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High body temperature induces micronuclei in mouse bone marrow
Mutation Research 390 Ž1997. 79–83
High body temperature induces micronuclei in mouse bone marrow S. Asanami ) , K. Shimono Naruto Research Institute, Otsuka Pharmaceutical Factory, Inc. 115 Muya-cho, Naruto, Tokushima 772, Japan Received 5 June 1996; revised 8 September 1996; accepted 26 November 1996
Abstract The mouse micronucleus test was conducted to investigate the effect of high body temperature on micronucleus induction. Groups of 10 male ddY mice were exposed to 308C for 1, 3 or 6 h, 378C for 0.5, 1, 2, 3 or 4 h, and 408C for 1 or 2 h. Bone marrow cells were sampled 24 h after heat exposure. Exposure of mice to 378C for 3 or 4 h and 408C for 1 or 2 h raised body temperature to approximately 40.58C and produced statistically significant increases in micronucleated polychromatic erythrocyte frequencies Ž8.1 " 4.5, 6.0 " 2.1, 5.3 " 3.3, 7.5 " 2.9‰, respectively; control frequencies, 2.0 " 1.1‰.. In addition, about 25% of the induced micronuclei were relatively large Ždiameter of micronucleus G 1r4 diameter of cytoplasm.. These results suggest that body temperatures of 39.58C or higher for more than 30 min induce micronuclei in bone marrow cells, and one possible mechanism is disturbance of the mitotic apparatus. Keywords: Micronucleus; Body temperature; Mouse; Bone marrow; Hyperthermia
1. Introduction The micronucleus test, an in vivo short-term screening test developed by Schmid w1,2x and Heddle w3x, is widely used to detect clastogens and microtubule poisons w4,5x. It is therefore important to clarify test conditions that might affect the results. Heat treatment exerts numerous effects on mammalian cells w6x, including chromosome aberrations w7x, alterations in chromosomal proteins w8x, membrane damage w9x, inhibition of DNA repair w10x, and
) Corresponding author. Tel.: 81 886-85-1151, ext. 406; Fax: 81 886-86-8176.
damage to the mitotic apparatus w7x. Recently, Cai and Jiang w11x reported that mild hyperthermia Ž418C for 1 h. can induce adaptation to cytogenetic damage caused by subsequent X irradiation. A possible mechanism for the induction of chromosome aberrations by heat treatment, indicated by many papers is the inhibition of spindle function. Coss et al. w12x showed that treatment of dividing cells at 45.58C Ž5 to 15 min. disassembled the spindle and disrupted both the contractile ring and the midbody-cytoplasmic bridge complex to varying degrees depending on the duration of the heat treatment. Yamada et al. w13x reported that polyploidy Žpredominantly tetraploidy. increased when cells were cultured at over 40.38C compared with cells cultured at 378C, and
1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 0 0 2 - 5
80
S. Asanami, K. Shimonor Mutation Research 390 (1997) 79–83
structural chromosome aberrations Žbreaks, stickiness, fragmentation, etc.. were observed at 41.58C. Furthermore, they showed that the transformed cells were more temperature-sensitive than normal cells with respect to chromosomal aberrations. Chrisman and Baumgartner w14x demonstrated a statistically significant increase in micronucleated erythrocytes ŽMNPCEs. in bone marrow cells of mice subjected to hyperthermia Ž35 " 18C for 3 h., and King and Wild w15x also reported that whole-body hyperthermia Ž35–368C for 20–32 h. induced a significant increase in MNPCEs in mouse bone marrow along with elevated rectal temperature. They did not comment about the possible mechanisms of micronuclei induction. We investigated the effects of high body temperature on the induction of micronuclei in mice and speculated on possible mechanisms on the basis of micronucleus size.
2. Materials and methods 2.1. Animals Six-week-old male ddY mice ŽSPF. were purchased from Japan, SLC, Inc. and acclimated for 10 days. They were housed in plastic cages in an airconditioned room and given food and water ad libitum. They were used at 9–12 weeks of age.
HDEN Co., Japan.; the probe was carefully inserted into the rectum to a depth of 1.5 cm and the temperature read about 10 s later. Bone marrow cells were sampled 24 h after heat treatment. Slides were fixed with methanol and stained with acridine orange w16x. After coding all slides, 1000 polychromatic erythrocytes ŽPCEs. were scored for each animal. The ratio of PCEs to total erythrocytes, based on 200 PCEs per animal, was also recorded. In a second microscopic examination, the size of each micronucleus was evaluated according to the morphological criteria of Yamamoto and Kikuchi w17x, i.e., 10 or 20 MNPCE per animal in the control and the treatment groups that showed a significant increase in MNPCE frequency were selected, the diameters of cytoplasm Ž D . and micronucleus Ž d . in each cell were compared directly under the microscope, and it was determined whether or not Ž d . exceeded Ž Dr4.. Kastenbaum and Bowman’s w18x table was used to test for significance. Animals kept at room temperature Ž23 " 28C. served as the control.
3. Results Fig. 1 shows the time course of rectal temperatures. The mean rectal temperature of the groups subjected to ambient temperature above 308C in-
2.2. Chemicals Colchicine ŽCASRN. 64-86-8, Wako pure chemical industries Ltd., Tokyo, Japan. and mitomycin C ŽCASRN. 50-07-7, Kyowa hakko kogyo Co. Ltd., Tokyo, Japan. were dissolved in distilled water to give treatment solutions of 10 mlrkg body weight. 2.3. Test procedure Groups of 10 male ddY mice were exposed to 308C at 29.0 " 2.6% relative humidity ŽRH. for 1, 3 or 6 h, 378C at 29.3 " 1.5% RH for 0.5, 1, 2, 3 or 4 h, and 408C at 28.7 " 2.1% RH for 1 or 2 h, using a hot air sterilizer ŽLCS-120, TABAI ESPEC Co., Japan.. Rectal temperature Ž8C. was taken with a thermistor thermometer ŽMGA3-219, NIHON KO-
Fig. 1. Time course of rectal temperature Ž8C. in mice. Mice were exposed to 308C for 1, 3, or 6 h, 378C for 0.5, 1, 2, 3, or 4 h, or 408C for 1 or 2 h. The solid lines indicate heat exposure and the dotted lines indicate room temperature. Error bars represent standard deviation of the mean value.
S. Asanami, K. Shimonor Mutation Research 390 (1997) 79–83
81
Table 1 Effects of heat exposure on induction of micronuclei in mouse bone marrow cells Treatment
n
a
Rectal temperature Žmean " SD, 8C.
% PCEs
MNPCEs based on 1000 PCEs assessed per animal
Pre-treatment
Žmean " SD.
Individual animal data
Mean " SD
54.6 " 6.6 45.1 " 8.6 49.8 " 10.1 49.9 " 9.4
3, 2, 1, 3, 3, 1, 2, 0, 2, 3 1, 2, 1, 3, 0, 2, 0, 1, 2, 1 2, 2, 3, 3, 2, 4, 1, 3, 1, 3 0, 1, 2, 3, 2, 1, 2, 2, 4, 1
2.0 " 1.1 1.3 " 0.9 2.4 " 1.0 1.8 " 1.1
Post-treatment b
Control 308C y -1 h 308C–3 h 308C–6 h
10 10 10 10
37.28 " 0.55 37.90 " 0.30 37.70 " 0.28 37.54 " 0.32
36.83 " 0.60 37.93 " 0.37 37.12 " 0.35 37.51 " 0.36
378C–0.5 h 378C–1 h 378C–2 h 378C–3 h 378C–4 h
10 10 10 10 10
37.78 " 0.51 37.73 " 0.31 37.38 " 0.33 37.89 " 0.59 37.40 " 0.30
39.07 " 0.23 38.96 " 0.25 39.09 " 0.44 40.63 " 0.47 40.42 " 0.78
47.0 " 7.8 41.3 " 7.7 48.4 " 6.5 44.2 " 10.6 47.7 " 10.2
4, 1, 2, 1, 3, 2, 2, 1, 0, 1 4, 3, 2, 2, 1, 4, 1, 0, 4, 2 2, 2, 3, 3, 1, 3, 2, 3, 4, 4 14, 5, 1, 13, 6, 4, 10, 10, 13, 5 5, 6, 7, 3, 7, 8, 6, 8, 2, 8
1.7 " 1.2 2.3 " 1.4 2.7 " 1.0 8.1 " 4.5 6.0 " 2.1
408C–1 h 408C–2 h
9c 10
37.46 " 0.35 37.95 " 0.45
40.21 " 0.80 40.50 " 0.60
47.6 " 5.8 46.6 " 7.2
2, 3, –, 2, 7, 2, 10, 7, 5, 10 8, 4, 13, 9, 9, 3, 8, 7, 5, 9
5.3 " 3.3 7.5 " 2.9
d
)) ))
)) ))
)) a
p - 0.01 by table of Kastenbaum and Bowman w18x. At room temperature Ž23 " 28C.; b Data at 24 h later; c One animals died;
creased to approximately 40.58C. The temperature returned to normal within an hour of the animals’ return to room temperature. Mean rectal temperature before heat exposure was 37.6 " 0.58C Ž n s 110., and the range from 9 : 00 a.m. to 8 : 00 p.m. was 36.5 " 0.58C to 37.9 " 0.38C Ž n s 10.. Table 1 shows the effects of heat treatment on rectal temperature and the induction of micronuclei. Exposure of mice to 378C for 3 or 4 h and to 408C for 1 or 2 h raised body temperature to approximately 40.58C and produced a statistically significant
d
n s 10.
increase in MNPCEs Ž8.1 " 4.5, 6.0 " 2.1, 5.3 " 3.3, 7.5 " 2.9‰, respectively.. The relationship between rectal temperature and the induction of micronuclei is shown in Fig. 2. The MNPCE frequency increased at body temperatures of 39.58C or higher. Table 2 shows the frequency in treated mice of MNPCEs containing relatively large Ž d G 1r4 D . micronuclei. In the treatment group, the frequency of relatively large micronuclei significantly increased Ž p - 0.01. and was approximately 25% of the total MNPCEs. The frequency of large micronuclei in the
Table 2 Frequency of micronucleated erythrocytes containing relatively large micronuclei Ž d G Dr4. Treatment
n
a
Control 378C–3 h 378C–4 h 408C–1 h 408C–2 h Colchicine b Mitomycin C
c
10 10 10 9 10 3 5
MNPCEs Ž‰.
2.1 " 1.1 8.1 " 4.5 6.0 " 2.1 5.3 " 3.3 7.5 " 2.9 10.7 " 1.5 36.4 " 7.6
Large MNPCEs based on 20 MNPCEs counted per animal Individual animal data
Mean " SD Ž%.
1, 1, 0, 0, 1, 1, 0, 1, 0, 0 9, 3, 2, 7, 8, 3, 5, 4, 6, 6 5, 5, 5, 2, 4, 7, 6, 5, 6, 6 5, 4, –, 3, 6, 3, 4, 4, 6, 3 4, 3, 7, 6, 5, 4, 4, 4, 5, 6 6, 10, 8 2, 1, 1, 0, 2
5.0 " 1.0 26.5 " 11.6 ) ) 25.5 " 6.9 ) ) 21.1 " 6.0 ) ) 24.0 " 6.1 ) ) 40.0 " 10.0 ) ) 6.0 " 4.2
D, d, the diameters of cytoplasm Ž D . and micronuclei Ž d .. 10 MNPCEs counted per animals; b Double dosing i.p. of 0.5 mgrkg and 24 h sampling; sampling.
a
c
Single dosing i.p. of 2 mgrkg and 24 h
82
S. Asanami, K. Shimonor Mutation Research 390 (1997) 79–83
Fig. 2. The relationship between rectal temperature Ž8C. and micronucleus frequency Ž‰. after heat exposure. Error bars represent standard deviation of the mean value.
control group and the colchicine-, and mitomycin C-treated groups were in agreement with the data of Yamamoto and Kikuchi w17x.
4. Discussion There are few reports on the relationship between chromosome aberrations and high body temperature, although there are many in vitro reports of the induction of chromosome aberration by heat treatment w12,13x. Here, we demonstrated the effect of high body temperature on the induction of micronuclei in a mammalian system. Exposure of mice to 378C for 3 or 4 h, and to 408C for 1 or 2 h, raised the rectal temperature to approximate 40.58C and produced a significant increase in MNPCEs ŽTable 1.. Chrisman and Baumgartner w14x demonstrated the induction of MNPCEs in mouse bone marrow by hyperthermia Ž35 " 18C for 3 h. without the body temperature data, and King and Wild w15x also reported that whole-body hyperthermia Ž35–368C for 20–32 h. induced a significant increase in MNPCE frequencies in mouse bone marrow with elevated rectal temperature. In contrast, some papers demonstrated that heat treatment alone did not induce chromosome aberrations. Weissenborn and Obe w19x showed that heat alone did not induce structural chromosomal aberrations but they did not count numerical abnormalities as aberrations. We included both structural and numerical chromosomal abnormalities as aberrations. Shen et al. w20x reported that treatment with whole body hyperther-
mia Ž40 " 0.28C for 1 h. alone did not significantly change the frequency of chromatin fragments by flow cytometry in the thymus and bone marrow cells when compared to that in the corresponding tissues of the untreated mice. Because MNPCE frequencies induced by hyperthermia, however, are not very high ŽTable 1., it may be difficult to assess a weak positive response for micronucleus induction by flow cytometry analysis. Concerning the possible mechanisms of chromosome aberration induction in mammalian cells by heat treatment, some investigators have concluded that high temperatures inhibit spindle function in vitro w12,13x. In general, micronuclei induced by spindle poisons are considerably larger than those induced by clastogens w2x. Yamamoto and Kikuchi w17x concluded that it is possible to deduce the action site of micronucleus-inducing agents on the basis of the relative size of the micronuclei, and Tinwell and Ashby suggested that large or crescent shaped micronuclei are strongly indicative of aneugenicity w21x. In this study, a significant Ž p - 0.01. increase of relatively large micronuclei was associated with hyperthermia, suggesting that hyperthermia also disturbs the mitotic apparatus in mammalian cells in vivo. Increased MNPCE frequencies were observed at rectal temperatures of 39.58C or higher ŽFig. 2.. There was no increase in MNPCE frequencies in the 378C–2 h treatment group Žthe mean rectal temperature was 39.09 " 0.448C., but significantly increased MNPCE frequencies were observed in the 378C–3 h treatment group Žrectal temperature, 40.63 " 0.478C; Table 1.. These results suggest that mouse rectal temperatures of over 39.58C for 30 min could induce micronuclei in bone marrow. We therefore speculate that the threshold rectal temperature for micronucleus induction is 39.58C for 30 min. Regarding the duration of hyperthermia, King and Wild w15x reported that no significant increase in the incidence of MNPCEs in bone marrow was observed when mice were exposed to hyperthermia for less than 10 h. We demonstrated here, however, that even short-term hyperthermia Žfor example 408C for 1 h. could induce a significant increase in MNPCE and elevated rectal temperature of approximately 408C or higher, although King and Wild found that the mean rectal temperature of mice subjected to hyperthermia was 39.18C. Coss et al. w12x reported that treatment of
S. Asanami, K. Shimonor Mutation Research 390 (1997) 79–83
dividing CHO cells at 45.58C for 5 to 15 min disassembled the spindle to varying degrees depending on the length of heat exposure, and Hinkley and Samson w22x reported that microtubules in a nonmammalian system rapidly dissociate in vivo at 408C. Several reports have shown that temperatures above 378C will induce the disassembly of microtubules in mammalian systems w23x, and Fiorenza and Mangia w24x demonstrated that abnormal chromosome numbers were observed in mouse oocytes matured at either 39.08C or 40.08C, but not at 37.08C. Thus, it appears that the formation of a functional mitotic spindle can only occur within narrow temperature limits. In conclusion, body temperatures of 39.58C or higher for more than 30 min induced micronuclei in mouse bone marrow cells, and one possible mechanism was the disturbance of the mitotic apparatus. Monitoring body temperature therefore may be advisable when chemicals that give negative results in vitro produce positive results in vivo.
w9x
w10x
w11x
w12x
w13x
w14x
w15x
w16x
w17x
References w18x w1x Schmid, W. Ž1973. Chemical mutagen testing on in vivo somatic mammalian cells, Agents Actions, 3, 77–85. w2x Schmid, W. Ž1975. The micronucleus test, Mutation Res., 31, 9–15. w3x Heddle, J.A. Ž1973. A rapid in vivo test for chromosomal damage. Mutation Res., 18, 187–190. w4x Mavournin, K.H., D.H. Blakey, M.C. Cimino, M.F. Salamone and J.A. Heddle Ž1990. 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. w5x Hayashi, M., R. Tice, J.T. MacGregor, D. Anderson, D.H. Blakey, M. Kirsh-Volders, F.B. Oleson, Jr., F. Pacchierotti, F. Romagna, H. Shimada, S. Sutou and B. Vannier Ž1994. In vivo rodent erythrocyte micronucleus assay, Mutation Res., 312, 293–304. w6x Dewey, W.C. Ž1989. The search for critical cellular targets damaged by heat, Radiat. Res., 120, 191–204. w7x Dewey, W.C., A. Westra, H.H. Miller and H. Nagasawa Ž1971. Heat-induced lethality and chromosomal damage in synchronized Chinese hamster cells treated with 5bromodeoxyuridine, Int. J. Radiat. Biol., 20, 505–520. w8x Tomasovic, S.P., G.N. Turner and W.C. Dewey Ž1978. Ef-
w19x
w20x
w21x
w22x
w23x w24x
83
fect of hyperthermia on nonhistone proteins isolated with DNA, Radiat. Res., 73, 535–552. Yatvin, M.B. Ž1977. The influence of membrane lipid composition and procaine on hyperthermic death of cells, Int. J. Radiat. Biol., 32, 513–521. Jorritsma, J.B.M. and A.W.T. Konings Ž1983. Inhibition of repair of radiation-induced strand breaks by hyperthermia, and its relationship to cell survival after hyperthermia alone, Int. J. Radiat. Biol., 43, 505–516. Cai, L and J. Jiang Ž1995. Mild hyperthermia can induce adaptation to cytogenetic damage caused by subsequent X irradiation, Radiat. Res., 143, 26–33. Coss, R.A., W.C. Dewey and J.R. Bamburg Ž1982. Effects of hyperthermia on dividing Chinese hamster ovary cells and on microtubules in vitro, Cancer Res., 42, 1059–1071. Yamada K., Y. Ichikawa and H. Okumura Ž1989. Effects of high temperatures on chromosomes of normal and transformed human cells, Human Cell, 2, 80–85. Chrisman, C.L. and A.P. Baumgartner Ž1980. Micronuclei in bone–marrow cells of mice subjected to hyperthermia, Mutation Res., 77, 95–97. King, M.T. and D. Wild Ž1983. The mutagenic potential of hyperthermia and fever in mice, Mutation Res., 111, 219– 226. Hayashi, M., T. Sofuni and M. Ishidate, Jr. Ž1983. An application of acridine orange fluorescent staining to the micronucleus test, Mutation Res., 120, 241–247. Yamamoto, K. and Y. Kikuchi Ž1980. A comparison of diameters of micronuclei induced by clastogens and by spindle poisons, Mutation Res., 71, 127–131. Kastenbaum, M.A. and K.O. Bowman Ž1970. Tables for determining the statistical significance of mutation frequencies, Mutation. Res., 9, 527–549. Weissenborn, U. and G. Obe Ž1991. Modification of X-ray induced chromosome aberration frequency by pre- and postirradiation hyperthermia of human peripheral lymphocytes, Int. J. Radiat. Biol., 59, 973–984. Shen, R.N., W.N. Crabtree, B. Wu, P. Young, G.A. Sandison, N.B. Hornback and H. Shidnia Ž1992. A reliable method for quantitating chromatin fragments by flow cytometry to predict the effect of total body irradiation and hyperthermia on mice, Int. J. Radiat. Oncol. Biol. Phys. 24, 139–143. Tinwell, H. and J. Ashby Ž1991. Micronucleus morphology as a means to distinguish aneugens and clastogens in the mouse bone marrow micronucleus assay, Mutagenesis, 6, 193–198. Hinkley, R.E. Jr. and F.E. Samson, Jr. Ž1974. The effects of an elevated temperature, colchicine, and vinblastine on axonal microtubules of the crayfish Ž Procambarus clarkii ., J. Exp. Zool., 188, 321–336. Stephens, R.E. and K.T. Edds Ž1976. Microtubules: structure, chemistry, and function, Physiol. Rev., 56, 709–777. Fiorenza, M.T. and F. Mangia Ž1992. Hyperthermia specifically inhibits bivalent chromosome disjunction in maturing mouse oocytes, Biol. Reprod., 46, 658–664.
High body temperature induces micronuclei in mouse bone marrow S. Asanami ) , K. Shimono Naruto Research Institute, Otsuka Pharmaceutical Factory, Inc. 115 Muya-cho, Naruto, Tokushima 772, Japan Received 5 June 1996; revised 8 September 1996; accepted 26 November 1996
Abstract The mouse micronucleus test was conducted to investigate the effect of high body temperature on micronucleus induction. Groups of 10 male ddY mice were exposed to 308C for 1, 3 or 6 h, 378C for 0.5, 1, 2, 3 or 4 h, and 408C for 1 or 2 h. Bone marrow cells were sampled 24 h after heat exposure. Exposure of mice to 378C for 3 or 4 h and 408C for 1 or 2 h raised body temperature to approximately 40.58C and produced statistically significant increases in micronucleated polychromatic erythrocyte frequencies Ž8.1 " 4.5, 6.0 " 2.1, 5.3 " 3.3, 7.5 " 2.9‰, respectively; control frequencies, 2.0 " 1.1‰.. In addition, about 25% of the induced micronuclei were relatively large Ždiameter of micronucleus G 1r4 diameter of cytoplasm.. These results suggest that body temperatures of 39.58C or higher for more than 30 min induce micronuclei in bone marrow cells, and one possible mechanism is disturbance of the mitotic apparatus. Keywords: Micronucleus; Body temperature; Mouse; Bone marrow; Hyperthermia
1. Introduction The micronucleus test, an in vivo short-term screening test developed by Schmid w1,2x and Heddle w3x, is widely used to detect clastogens and microtubule poisons w4,5x. It is therefore important to clarify test conditions that might affect the results. Heat treatment exerts numerous effects on mammalian cells w6x, including chromosome aberrations w7x, alterations in chromosomal proteins w8x, membrane damage w9x, inhibition of DNA repair w10x, and
) Corresponding author. Tel.: 81 886-85-1151, ext. 406; Fax: 81 886-86-8176.
damage to the mitotic apparatus w7x. Recently, Cai and Jiang w11x reported that mild hyperthermia Ž418C for 1 h. can induce adaptation to cytogenetic damage caused by subsequent X irradiation. A possible mechanism for the induction of chromosome aberrations by heat treatment, indicated by many papers is the inhibition of spindle function. Coss et al. w12x showed that treatment of dividing cells at 45.58C Ž5 to 15 min. disassembled the spindle and disrupted both the contractile ring and the midbody-cytoplasmic bridge complex to varying degrees depending on the duration of the heat treatment. Yamada et al. w13x reported that polyploidy Žpredominantly tetraploidy. increased when cells were cultured at over 40.38C compared with cells cultured at 378C, and
1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 0 0 2 - 5
80
S. Asanami, K. Shimonor Mutation Research 390 (1997) 79–83
structural chromosome aberrations Žbreaks, stickiness, fragmentation, etc.. were observed at 41.58C. Furthermore, they showed that the transformed cells were more temperature-sensitive than normal cells with respect to chromosomal aberrations. Chrisman and Baumgartner w14x demonstrated a statistically significant increase in micronucleated erythrocytes ŽMNPCEs. in bone marrow cells of mice subjected to hyperthermia Ž35 " 18C for 3 h., and King and Wild w15x also reported that whole-body hyperthermia Ž35–368C for 20–32 h. induced a significant increase in MNPCEs in mouse bone marrow along with elevated rectal temperature. They did not comment about the possible mechanisms of micronuclei induction. We investigated the effects of high body temperature on the induction of micronuclei in mice and speculated on possible mechanisms on the basis of micronucleus size.
2. Materials and methods 2.1. Animals Six-week-old male ddY mice ŽSPF. were purchased from Japan, SLC, Inc. and acclimated for 10 days. They were housed in plastic cages in an airconditioned room and given food and water ad libitum. They were used at 9–12 weeks of age.
HDEN Co., Japan.; the probe was carefully inserted into the rectum to a depth of 1.5 cm and the temperature read about 10 s later. Bone marrow cells were sampled 24 h after heat treatment. Slides were fixed with methanol and stained with acridine orange w16x. After coding all slides, 1000 polychromatic erythrocytes ŽPCEs. were scored for each animal. The ratio of PCEs to total erythrocytes, based on 200 PCEs per animal, was also recorded. In a second microscopic examination, the size of each micronucleus was evaluated according to the morphological criteria of Yamamoto and Kikuchi w17x, i.e., 10 or 20 MNPCE per animal in the control and the treatment groups that showed a significant increase in MNPCE frequency were selected, the diameters of cytoplasm Ž D . and micronucleus Ž d . in each cell were compared directly under the microscope, and it was determined whether or not Ž d . exceeded Ž Dr4.. Kastenbaum and Bowman’s w18x table was used to test for significance. Animals kept at room temperature Ž23 " 28C. served as the control.
3. Results Fig. 1 shows the time course of rectal temperatures. The mean rectal temperature of the groups subjected to ambient temperature above 308C in-
2.2. Chemicals Colchicine ŽCASRN. 64-86-8, Wako pure chemical industries Ltd., Tokyo, Japan. and mitomycin C ŽCASRN. 50-07-7, Kyowa hakko kogyo Co. Ltd., Tokyo, Japan. were dissolved in distilled water to give treatment solutions of 10 mlrkg body weight. 2.3. Test procedure Groups of 10 male ddY mice were exposed to 308C at 29.0 " 2.6% relative humidity ŽRH. for 1, 3 or 6 h, 378C at 29.3 " 1.5% RH for 0.5, 1, 2, 3 or 4 h, and 408C at 28.7 " 2.1% RH for 1 or 2 h, using a hot air sterilizer ŽLCS-120, TABAI ESPEC Co., Japan.. Rectal temperature Ž8C. was taken with a thermistor thermometer ŽMGA3-219, NIHON KO-
Fig. 1. Time course of rectal temperature Ž8C. in mice. Mice were exposed to 308C for 1, 3, or 6 h, 378C for 0.5, 1, 2, 3, or 4 h, or 408C for 1 or 2 h. The solid lines indicate heat exposure and the dotted lines indicate room temperature. Error bars represent standard deviation of the mean value.
S. Asanami, K. Shimonor Mutation Research 390 (1997) 79–83
81
Table 1 Effects of heat exposure on induction of micronuclei in mouse bone marrow cells Treatment
n
a
Rectal temperature Žmean " SD, 8C.
% PCEs
MNPCEs based on 1000 PCEs assessed per animal
Pre-treatment
Žmean " SD.
Individual animal data
Mean " SD
54.6 " 6.6 45.1 " 8.6 49.8 " 10.1 49.9 " 9.4
3, 2, 1, 3, 3, 1, 2, 0, 2, 3 1, 2, 1, 3, 0, 2, 0, 1, 2, 1 2, 2, 3, 3, 2, 4, 1, 3, 1, 3 0, 1, 2, 3, 2, 1, 2, 2, 4, 1
2.0 " 1.1 1.3 " 0.9 2.4 " 1.0 1.8 " 1.1
Post-treatment b
Control 308C y -1 h 308C–3 h 308C–6 h
10 10 10 10
37.28 " 0.55 37.90 " 0.30 37.70 " 0.28 37.54 " 0.32
36.83 " 0.60 37.93 " 0.37 37.12 " 0.35 37.51 " 0.36
378C–0.5 h 378C–1 h 378C–2 h 378C–3 h 378C–4 h
10 10 10 10 10
37.78 " 0.51 37.73 " 0.31 37.38 " 0.33 37.89 " 0.59 37.40 " 0.30
39.07 " 0.23 38.96 " 0.25 39.09 " 0.44 40.63 " 0.47 40.42 " 0.78
47.0 " 7.8 41.3 " 7.7 48.4 " 6.5 44.2 " 10.6 47.7 " 10.2
4, 1, 2, 1, 3, 2, 2, 1, 0, 1 4, 3, 2, 2, 1, 4, 1, 0, 4, 2 2, 2, 3, 3, 1, 3, 2, 3, 4, 4 14, 5, 1, 13, 6, 4, 10, 10, 13, 5 5, 6, 7, 3, 7, 8, 6, 8, 2, 8
1.7 " 1.2 2.3 " 1.4 2.7 " 1.0 8.1 " 4.5 6.0 " 2.1
408C–1 h 408C–2 h
9c 10
37.46 " 0.35 37.95 " 0.45
40.21 " 0.80 40.50 " 0.60
47.6 " 5.8 46.6 " 7.2
2, 3, –, 2, 7, 2, 10, 7, 5, 10 8, 4, 13, 9, 9, 3, 8, 7, 5, 9
5.3 " 3.3 7.5 " 2.9
d
)) ))
)) ))
)) a
p - 0.01 by table of Kastenbaum and Bowman w18x. At room temperature Ž23 " 28C.; b Data at 24 h later; c One animals died;
creased to approximately 40.58C. The temperature returned to normal within an hour of the animals’ return to room temperature. Mean rectal temperature before heat exposure was 37.6 " 0.58C Ž n s 110., and the range from 9 : 00 a.m. to 8 : 00 p.m. was 36.5 " 0.58C to 37.9 " 0.38C Ž n s 10.. Table 1 shows the effects of heat treatment on rectal temperature and the induction of micronuclei. Exposure of mice to 378C for 3 or 4 h and to 408C for 1 or 2 h raised body temperature to approximately 40.58C and produced a statistically significant
d
n s 10.
increase in MNPCEs Ž8.1 " 4.5, 6.0 " 2.1, 5.3 " 3.3, 7.5 " 2.9‰, respectively.. The relationship between rectal temperature and the induction of micronuclei is shown in Fig. 2. The MNPCE frequency increased at body temperatures of 39.58C or higher. Table 2 shows the frequency in treated mice of MNPCEs containing relatively large Ž d G 1r4 D . micronuclei. In the treatment group, the frequency of relatively large micronuclei significantly increased Ž p - 0.01. and was approximately 25% of the total MNPCEs. The frequency of large micronuclei in the
Table 2 Frequency of micronucleated erythrocytes containing relatively large micronuclei Ž d G Dr4. Treatment
n
a
Control 378C–3 h 378C–4 h 408C–1 h 408C–2 h Colchicine b Mitomycin C
c
10 10 10 9 10 3 5
MNPCEs Ž‰.
2.1 " 1.1 8.1 " 4.5 6.0 " 2.1 5.3 " 3.3 7.5 " 2.9 10.7 " 1.5 36.4 " 7.6
Large MNPCEs based on 20 MNPCEs counted per animal Individual animal data
Mean " SD Ž%.
1, 1, 0, 0, 1, 1, 0, 1, 0, 0 9, 3, 2, 7, 8, 3, 5, 4, 6, 6 5, 5, 5, 2, 4, 7, 6, 5, 6, 6 5, 4, –, 3, 6, 3, 4, 4, 6, 3 4, 3, 7, 6, 5, 4, 4, 4, 5, 6 6, 10, 8 2, 1, 1, 0, 2
5.0 " 1.0 26.5 " 11.6 ) ) 25.5 " 6.9 ) ) 21.1 " 6.0 ) ) 24.0 " 6.1 ) ) 40.0 " 10.0 ) ) 6.0 " 4.2
D, d, the diameters of cytoplasm Ž D . and micronuclei Ž d .. 10 MNPCEs counted per animals; b Double dosing i.p. of 0.5 mgrkg and 24 h sampling; sampling.
a
c
Single dosing i.p. of 2 mgrkg and 24 h
82
S. Asanami, K. Shimonor Mutation Research 390 (1997) 79–83
Fig. 2. The relationship between rectal temperature Ž8C. and micronucleus frequency Ž‰. after heat exposure. Error bars represent standard deviation of the mean value.
control group and the colchicine-, and mitomycin C-treated groups were in agreement with the data of Yamamoto and Kikuchi w17x.
4. Discussion There are few reports on the relationship between chromosome aberrations and high body temperature, although there are many in vitro reports of the induction of chromosome aberration by heat treatment w12,13x. Here, we demonstrated the effect of high body temperature on the induction of micronuclei in a mammalian system. Exposure of mice to 378C for 3 or 4 h, and to 408C for 1 or 2 h, raised the rectal temperature to approximate 40.58C and produced a significant increase in MNPCEs ŽTable 1.. Chrisman and Baumgartner w14x demonstrated the induction of MNPCEs in mouse bone marrow by hyperthermia Ž35 " 18C for 3 h. without the body temperature data, and King and Wild w15x also reported that whole-body hyperthermia Ž35–368C for 20–32 h. induced a significant increase in MNPCE frequencies in mouse bone marrow with elevated rectal temperature. In contrast, some papers demonstrated that heat treatment alone did not induce chromosome aberrations. Weissenborn and Obe w19x showed that heat alone did not induce structural chromosomal aberrations but they did not count numerical abnormalities as aberrations. We included both structural and numerical chromosomal abnormalities as aberrations. Shen et al. w20x reported that treatment with whole body hyperther-
mia Ž40 " 0.28C for 1 h. alone did not significantly change the frequency of chromatin fragments by flow cytometry in the thymus and bone marrow cells when compared to that in the corresponding tissues of the untreated mice. Because MNPCE frequencies induced by hyperthermia, however, are not very high ŽTable 1., it may be difficult to assess a weak positive response for micronucleus induction by flow cytometry analysis. Concerning the possible mechanisms of chromosome aberration induction in mammalian cells by heat treatment, some investigators have concluded that high temperatures inhibit spindle function in vitro w12,13x. In general, micronuclei induced by spindle poisons are considerably larger than those induced by clastogens w2x. Yamamoto and Kikuchi w17x concluded that it is possible to deduce the action site of micronucleus-inducing agents on the basis of the relative size of the micronuclei, and Tinwell and Ashby suggested that large or crescent shaped micronuclei are strongly indicative of aneugenicity w21x. In this study, a significant Ž p - 0.01. increase of relatively large micronuclei was associated with hyperthermia, suggesting that hyperthermia also disturbs the mitotic apparatus in mammalian cells in vivo. Increased MNPCE frequencies were observed at rectal temperatures of 39.58C or higher ŽFig. 2.. There was no increase in MNPCE frequencies in the 378C–2 h treatment group Žthe mean rectal temperature was 39.09 " 0.448C., but significantly increased MNPCE frequencies were observed in the 378C–3 h treatment group Žrectal temperature, 40.63 " 0.478C; Table 1.. These results suggest that mouse rectal temperatures of over 39.58C for 30 min could induce micronuclei in bone marrow. We therefore speculate that the threshold rectal temperature for micronucleus induction is 39.58C for 30 min. Regarding the duration of hyperthermia, King and Wild w15x reported that no significant increase in the incidence of MNPCEs in bone marrow was observed when mice were exposed to hyperthermia for less than 10 h. We demonstrated here, however, that even short-term hyperthermia Žfor example 408C for 1 h. could induce a significant increase in MNPCE and elevated rectal temperature of approximately 408C or higher, although King and Wild found that the mean rectal temperature of mice subjected to hyperthermia was 39.18C. Coss et al. w12x reported that treatment of
S. Asanami, K. Shimonor Mutation Research 390 (1997) 79–83
dividing CHO cells at 45.58C for 5 to 15 min disassembled the spindle to varying degrees depending on the length of heat exposure, and Hinkley and Samson w22x reported that microtubules in a nonmammalian system rapidly dissociate in vivo at 408C. Several reports have shown that temperatures above 378C will induce the disassembly of microtubules in mammalian systems w23x, and Fiorenza and Mangia w24x demonstrated that abnormal chromosome numbers were observed in mouse oocytes matured at either 39.08C or 40.08C, but not at 37.08C. Thus, it appears that the formation of a functional mitotic spindle can only occur within narrow temperature limits. In conclusion, body temperatures of 39.58C or higher for more than 30 min induced micronuclei in mouse bone marrow cells, and one possible mechanism was the disturbance of the mitotic apparatus. Monitoring body temperature therefore may be advisable when chemicals that give negative results in vitro produce positive results in vivo.
w9x
w10x
w11x
w12x
w13x
w14x
w15x
w16x
w17x
References w18x w1x Schmid, W. Ž1973. Chemical mutagen testing on in vivo somatic mammalian cells, Agents Actions, 3, 77–85. w2x Schmid, W. Ž1975. The micronucleus test, Mutation Res., 31, 9–15. w3x Heddle, J.A. Ž1973. A rapid in vivo test for chromosomal damage. Mutation Res., 18, 187–190. w4x Mavournin, K.H., D.H. Blakey, M.C. Cimino, M.F. Salamone and J.A. Heddle Ž1990. 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. w5x Hayashi, M., R. Tice, J.T. MacGregor, D. Anderson, D.H. Blakey, M. Kirsh-Volders, F.B. Oleson, Jr., F. Pacchierotti, F. Romagna, H. Shimada, S. Sutou and B. Vannier Ž1994. In vivo rodent erythrocyte micronucleus assay, Mutation Res., 312, 293–304. w6x Dewey, W.C. Ž1989. The search for critical cellular targets damaged by heat, Radiat. Res., 120, 191–204. w7x Dewey, W.C., A. Westra, H.H. Miller and H. Nagasawa Ž1971. Heat-induced lethality and chromosomal damage in synchronized Chinese hamster cells treated with 5bromodeoxyuridine, Int. J. Radiat. Biol., 20, 505–520. w8x Tomasovic, S.P., G.N. Turner and W.C. Dewey Ž1978. Ef-
w19x
w20x
w21x
w22x
w23x w24x
83
fect of hyperthermia on nonhistone proteins isolated with DNA, Radiat. Res., 73, 535–552. Yatvin, M.B. Ž1977. The influence of membrane lipid composition and procaine on hyperthermic death of cells, Int. J. Radiat. Biol., 32, 513–521. Jorritsma, J.B.M. and A.W.T. Konings Ž1983. Inhibition of repair of radiation-induced strand breaks by hyperthermia, and its relationship to cell survival after hyperthermia alone, Int. J. Radiat. Biol., 43, 505–516. Cai, L and J. Jiang Ž1995. Mild hyperthermia can induce adaptation to cytogenetic damage caused by subsequent X irradiation, Radiat. Res., 143, 26–33. Coss, R.A., W.C. Dewey and J.R. Bamburg Ž1982. Effects of hyperthermia on dividing Chinese hamster ovary cells and on microtubules in vitro, Cancer Res., 42, 1059–1071. Yamada K., Y. Ichikawa and H. Okumura Ž1989. Effects of high temperatures on chromosomes of normal and transformed human cells, Human Cell, 2, 80–85. Chrisman, C.L. and A.P. Baumgartner Ž1980. Micronuclei in bone–marrow cells of mice subjected to hyperthermia, Mutation Res., 77, 95–97. King, M.T. and D. Wild Ž1983. The mutagenic potential of hyperthermia and fever in mice, Mutation Res., 111, 219– 226. Hayashi, M., T. Sofuni and M. Ishidate, Jr. Ž1983. An application of acridine orange fluorescent staining to the micronucleus test, Mutation Res., 120, 241–247. Yamamoto, K. and Y. Kikuchi Ž1980. A comparison of diameters of micronuclei induced by clastogens and by spindle poisons, Mutation Res., 71, 127–131. Kastenbaum, M.A. and K.O. Bowman Ž1970. Tables for determining the statistical significance of mutation frequencies, Mutation. Res., 9, 527–549. Weissenborn, U. and G. Obe Ž1991. Modification of X-ray induced chromosome aberration frequency by pre- and postirradiation hyperthermia of human peripheral lymphocytes, Int. J. Radiat. Biol., 59, 973–984. Shen, R.N., W.N. Crabtree, B. Wu, P. Young, G.A. Sandison, N.B. Hornback and H. Shidnia Ž1992. A reliable method for quantitating chromatin fragments by flow cytometry to predict the effect of total body irradiation and hyperthermia on mice, Int. J. Radiat. Oncol. Biol. Phys. 24, 139–143. Tinwell, H. and J. Ashby Ž1991. Micronucleus morphology as a means to distinguish aneugens and clastogens in the mouse bone marrow micronucleus assay, Mutagenesis, 6, 193–198. Hinkley, R.E. Jr. and F.E. Samson, Jr. Ž1974. The effects of an elevated temperature, colchicine, and vinblastine on axonal microtubules of the crayfish Ž Procambarus clarkii ., J. Exp. Zool., 188, 321–336. Stephens, R.E. and K.T. Edds Ž1976. Microtubules: structure, chemistry, and function, Physiol. Rev., 56, 709–777. Fiorenza, M.T. and F. Mangia Ž1992. Hyperthermia specifically inhibits bivalent chromosome disjunction in maturing mouse oocytes, Biol. Reprod., 46, 658–664.