and micronucleated NCE (MnNCEs) observed in spleen and bone marrow of mice treated with a single dose of 7,12-DMBA, NEU, NDMA, and 4-AAF. The dose levels employed of 7,12-DMBA (Mizuhashi et al., 1989) and NEU (Sutou et al., 1989) have been previously found to be subtoxic but capable of inducing a positive response in the micronucleus assay; the one of NDMA was the same as used by Krishna et al. (1990); that of 4-AAF was derived from Ashby et al. (1986). A comparison of data shows that the treated/control ratio of MnPCEs was similar in the two tissues for NDMA and the 48-h sampling time of 7,12-DMBA, while for NEU and the 24-h sam-
TABLE 2 COMPARISON IN YOUNG AND OLD MICE OF THE FREQUENCIES OF MICRONUCLEI INDUCED BY NEU IN PCEs AND NCEs OF SPLEEN AND BONE MARROW Each dose group consisted of four mice; the values are the means of individual mouse data. Treatment
Time of death (h)
Age of mice
Number of PCEs observed
Frequency of PCEs (% -I-SD)
Frequency of MnPCEs (%o + SD)
Number of NCEs observed
Frequency of MnNCEs (%o + SD)
24 48 24 48 24 48 24 48
young
4093 4182 4172 4034 4342 2117 4069 219
19.4+ 17.1_+ 18.0_+ 16.5_+ 12.9_+ 8.8+ 8.4_+ 1.0_+
1.22+ 0.94 0.75_+ 1.49 1.42_+ 0.95 0.75_+ 0.96 32.67_+12.15 1.97_+ 2.67 31.13_+15.21 0
19327 24273 21578 21066 31010 18213 47106 13037
0.54_+0.62 0.10_+0.12 0.12_+0.17 0.14_+0.16 0.47_+0.39 0.30_+0.41 0.18_+0.17 0
24 48 24 48 24 48 24 48
young
4129 4571 4145 4239 4282 4197 4251 4038
47.6+23.5 48.0+ 11.5 40.7+18.1 39.1+ 4.2 37.4+ 6.0 20.8+ 9.6 38.6+ 2.5 10.8+ 5.1
0.96+ 1.11 0.59+ 0.73 1.15+ 0.88 0.74+ 0.94 53.32+34.47 9.66+ 8.60 45.83+47.04 3.20+ 3.95
6325 5347 7971 6739 7399 20407 6848 39459
0 0.42+0.84 0.14-1-0.27 0.14-1-0.28 2.48+1.66 2.81+2.22 2.56+4.27 0.94+0.83
Spleen
Control
NEU 100mg/kgi.p.
old young old
6.4 9.0 6.9 3.3 3.8 9.9 2.0 1.6
Bone marrow
Control
NEU 100 mg/kg i.p.
old young old
66 pling time of 7,12-DMBA the same ratio was higher in the bone marrow than in the spleen. Concerning NDMA, toxicity-induced lethality precluded any evaluation at 48 h. Negative results were obtained with 4-AAF in both tissues. The frequency of PCEs was constantly lower in the spleen than in the bone marrow. Moreover, at 48 h the toxicity-induced reduction of PCE frequencies for both 7,12-DMBA and NEU was greater in the spleen than in the bone marrow. 4-AAF produced, presumably because of its mitogenic activity, an increase in PCE frequency in both organs. The incidences of MnNCEs were markedly lower than those of MnPCEs in both tissues. However, while any increase over controls was absent in the spleen, a definite increment of MnNCEs was observed in the bone marrow at both sampling times with N E U and at 48 h with 7,12-DMBA. In order to examine the influence of age on both the erythropoietic activity of the spleen and the frequency of micronucleated cells, the clastogenic effect of N E U was evaluated in young and old mice. The results shown in Table 2 indicate that no meaningful differences exist between young and old animals in the absence of treatment, while the old mice exhibited after exposure to NEU a greater sensitivity to the toxic effect. A reduction of PCE frequencies was observed in both tissues being particularly evident at 48 h, but, due to the lower number of PCEs in the spleen, this toxic effect was more evident in this organ. Discussion The present study was undertaken to verify whether the spleen may represent, as suggested by Krishna et al. (1990), an additional or alternative source of PCEs in the micronucleus test, usually carried out on bone marrow cells. In fact in the adult mouse both spleen and bone marrow are hemopoietic organs; moreover, Krishna et al. (1990) formulated the hypothesis that, the spleen being the main site of erythrocyte destruction, it is possible that MnPCEs, which arise from the bone marrow, may be sequestered in the spleen. Our results demonstrate that counting of Mn-
PCEs in the spleen of Swiss albino mice is not a useful alternative to the bone marrow because of the following reasons. First of all in our experiments the frequency of MnPCEs, depending on the carcinogen, was equal to or lower than that observed in the bone marrow, thus excluding that the spleen sequesters micronucleated erythrocytes. Moreover, considering that the spleen is the main site of erythrocyte destruction, it could be expected that an increase of MnNCEs occurred due to the fact that PCEs released from bone marrow mature in the peripheral blood into NCEs (Mavournin et al., 1990). In contrast, the frequency of MnNCEs in the spleen was consistently lower than in the bone marrow. A double interpretation of this phenomenon could be advanced: (1) MnPCEs produced in the bone marrow are presumably destroyed in the same organ with no releasing of damaged cells in the general circulation; (2) the spleen sequesters and destroys these cells at an extremely fast rate. In any case the clastogenic effect of the tested compound cannot be monitored by the increase of the frequency of MnNCEs. In the second place, the number of PCEs per 1000 erythrocytes is always markedly lower in the spleen than in the bone marrow; in our experiment the ratio bone marrow P C E s / s p l e e n PCEs was about 2.5. Moreover, the spleen seems to be more sensitive to toxic effects than bone marrow; this implies a further reduction of PCEs. For instance, in mice treated with 7,12-DMBA we found at 48 h only 47 spleen PCEs in 16 slides from four mice. We have provocatively reported this finding in order to underline that in similar cases the significance of MnPCE frequency is questionable; the mean value of 36.5%c. MnPCEs reported in Table 1 comes from two MnPCEs found in one slide of a single mouse. This unfavorable event may be amplified by the use of old animals. Coming specifically to a comparison of our results with those published by Krishna et al. (1990), the main discrepancy is that in the spleen of mice treated with 50 m g / k g N D M A we did not observe a frequency of MnPCEs higher than in the bone marrow, but of the same order. At 24 h this frequency was lower in both tissues than that reported by Krishna et al. (1990); the evalua-
67
tion at subsequent times was precluded by the death of the majority of mice. At any rate we think that it is unwise to assume, as Krishna et al. (1990) did, that the spleen may be a useful additional source of PCEs on the basis of effects produced by N D M A dosages 2.5-5-fold higher than its LDs0, particularly if it is taken into account that present guidelines indicate the use in this test of a maximal dose between LDs0 and LD 100Taking into account that mice of a Swiss albino strain were used in this study whereas Krishna et al. (1990) employed mice of the CD-1 strain, our findings should be conservatively interpreted as indicating that the spleen of the former has a lower capability of sequestering micronucleated erythrocytes than that of the latter. Consequently the only reasonable conclusion which can at present be drawn is that micronucleus formation in mouse spleen cannot be recommended as a routine alternative procedure to the bone marrow micronucleus assay.
Acknowledgement This work was supported by funds from M.U.R.S.T. (quota 40%), Italy.
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