Mouse strain differences in the induction of micronuclei by polycyclic aromatic hydrocarbons

Mutation Research, 192 (1987) 185-189 Elsevier

185

MTRL 060

Mouse strain differences in the induction of micronuclei by polycyclic aromatic hydrocarbons Sei-ichi Sato, Hirono Kitajima, Sumie Konishi, Haruo Takizawa and Naomichi Inui Biological Research Center, Japan Tobacco Inc.. 23 Nakogi, Hatano, Kanagawa 257 (Japan) (Accepted 9 July 1987)

Keywordsv Polycyclic aromatic hydrocarbon; Micronucleated erythrocyte; (Mouse).

Summary The frequency of micronucleated erythrocytes (MNE) in 3 inbred mouse strains and 2 of their hybrids (C57BL/6, BALB/c, DBA/2, BDFI and CDF1) were examined after polycyclic aromatic hydrocarbons (PAHs; 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (3-MC), benzo[a]pyrene (BaP), benzo[e]pyrene (BeP) and anthracene (ANT)) were injected i.p. PAHs are thought to form active metabolites after being administered to mammals. In mouse strains with inducible PAH activating enzymes, such as C57BL/6 or BALB/c, M N E were significantly induced, as compared to control mice, 48 h after DMBA, BaP, or 3-MC was injected. No increase in the frequency of M N E occurred in the DBA/2 strain which cannot induce the activating enzymes. BeP and ANT did not increase the frequency of M N E in any mouse used. The levels of M N E induction in BDF1 or CDF1 hybrids were similar to those in C57BL/6 or BALB/c. These results support the view that the genetic capacity to metabolize PAHs is strongly associated with micronucleus induction as in the case of PAH carcinogenesis.

Polycyclic aromatic hydrocarbons (PAHs) are a major concern in public health, because they are highly mutagenic and carcinogenic. PAHs are chemical compounds that require metabolic activation by microsomal monooxygenase enzymes to become mutagenic or carcinogenic (Ames et al., 1973, 1975; McCann et al., 1975; Krahn and Heidelberger, 1977). PAHs, after being metabolized by microsome monooxygenase, bind covalently to cellular Correspondence: S. Sato, Biological Research Center, Japan Tobacco Inc., 23 Nakogi, Hatano, Kanagawa 257 (Japan).

macromolecules (Gelboin, 1969; Oesch et al., 1977). Kouri and Nebert (1977) reported that some mouse strains possess PAH-inducible metabolic enzymes whereas other strains do not. Basal or induced levels of PAH-activating enzymes are different among mouse strains (Kodama and Bock, 1970). Kouri et al. (1972) found a good positive correlation between the inducibility of the PAHactivating enzymes and the induction of sub~ cutaneous tumors with 3-methylcholanthrene (3-MC) treatment. In certain short-term assays, it was found that 7,8-benzoflavone (7,8-BF) inhibited the induction of bone marrow micronuclei

0165-7992/87/$ 03.50 (o 1987 Elsevier Science Publishers B.V. (Biomedical Division)

186

in mice with 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (BaP), and of sperm anomalies in mice with DMBA (Raj and Katz, 1983, 1984). These findings suggest that P A H metabolic enzyme activity is also involved in the induction of micronuclei by PAHs in the bone marrow. Based on this assumption, we investigated the PAH-induced micronuclei in representative mouse strains which differ in their ability to induce PAHactivating enzymes.

From each animal 1000 erythrocytes (polychromatic and normochromatic erythrocytes) were examined to determine the frequencies of micronucleated erythrocytes (MNEs) according to Yamamoto and Kikuchi (1981). To examine the toxicity of the chemicals on the bone marrow, the appearance of polychromatic erythrocytes (PCEs) among 1000 erythrocytes was also examined. The results were analyzed statistically with the method described by Kastenbaum and Bowman (1970).

Material and methods Results and discussion

Female mice of the C57BL/6 Cr, BALB/c Cr, and DBA/2 Cr strains and BDF1 (C57BL/6 x DBA/2) and CDFI (BALB/c x DBA/2) hybrids were obtained from Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka. They were 10 weeks old when used in the experiment. DMBA and 3-MC were obtained from Tokyo Kasei Kogyo Co., Tokyo. BaP was from Sigma Chemical Co., Saint Louis. Benzo[e]pyrene (BeP) was from Aldrich Chemical Co., Milwaukee. Anthracene (ANT) was from Wako Pure Chemical Industries, Osaka. Cyclophosphamide (CP) was from Shionogi Co., Osaka. Thiotepa (TT) was from Sumitomo Chemical Kogyo Co., Osaka. Mitomycin C (MMC) was from Kyowa Hakko Kogyo Co., Tokyo. The PAHs were suspended in olive oil (Wako Pure Chemical, Osaka) to give a dose of 100 mg/kg body weight. CP, TT, and MMC were dissolved in distilled water to give a dose of 100, 10 and 5 mg/kg body weight, respectively. Each group was comprised of 3 mice, which received a single i.p. injection of a volume corresponding to 10 ml/kg body weight. The control groups received olive oil or distilled water by i.p. injection. The preparation and staining of bone marrow cells were carried out according to Schmid (1975). The mice were killed at 24, 48, or 72 h after treatment. The bone marrow cells were flushed out with fetal calf serum, centrifuged and smeared, and stained with May-Gr~inwald and Giemsa solutions.

The effect of sampling time on the frequency of MNEs induced by PAHs is presented in Figs. 1 and 2. C57BL/6 and BALB/c mice showed significant (p < 5%) increases in the frequency of MNEs 48 h after treatment with DMBA, BaP, or 3-MC as compared with the corresponding olive oil control; the frequency of MNEs did not increase in mice treated with BeP or ANT. In contrast, in DBA/2 mice no PAH produced a significant increase of MNEs (Table 1). The frequency of MNEs in hybrid mice (BDF1 and CDF1) increased 48 h after DMBA, 3-MC, or BaP treatment as it did in C57BL/6 or BALB/c mice (Table 1, Fig. 2). The effect of CP, TT, and MMC on the frequency of MNEs is shown in Fig. 3. In the 5 mouse

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Fig. 1. Frequency of micronucleated erythrocytes for P A H s in the bone marrow of C57BL/6, B A L B / c or D B A / 2 mice. DMBA: I ; 3-MC: O; BaP; A; BeP; El; ANT: ZX; dotted line indicates control level. Calculated from at least 1000 erythrocytes per mouse and 3 mice per group.

187

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72

Fig. 2. Frequency of micronucleated erythrocytes for PAHs in the bone marrow of BDFI or CDF1 mice. DMBA: II; 3-MC: O; BaP: A; BeP; El; ANT: A; dotted line indicates control level. Calculated from at least 1000 erythrocytes per mouse and 3 mice per group. strains used, frequencies of M N E s increased 24-48 h after t r e a t m e n t a n d rapidly decreased between 48 a n d 72 h. In the case of C P , T T , a n d M M C , the f r e q u e n c y of M N E s significantly (p < 1%) increased as c o m p a r e d with that p r o d u c e d by distilled water. The frequency range of M N E s in distilled water was 0 . 1 2 - 0 . 2 3 % , a n d in olive oil 0 . 1 9 - 0 . 2 3 % (Table 1). T h e frequency of P C E s decreased timed e p e n d e n t l y after t r e a t m e n t with all chemicals ex-

o f BaP, the i n d u c t i o n of c h r o m o s o m e a b e r r a t i o n s a n d m i c r o n u c l e i in the B6C3F1 mouse are positively correlated (Kliesch et al., 1982). Raj a n d Katz (1983, 1984) reported that m u t a g e n i c i t y a n d metabolic enzyme activation of P A H in B6C3F1 m o u s e are closely correlated. 7,8-BF inhibits the i n d u c t i o n of micronuclei in b o n e m a r r o w by D M B A a n d BaP. 7,8-BF is k n o w n to inhibit the enzyme involved in the activation of P A H s . Thus there are close correlations between enzyme activities and the i n d u c t i o n of c h r o m o s o m e m u t a t i o n s , such as micronuclei. Studies show that there are differences in susceptibility to P A H t r e a t m e n t a m o n g various mouse strains in vivo. Some m o u s e strains do a n d others do not induce P A H metabolic enzymes ( K o d a m a a n d Bock, 1970; Kouri a n d Nebert, 1977).

TABLE 1 MOUSE STRAIN DIFFERENCES IN THE FREQUENCY OF MICRONUCLEATED ERYTHROCYTES (MNE) IN BONE MARROW 48 h AFTER TREATMENT WITH PAHs Chemical

Olive oil DMBA 3-MC BaP BeP ANT

C57BL/6

DBA/2

BALB/c

BDFI

CDF1

%MNE"

(S.D.)

% M N E " (S.D.)

%MNEa

(S.D.)

%MNEa

(S.D.)

%MNEa

(S.D.)

0.21 0.93** 0.60** 0.57* 0.30 0.17

(0.07) (0.15) (0.20) (0.15) (0.10) (0.06)

0.20 0.33 0.27 0.33 0.07 0.10

0.21 1.13"* 0.43* 0.77** 0.30 0.33

(0.09) (0.51) (0.06) (0.12) (0.10) (0.06)

0.19 0.83 0.63** 0.57* 0.37 0.13

(0.06) (0.06) (0.15) (0.60) (0.29) (0.06)

0.23 0.73** 0.60** 0.63** 0.20 0.13

(0.08) (0.15) (0.10) (0.42) (0.10) (0.06)

(0.09) (0.06) (0.06) (0.12) (0.06) (0.10)

a Percentage of micronucleated erythrocytes in bone marrow, calculated from at least 1000 erythrocytes per mouse and 3 mice per group. * Significantly different from olive oil (p <0.05). ** Significantly different from olive oil (p <0.01).

188

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Fig. 3. Frequency of micronucleated erythrocytes for CP, TT, or MMC in the bone marrow of mice. CP: O; TT: A; MMC: II; dotted line indicates control level. Calculated from at least 1000 erythrocytes per mouse and 3 mice per group.

Hybrid mice derived from mating between inducible and non-inducible strains are generally inducible (Kouri and Nebert, 1977). An inducible mouse strain was more highly sensitive to subcutaneous tumor induction with PAH treatment than a non-inducible strain (Kouri et al., 1973). This study clearly showed that DMBA, 3-MC, and BaP induce micronuclei in strains, such as C57BL/6 or BALB/c, that possess inducible PAHactivating enzymes; the BDF1 and CDF1 hybrids are also sensitive. These compounds do not induce micronuclei in DBA/2 mice which lack inducible enzymes. CP, TT, and MMC produced similar increases in the frequency of MNEs in all strains. The strain differences were observed with the average numbers of induced MNEs with DMBA, 3-MC or BaP, that is, MNEs increased in mice

with the inducible PAH-activating enzyme but not in mice lacking the enzyme. Thus, the induction of micronuclei is strongly correlated with the presence of the metabolic activation enzyme of PAHs, as in the case of tumor induction.

Acknowledgement We are grateful to Dr. James R. Miller, Takeda Chemical Industries Ltd., Osaka (Japan) for critically reading the manuscript.

References Ames, B.N., W.E. Durston, E. Yamasaki and F.D. Lee (1973) Carcinogens are mutagens: a simple test system combining

189 liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. (U.S.A.), 70, 2281-2285. Dipaolo, J.A., P. Donovan and R. Nelson (1969) Quantitative studies of in vitro transformation by chemical carcinogens, J. Natl. Cancer Inst., 42, 867-876. Heddle, J.A. (1973) A rapid in vivo test for chromosomal damage, Mutation Res., 18, 187-190. Kastenbaum, M.A., and K.O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549. Kliesch, U., 1. Roupova and I.-D. Adler (1982) Induction of chromosome damage in mouse bone marrow by benzo[a]pyrene, Mutation Res., 102, 265-273. Kodama, Y., and F.D. Bock (1970) Benzo[alpyrenemetabolizing enzyme activity of liver of various strains of mice, Cancer Res., 30, 1846-1849. Kouri, R.K., and D.W. Nebert (1977) Genetic regulation of susceptibility to polycyclic-hydrocarbon-induced tumor in the mouse, in: H.H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Cold Spring Harbor Laboratory, University of Tokyo Press, Tokyo, pp. 811-835. Krahn, D.F., and C. Heidelberger (1977) Liver homogenatemediated mutagenesis in Chinese hamster V79 cell by polycyclic aromatic hydrocarbons and aflatoxins, Mutation Res., 46, 27-44. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Detection of carcinogens as mutagens in the

Salmonella/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72, 5135-5139. Oesch, F., D. Raphael, H. Schwind and H.R. Glatt (1977) Species differences in activation and inactivation enzyme related to the control of mutagenic metabolites, Arch. Toxicol., 39, 97-108. Pienta, R.J., J.A. Poiley and W.B. Lebherz, I11 (1977) Morphological transformation of early passage golden Syrian hamster embryo cells derived from cryopreserved primary cultures as a reliable in vitro bioassay for identifying diverse carcinogens, Int. J. Cancer, 19, 642-655. Raj, A.J., 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. Raj, A.S., and M. Katz (1984) Inhibitory effect of ~- and 13naphtho flavones on DMBA-induced anomalies in germ cells assessed by sperm abnormality assay, Mutation Res., 136, 81-84. Schmid, W. (1973) Chemical mutagen testing on in vivo somatic mammalian cell, Agents and Actions, 3, 77-85. Schmid, W. (1975) The micronucleus test, Mutation Res., 31, 9-15. Yamamoto, K.I., and Y. Kikuchi (1981) Studies on micronuclei time response and on the effect of multiple treatments of mutagens on induction of micronuclei, Mutation Res., 90, 163-173. Communicated by R.J. Preston