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Mouse strain differences in induction of micronuclei by base analogues and nucleosides
Mutation Research, 301 (1993) 45-49
45
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-7992/93/$06.00
MUTLET 00745
M o u s e strain differences in induction of micronuclei by base analogues and nucleosides Sei-ichi Sato a, Haruo Takizawa a and Naomichi Inui b a Toxicology Research Laboratories, Japan Tobacco Inc., 23 Nakogi, Hatano, Kanagawa 257, Japan and b Preclinical Division, Bioiatric Center, The Kitazato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108, Japan
(Received 23 April 1992) (Revisionreceived 17 August 1992) (Accepted 19 August1992)
Keywords: Micronucleusinduction;Base analogue;Strain difference,mice
Summary The frequency of induced micronucleated polychromatic erythrocytes (MNPCEs) was compared in BALB/c, C57BL/6, and D B A / 2 mice after intraperitoneal (i.p.) injection of 5-bromodeoxyuridine (BUdR), 5-fluorodeoxyuridine (FUdR), cytosine arabinoside (Ara-C), 6-mercaptopurine (6-MP), 5bromouracil (5-BU), thymidine (TdR), uridine (UdR), adenosine (AdR) and guanosine (GdR). The experimental procedure was a single i.p. injection followed by harvest at 30 h. The frequency of MNPCEs was significantly increased in all strains by treatment with BUdR, FUdR, Ara-C and 6-MP compared to vehicle control. TdR and UdR induced MNPCEs slightly in BALB/c mice but showed no effect on C57BL/6 and D B A / 2 mice. 5-BU, AdR, and GdR did not increase the frequency of MNPCEs in any mouse strain used. These results suggest that BALB/c mice are more susceptible to induction of MNPCEs by clastogenic base analogues and nucleosides than are C57BL/6 or D B A / 2 mice.
In the micronucleus test, an in vivo short-term cytogenetic test widely used for detecting clastogenic substances (Schmid, 1973, 1975; Heddle, 1973), in- or out-bred mouse strains have been the organisms most widely used (Heddle et al., 1983). It is well known that these strains have different susceptibilities to micronucleus induction (Aeschbacher, 1979, 1986; CSGMT, 1988; Styles et al., 1983; Luke et al., 1988). We have previously reported that differences in induction
Correspondence: Dr. S.-i. Sato, ToxicologyResearch Laboratories, Japan Tobacco Inc., 23 Nakogi, Hatano, Kanagawa 257, Japan.
of micronuclei are evident among some inbred mouse strains (Sato et al., 1987, 1990). In the present study, we use five base analogues and four nucleosides to compare the susceptibility of three inbred mouse strains to micronucleus induction. Material and methods Female B A L B / c Cr, C57BL/6 Cr, and D B A / 2 Cr mice were obtained from Japan SLC Inc. (Shizuoka, Japan). During the acclimatization and experimental periods they were housed three per polypropylene cage with wood chip
46 bedding in a barrier system room controlled at 23 + I°C, 55 + 5% RH, and a 12-h light cycle. The animals were given commercial food and tap water ad libitum. As for chemicals, 5-bromodeoxyuridine (BUdR), 5-fluorodeoxyuridine (FUdR), cytosine arabinoside (Ara-C), 6-mercaptopurine (6-MP), 5-bromouracil (5-BU), and thymidine (TdR) were obtained from Sigma Chemical Co. (Saint Louis, MO, USA); uridine (UdR), adenosine (AdR), and guanosine (GdR) were from P-L Biochemical Inc. (Milwaukee, WI, USA); for the positive control, ethyl methanesulfonate (EMS) was purchased from Nakarai Tesque Inc. (Kyoto, Japan). BUdR, FUdR, Ara-C, TdR, UdR, and EMS were dissolved in distilled water (DW). 6-MP, 5-BU, AdR, and G d R were suspended in a 0.5% aqueous solution of sodium carboxymethylcellulose (0.5% CMC). The chemicals were tested at two dose levels, and each test group consisted of six randomly assigned 10-week-old mice, except the B U d R group, which consisted of nine mice. Each animal received a single intraperitoneal injection in a volume of 10 m l / k g body weight. Mice of the vehicle control group received a single injection of D W or 0.5% CMC intraperitoneally. The mice were killed by cervical dislocation 30 h after treatment. Bone marrow cells from a femur were flushed out with fetal calf serum, centrifuged, smeared on a glass slide, and stained
with 2.5% Giemsa solution. For each mouse, the number of micronucleated polychromatic erythrocytes (MNPCEs) in 1000 PCEs and also the number of PCEs in 1000 erythrocytes were counted. This protocol of a single injection with 30-h harvest was selected after Yamamoto and Kikuchi (1981), who demonstrated that a maximum response was generally observed 30 h after a single treatment, with various chemicals including 6-MP, and in accordance with our previous report (1990). "The frequency of MNPCEs and PCEs was statistically analyzed with the method of Kastenbaum and Bowman (1970) and the t-test, respectively. Results and discussion
The results for BUdR, FUdR, Ara-C, EMS, 6-MP and 5-BU are summarized in Fig. 1. The potent mutagens BUdR, FUdR, Ara-C, 6-MP, and EMS increased the frequency of MNPCEs in all three strains ( p <0.01), but 5-BU did not induce MNPCEs in any. Generally, the order of susceptibility of these three strains was B A L B / c > C 5 7 B L / 6 > D B A / 2 , which order was also shown with EMS, the alkylating clastogen used as the positive control. The results for TdR, UdR, AdR, and G d R are summarized in Table 1. T d R and U d R increased the frequency of MNPCEs significantly in the high dose group only of B A L B / c mice ( p < 0.05), and not in any group of
TABLE 1 FREQUENCY OF MNPCEs INDUCED BY THYMIDINE (TdR), URIDINE (UdR), ADENOSINE (AdR), AND GUANOSINE (GdR) IN MOUSE BONE MARROW, BASED ON AN EXAMINATION OF AT LEAST 1000 PCEs Compound DW TdR UdR 0.5% CMC AdR GdR
Dose (mg/kg)
Number of mice
BALB/c %mean
(SD)
C57BL/6 %mean
(SD)
DBA/2 %mean
(SD)
370 740 370 740
6 6 6 6 6
0.24 0.33 0.43 0.33 0.46
(0.08) (0.08) (0.05) * (0.08) (0.05) *
0.22 0.28 0.35 0.30 0.35
(0.09) (0.08) (0.05) (0.09) (0.05)
0.18 0.23 0.22 0.18 0.23
(0.12) (0.10) (0.04) (0.04) (0.14)
370 740 370 740
6 6 6 6 6
0.25 0.30 0.33 0.27 0.32
(0.12) (0.09) (0.12) (0.12) (0.10)
0.24 0.20 0.20 0.20 0.22
(0.09) (0.09) (0.06) (0.06) (0.15)
0.15 0.15 0.17 0.15 0.18
(0.08) (0.05) (0.06) (0.05) (0.08)
* Significantlydifferent from vehicle control group (p < 0.05).
47
C57BL/6 or D B A / 2 mice. AdR and GdR did not induce MNPCEs in any strain used. The frequencies of PCEs are shown in Table 2. FUdR decreased the percentage of PCEs in all strains, but the other chemicals generally showed no marked influence on bone marrow cell proliferation. Thus, an order of strain susceptibility for bone marrow cytotoxicity by these chemicals was not shown in this study. The present results show strain differences in the induction of micronuclei by the base analogues and nucleosides used. Styles et al. (1983) showed strain differences among C57BL/6, BALBc/CBA hybrid, and C3H/C57 hybrid strains treated with cyclophosphamide and hexamethylphosphoramide. Aeschbacher (1986) showed that except for M S / A e mice, BALB/c mice were most sensitive among six strains to micronucleus induction by two alkylating agents.
The M S / A e mouse was originally selected as a mutagen-sensitive strain (Aeschbacher et al., 1979), and showed higher susceptibility than various other strains to as many as 17 different mutagens (Aeschbacher et al., 1979; Aeschbacher, 1986; Hayashi et al., 1982). The mechanism for the elevated sensitivity of the M S / A e strain is not well understood. Hayashi et al. (1982) and Sutou and Sato (1990) showed that this characteristic might be related to DNA repair capacity and be genetically controlled. The CSGMT (1988) studied strain differences in the micronucleus test with various classes of micronucleus inducers, i.e., colchicine, EMS, 7,12-dimethylbenz[a]anthracene, ethylnitrosourea, 6-MP, and potassium chromate. They used four mouse strains commonly used in this test in Japan. All four strains reacted positively to all chemicals tested in the order M S / A e > BDF 1 > d d Y = CD-1. Sato et al.
TABLE 2 F R E Q U E N C Y O F PCEs IN BONE M A R R O W , BASED ON AN E X A M I N A T I O N O F AT LEAST 1000 E R Y T H R O C Y T E S Compound
DW TdR UdR 0.5% CMC AdR GdR DW BUdR FUdR Ara-C EMS 0.5% CMC 6-MP 5-BU
Dose (mg/kg)
Number of mice
BALB/c %mean
C57BL/6 (SD)
DBA/2
%mean
(SD)
%mean
(SD)
48.8 53.2 52.8 49.7 54.0
(7.5) (6.7) (11.2) (9.2) (11.2)
52.8 62.3 55.1 65.8 59.8
(8.9) (6.1) (14.0) (9.4) * (6.3)
370 740 370 740
6 6 6 6 6
52.0 52.3 48.3 50.4 54.5
(6.9) (5.1) (9.1) (6.3) (7.5)
370 740 370 740
6 6 6 6 6
53.8 40.7 43.2 35.9 34.1
(5.1) (9.4) (6.2) (12.2) (10.4)
* ** ** **
44.9 43.6 47.3 39.4 32.3
(10.4) (10.4) (6.7) (7.4) (9.4)
51.5 59.3 58.4 47.1 39.7
(10.9) (7.9) (11.8) (8.1) (6.5) *
500 1000 500 1000 5 10 30 60
12 9 9 6 6 6 6 6 6
49.1 49.9 40.0 17.0 15.3 40.9 47.0 51.8 51.0
(6.3) (4.7) (5.7) * (2.1) ** (2.1) ** (6.7) (9.9) (8.1) (15.1)
44.7 42.5 34.3 18.6 20.7 49.8 44.2 52.3 49.7
(8.8) (4.6) (7.3) * (4.6) * * (9.9) * * (14.0) (9.6) (10.4) (8.8)
51.3 53.2 45.4 25.3 20.8 48.7 52.2 63.8 57.3
(4.7) (1.2) (8.1) (3.1) * * (3.2) * * (4.5) (9.6) (5.9) ** (14.6)
25 50 25 50
6 6 6 6 6
53.8 45.8 37.6 50.7 47.4
(5.1) (4.2) * (6.0) ** (5.1) (6.8)
44.9 43.6 30.3 37.3 38.2
(10.4) (17.3) (9.7) * (8.0) (7.1)
51.5 50.9 33.1 49.3 49.0
(10.9) (7.9) (12.2) * (8.10 (2.4)
*'* * Significantly different from vehicle control group ( p < 0.05, p < 0.01).
48 [] BALB/c
2t
0
BUdR
0
~ 2] I
500 1000 Dose (mg/kg)
D
Ara-C
FUdR
0
at PA DBA/2 C57BL/6
500 1000 Dose (mg/kg)
EMS
3'
0
0
5 Dose
10
0
(mg/kg)
30 60 Dose (mg/kg)
2"
41
6-MP
5-BU
(UDS) in mouse prespermiogenic cells was altered strain dependently. In their results C57BL/ 6 mice exhibited the highest UDS activity while B A L B / c showed the lowest. This lower sensitivity of BALB/c mice in the UDS assay seemed to suggest a lower D N A repair activity in BALB/c. The present and previous studies (Sato et al., 1987, 1990) showed that B A L B / c mice were more sensitive to induction of micronuclei than two other inbred mouse strains. This phenomenon was shown by some base analogues, nucleosides, PAHs, alkylating agents, a spindle poison, and other miscellaneous chemicals. This tendency might be related to a lower rate of D N A repair and also to metabolic activation which are genetically controlled. Cell kinetics may also contribute to apparent strain differences, if the sampling time at which the optimal yield of micronucleated PCEs is found differs among strains. Such studies are the subject of a future report. References
z
21
0
**
0
~ 25 50 Dose (mg/kg)
o [ - I0 h
25 50 Dose (mg/kg)
Fig. l. Frequency of MNPCEs for 5-bromodeoxyuridine (BUdR), 5-fluorodeoxyuridine (FUdR), cytosine arabinoside (Are-C), ethyl methanesulfonate (EMS), 6-mercaptopurine (6MP), and 5-bromouracil (5-BU) in BALB/c, C57BL/6, and DBA/2 mice. *, * *: Significantly different from vehicle control group (p < 0.05, p .< 0.01).
(1987) and Raj and Katz (1983) reported that micronucleus induction and metabolic activation of polycyclic aromatic hydrocarbons (PAHs) were closely correlated in several mouse strains. Some mutagens in some other in vivo short-term assays also affected strains differently. Dragani et al. (1981, 1983) studied sister-chromatid exchange (SCE) induction in bone marrow cells using several mouse strains. They indicated that D B A / 2 mice were most sensitive to SCE induction with cyclophosphamide and urethane. Lee and Suzuki (1981) demonstrated that methyl methanesulfonate-induced unscheduled D N A synthesis
Aeschbacher, H.U. (1986) Rates of micronudei induction in different mouse strains, Mutation Res., 164, 109-115. Aeschbacher, H.U., D. Gottwick, H. Meier and A.W. Poot (1979) Mutagen-sensitive strain of mice, Mutation Res., 59, 301-304. Collaborative Study Group for the Micronucleus Test (CSGMT) (1988) Strain difference in the micronucleus test, Mutation Res., 204, 307-316. Dragani, T.A., A. Zunino and G. Sozzi (1981) Differences in sister chromatid exchange (SCE) induction in vivo by cyclophosphamide in murine strains, Carcinogenesis, 2, 219222. Dragani, T.A., G. Sozzi and G.D. Porte (1983) Comparison of urethane-induced sister-chromatid exchange in various murine strains, and the effect of enzyme inducers, Mutation Res., 121, 233-239. Hayashi, M., T. Sofuni and M. Ishidate Jr. (1982) High sensitivity in micronucleus induction of a mouse strain (MS), Mutation Res., 105, 253-256. Heddle, J.A. (1973) A rapid in vivo test for chromosomal damage, Mutation Res., 18, 187-190. Heddle, J.A., M. Hite, B. Kirkhart, K. Mavournin, J.T. MacGregor, G.W. Newell and M.F. Salamone (1983) The induction of micronuclei as a measure of genotoxicity: a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 123, 61-118. Kastenbaum, M.A., and K.O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549. Lee, I.P., and K. Suzuki (1981) Differential DNA-repair activity in prespermiogenic cells of various mouse strains, Mutation Res., 80, 201-211.
49 Luke, C.A., R.R. Tice and R.T. Drew (1988) The effect of exposure regimen and duration on benzene-induced bone marrow damage in mice; II. Strain comparisons involving B3C6F1, C57B1/6 and D B A / 2 male mice, Mutation Res., 203, 272-295. Raj, A.S., and M. Katz (1983) Inhibitory effect of 7,8-benzoflavone on DMBA- and BaP-induced bone marrow micronuclei in mouse, Mutation Res., 110, 337-342. Sato, S., H. Kitajima, S. Konishi, H. Takizawa and N. Inui (1987) Mouse strain difference in the induction of micronuclei by polycyclic aromatic hydrocarbons, Mutation Res., 192, 185-189. Sato, S., H. Takizawa and N. Inui (1990) A comparison of micronucleus induction in 3 mouse strains with representative clastogens, Toxicol. Lett., 52, 215-220. Schmid, W. (1973) Chemical mutagen testing on in vivo somatic mammalian cells, Agents Actions, 3, 77-85.
Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Styles, J.A., C.R. Richardson and B. Burlinson (1983) A comparison of the incidence of micronuclei in blood and bone marrow in 3 strains of mouse dosed with cyciophosphamide or hexamethylphosphoramide (HMPA), Mutation Res., 122, 143-147. Sutou, S., and S. Sato (1~)90) Maternal effect of micronucleus induction in M S / A e mice, Environ. Mol. Mutagen., 15, 125-130. Yamamoto, K.I., and Y. Kikuchi (1981) Studies on micronuclei time response and on the effects of multiple treatment of mutagens on induction of micronuclei, Mutation Res., 90, 163-173.
Communicated by S.M. Galloway
45
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-7992/93/$06.00
MUTLET 00745
M o u s e strain differences in induction of micronuclei by base analogues and nucleosides Sei-ichi Sato a, Haruo Takizawa a and Naomichi Inui b a Toxicology Research Laboratories, Japan Tobacco Inc., 23 Nakogi, Hatano, Kanagawa 257, Japan and b Preclinical Division, Bioiatric Center, The Kitazato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108, Japan
(Received 23 April 1992) (Revisionreceived 17 August 1992) (Accepted 19 August1992)
Keywords: Micronucleusinduction;Base analogue;Strain difference,mice
Summary The frequency of induced micronucleated polychromatic erythrocytes (MNPCEs) was compared in BALB/c, C57BL/6, and D B A / 2 mice after intraperitoneal (i.p.) injection of 5-bromodeoxyuridine (BUdR), 5-fluorodeoxyuridine (FUdR), cytosine arabinoside (Ara-C), 6-mercaptopurine (6-MP), 5bromouracil (5-BU), thymidine (TdR), uridine (UdR), adenosine (AdR) and guanosine (GdR). The experimental procedure was a single i.p. injection followed by harvest at 30 h. The frequency of MNPCEs was significantly increased in all strains by treatment with BUdR, FUdR, Ara-C and 6-MP compared to vehicle control. TdR and UdR induced MNPCEs slightly in BALB/c mice but showed no effect on C57BL/6 and D B A / 2 mice. 5-BU, AdR, and GdR did not increase the frequency of MNPCEs in any mouse strain used. These results suggest that BALB/c mice are more susceptible to induction of MNPCEs by clastogenic base analogues and nucleosides than are C57BL/6 or D B A / 2 mice.
In the micronucleus test, an in vivo short-term cytogenetic test widely used for detecting clastogenic substances (Schmid, 1973, 1975; Heddle, 1973), in- or out-bred mouse strains have been the organisms most widely used (Heddle et al., 1983). It is well known that these strains have different susceptibilities to micronucleus induction (Aeschbacher, 1979, 1986; CSGMT, 1988; Styles et al., 1983; Luke et al., 1988). We have previously reported that differences in induction
Correspondence: Dr. S.-i. Sato, ToxicologyResearch Laboratories, Japan Tobacco Inc., 23 Nakogi, Hatano, Kanagawa 257, Japan.
of micronuclei are evident among some inbred mouse strains (Sato et al., 1987, 1990). In the present study, we use five base analogues and four nucleosides to compare the susceptibility of three inbred mouse strains to micronucleus induction. Material and methods Female B A L B / c Cr, C57BL/6 Cr, and D B A / 2 Cr mice were obtained from Japan SLC Inc. (Shizuoka, Japan). During the acclimatization and experimental periods they were housed three per polypropylene cage with wood chip
46 bedding in a barrier system room controlled at 23 + I°C, 55 + 5% RH, and a 12-h light cycle. The animals were given commercial food and tap water ad libitum. As for chemicals, 5-bromodeoxyuridine (BUdR), 5-fluorodeoxyuridine (FUdR), cytosine arabinoside (Ara-C), 6-mercaptopurine (6-MP), 5-bromouracil (5-BU), and thymidine (TdR) were obtained from Sigma Chemical Co. (Saint Louis, MO, USA); uridine (UdR), adenosine (AdR), and guanosine (GdR) were from P-L Biochemical Inc. (Milwaukee, WI, USA); for the positive control, ethyl methanesulfonate (EMS) was purchased from Nakarai Tesque Inc. (Kyoto, Japan). BUdR, FUdR, Ara-C, TdR, UdR, and EMS were dissolved in distilled water (DW). 6-MP, 5-BU, AdR, and G d R were suspended in a 0.5% aqueous solution of sodium carboxymethylcellulose (0.5% CMC). The chemicals were tested at two dose levels, and each test group consisted of six randomly assigned 10-week-old mice, except the B U d R group, which consisted of nine mice. Each animal received a single intraperitoneal injection in a volume of 10 m l / k g body weight. Mice of the vehicle control group received a single injection of D W or 0.5% CMC intraperitoneally. The mice were killed by cervical dislocation 30 h after treatment. Bone marrow cells from a femur were flushed out with fetal calf serum, centrifuged, smeared on a glass slide, and stained
with 2.5% Giemsa solution. For each mouse, the number of micronucleated polychromatic erythrocytes (MNPCEs) in 1000 PCEs and also the number of PCEs in 1000 erythrocytes were counted. This protocol of a single injection with 30-h harvest was selected after Yamamoto and Kikuchi (1981), who demonstrated that a maximum response was generally observed 30 h after a single treatment, with various chemicals including 6-MP, and in accordance with our previous report (1990). "The frequency of MNPCEs and PCEs was statistically analyzed with the method of Kastenbaum and Bowman (1970) and the t-test, respectively. Results and discussion
The results for BUdR, FUdR, Ara-C, EMS, 6-MP and 5-BU are summarized in Fig. 1. The potent mutagens BUdR, FUdR, Ara-C, 6-MP, and EMS increased the frequency of MNPCEs in all three strains ( p <0.01), but 5-BU did not induce MNPCEs in any. Generally, the order of susceptibility of these three strains was B A L B / c > C 5 7 B L / 6 > D B A / 2 , which order was also shown with EMS, the alkylating clastogen used as the positive control. The results for TdR, UdR, AdR, and G d R are summarized in Table 1. T d R and U d R increased the frequency of MNPCEs significantly in the high dose group only of B A L B / c mice ( p < 0.05), and not in any group of
TABLE 1 FREQUENCY OF MNPCEs INDUCED BY THYMIDINE (TdR), URIDINE (UdR), ADENOSINE (AdR), AND GUANOSINE (GdR) IN MOUSE BONE MARROW, BASED ON AN EXAMINATION OF AT LEAST 1000 PCEs Compound DW TdR UdR 0.5% CMC AdR GdR
Dose (mg/kg)
Number of mice
BALB/c %mean
(SD)
C57BL/6 %mean
(SD)
DBA/2 %mean
(SD)
370 740 370 740
6 6 6 6 6
0.24 0.33 0.43 0.33 0.46
(0.08) (0.08) (0.05) * (0.08) (0.05) *
0.22 0.28 0.35 0.30 0.35
(0.09) (0.08) (0.05) (0.09) (0.05)
0.18 0.23 0.22 0.18 0.23
(0.12) (0.10) (0.04) (0.04) (0.14)
370 740 370 740
6 6 6 6 6
0.25 0.30 0.33 0.27 0.32
(0.12) (0.09) (0.12) (0.12) (0.10)
0.24 0.20 0.20 0.20 0.22
(0.09) (0.09) (0.06) (0.06) (0.15)
0.15 0.15 0.17 0.15 0.18
(0.08) (0.05) (0.06) (0.05) (0.08)
* Significantlydifferent from vehicle control group (p < 0.05).
47
C57BL/6 or D B A / 2 mice. AdR and GdR did not induce MNPCEs in any strain used. The frequencies of PCEs are shown in Table 2. FUdR decreased the percentage of PCEs in all strains, but the other chemicals generally showed no marked influence on bone marrow cell proliferation. Thus, an order of strain susceptibility for bone marrow cytotoxicity by these chemicals was not shown in this study. The present results show strain differences in the induction of micronuclei by the base analogues and nucleosides used. Styles et al. (1983) showed strain differences among C57BL/6, BALBc/CBA hybrid, and C3H/C57 hybrid strains treated with cyclophosphamide and hexamethylphosphoramide. Aeschbacher (1986) showed that except for M S / A e mice, BALB/c mice were most sensitive among six strains to micronucleus induction by two alkylating agents.
The M S / A e mouse was originally selected as a mutagen-sensitive strain (Aeschbacher et al., 1979), and showed higher susceptibility than various other strains to as many as 17 different mutagens (Aeschbacher et al., 1979; Aeschbacher, 1986; Hayashi et al., 1982). The mechanism for the elevated sensitivity of the M S / A e strain is not well understood. Hayashi et al. (1982) and Sutou and Sato (1990) showed that this characteristic might be related to DNA repair capacity and be genetically controlled. The CSGMT (1988) studied strain differences in the micronucleus test with various classes of micronucleus inducers, i.e., colchicine, EMS, 7,12-dimethylbenz[a]anthracene, ethylnitrosourea, 6-MP, and potassium chromate. They used four mouse strains commonly used in this test in Japan. All four strains reacted positively to all chemicals tested in the order M S / A e > BDF 1 > d d Y = CD-1. Sato et al.
TABLE 2 F R E Q U E N C Y O F PCEs IN BONE M A R R O W , BASED ON AN E X A M I N A T I O N O F AT LEAST 1000 E R Y T H R O C Y T E S Compound
DW TdR UdR 0.5% CMC AdR GdR DW BUdR FUdR Ara-C EMS 0.5% CMC 6-MP 5-BU
Dose (mg/kg)
Number of mice
BALB/c %mean
C57BL/6 (SD)
DBA/2
%mean
(SD)
%mean
(SD)
48.8 53.2 52.8 49.7 54.0
(7.5) (6.7) (11.2) (9.2) (11.2)
52.8 62.3 55.1 65.8 59.8
(8.9) (6.1) (14.0) (9.4) * (6.3)
370 740 370 740
6 6 6 6 6
52.0 52.3 48.3 50.4 54.5
(6.9) (5.1) (9.1) (6.3) (7.5)
370 740 370 740
6 6 6 6 6
53.8 40.7 43.2 35.9 34.1
(5.1) (9.4) (6.2) (12.2) (10.4)
* ** ** **
44.9 43.6 47.3 39.4 32.3
(10.4) (10.4) (6.7) (7.4) (9.4)
51.5 59.3 58.4 47.1 39.7
(10.9) (7.9) (11.8) (8.1) (6.5) *
500 1000 500 1000 5 10 30 60
12 9 9 6 6 6 6 6 6
49.1 49.9 40.0 17.0 15.3 40.9 47.0 51.8 51.0
(6.3) (4.7) (5.7) * (2.1) ** (2.1) ** (6.7) (9.9) (8.1) (15.1)
44.7 42.5 34.3 18.6 20.7 49.8 44.2 52.3 49.7
(8.8) (4.6) (7.3) * (4.6) * * (9.9) * * (14.0) (9.6) (10.4) (8.8)
51.3 53.2 45.4 25.3 20.8 48.7 52.2 63.8 57.3
(4.7) (1.2) (8.1) (3.1) * * (3.2) * * (4.5) (9.6) (5.9) ** (14.6)
25 50 25 50
6 6 6 6 6
53.8 45.8 37.6 50.7 47.4
(5.1) (4.2) * (6.0) ** (5.1) (6.8)
44.9 43.6 30.3 37.3 38.2
(10.4) (17.3) (9.7) * (8.0) (7.1)
51.5 50.9 33.1 49.3 49.0
(10.9) (7.9) (12.2) * (8.10 (2.4)
*'* * Significantly different from vehicle control group ( p < 0.05, p < 0.01).
48 [] BALB/c
2t
0
BUdR
0
~ 2] I
500 1000 Dose (mg/kg)
D
Ara-C
FUdR
0
at PA DBA/2 C57BL/6
500 1000 Dose (mg/kg)
EMS
3'
0
0
5 Dose
10
0
(mg/kg)
30 60 Dose (mg/kg)
2"
41
6-MP
5-BU
(UDS) in mouse prespermiogenic cells was altered strain dependently. In their results C57BL/ 6 mice exhibited the highest UDS activity while B A L B / c showed the lowest. This lower sensitivity of BALB/c mice in the UDS assay seemed to suggest a lower D N A repair activity in BALB/c. The present and previous studies (Sato et al., 1987, 1990) showed that B A L B / c mice were more sensitive to induction of micronuclei than two other inbred mouse strains. This phenomenon was shown by some base analogues, nucleosides, PAHs, alkylating agents, a spindle poison, and other miscellaneous chemicals. This tendency might be related to a lower rate of D N A repair and also to metabolic activation which are genetically controlled. Cell kinetics may also contribute to apparent strain differences, if the sampling time at which the optimal yield of micronucleated PCEs is found differs among strains. Such studies are the subject of a future report. References
z
21
0
**
0
~ 25 50 Dose (mg/kg)
o [ - I0 h
25 50 Dose (mg/kg)
Fig. l. Frequency of MNPCEs for 5-bromodeoxyuridine (BUdR), 5-fluorodeoxyuridine (FUdR), cytosine arabinoside (Are-C), ethyl methanesulfonate (EMS), 6-mercaptopurine (6MP), and 5-bromouracil (5-BU) in BALB/c, C57BL/6, and DBA/2 mice. *, * *: Significantly different from vehicle control group (p < 0.05, p .< 0.01).
(1987) and Raj and Katz (1983) reported that micronucleus induction and metabolic activation of polycyclic aromatic hydrocarbons (PAHs) were closely correlated in several mouse strains. Some mutagens in some other in vivo short-term assays also affected strains differently. Dragani et al. (1981, 1983) studied sister-chromatid exchange (SCE) induction in bone marrow cells using several mouse strains. They indicated that D B A / 2 mice were most sensitive to SCE induction with cyclophosphamide and urethane. Lee and Suzuki (1981) demonstrated that methyl methanesulfonate-induced unscheduled D N A synthesis
Aeschbacher, H.U. (1986) Rates of micronudei induction in different mouse strains, Mutation Res., 164, 109-115. Aeschbacher, H.U., D. Gottwick, H. Meier and A.W. Poot (1979) Mutagen-sensitive strain of mice, Mutation Res., 59, 301-304. Collaborative Study Group for the Micronucleus Test (CSGMT) (1988) Strain difference in the micronucleus test, Mutation Res., 204, 307-316. Dragani, T.A., A. Zunino and G. Sozzi (1981) Differences in sister chromatid exchange (SCE) induction in vivo by cyclophosphamide in murine strains, Carcinogenesis, 2, 219222. Dragani, T.A., G. Sozzi and G.D. Porte (1983) Comparison of urethane-induced sister-chromatid exchange in various murine strains, and the effect of enzyme inducers, Mutation Res., 121, 233-239. Hayashi, M., T. Sofuni and M. Ishidate Jr. (1982) High sensitivity in micronucleus induction of a mouse strain (MS), Mutation Res., 105, 253-256. Heddle, J.A. (1973) A rapid in vivo test for chromosomal damage, Mutation Res., 18, 187-190. Heddle, J.A., M. Hite, B. Kirkhart, K. Mavournin, J.T. MacGregor, G.W. Newell and M.F. Salamone (1983) The induction of micronuclei as a measure of genotoxicity: a report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 123, 61-118. Kastenbaum, M.A., and K.O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549. Lee, I.P., and K. Suzuki (1981) Differential DNA-repair activity in prespermiogenic cells of various mouse strains, Mutation Res., 80, 201-211.
49 Luke, C.A., R.R. Tice and R.T. Drew (1988) The effect of exposure regimen and duration on benzene-induced bone marrow damage in mice; II. Strain comparisons involving B3C6F1, C57B1/6 and D B A / 2 male mice, Mutation Res., 203, 272-295. Raj, A.S., and M. Katz (1983) Inhibitory effect of 7,8-benzoflavone on DMBA- and BaP-induced bone marrow micronuclei in mouse, Mutation Res., 110, 337-342. Sato, S., H. Kitajima, S. Konishi, H. Takizawa and N. Inui (1987) Mouse strain difference in the induction of micronuclei by polycyclic aromatic hydrocarbons, Mutation Res., 192, 185-189. Sato, S., H. Takizawa and N. Inui (1990) A comparison of micronucleus induction in 3 mouse strains with representative clastogens, Toxicol. Lett., 52, 215-220. Schmid, W. (1973) Chemical mutagen testing on in vivo somatic mammalian cells, Agents Actions, 3, 77-85.
Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Styles, J.A., C.R. Richardson and B. Burlinson (1983) A comparison of the incidence of micronuclei in blood and bone marrow in 3 strains of mouse dosed with cyciophosphamide or hexamethylphosphoramide (HMPA), Mutation Res., 122, 143-147. Sutou, S., and S. Sato (1~)90) Maternal effect of micronucleus induction in M S / A e mice, Environ. Mol. Mutagen., 15, 125-130. Yamamoto, K.I., and Y. Kikuchi (1981) Studies on micronuclei time response and on the effects of multiple treatment of mutagens on induction of micronuclei, Mutation Res., 90, 163-173.
Communicated by S.M. Galloway