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Multiple-dosing effects of benzo[a]pyrene in the mouse bone marrow micronucleus test
Mutation Research, 234 (1990) 179-181
179
Elsevier MUTREV 02912
Multiple-dosing effects of benzo[ a]pyrene in the mouse bone marrow micronucleus test H i r o y a s u S h i m a d a 1, S a c h i k o S a t a k e 1, S a t o r u I t o h 1, C h i h a l ' u H a t t o r i M a k o t o H a y a s h i 2 a n d M o t o i I s h i d a t e , Jr. 2
1,
Research Institute of Daiichi Pharmaceutical Co., Tokyo (Japan) and 2 National Institute of Hygienic Sciences, Tokyo (Japan)
(Accepted25 January1990)
Keywords: Benzo[a]pyrene;Micronucleustest; Multipledosing
Summary Multiple-dosing effects of benzo[a]pyrene (B[a]P) in the micronucleus test were studied using CD-1 male mice. Mice were treated orally once, twice or 3 times with 250, 500, 1000 or 2000 mg/kg, at 24-h intervals. Bone marrow cells were sampled 24 h after the last administration. The present study indicated that the incidence of polychromatic erythrocytes with micronuclei significantly increased more in the group of animals that received B[a]P twice than in those receiving it one or 3 times. The dose of 500 mg/kg B[a]P yielded the greatest response of any dose regimen.
The micronucleus test has been widely used as a sensitive in vivo genotoxicity test and has been recommended for use in regulatory guidelines for the safety evaluation of new drugs or industrial chemicals in Japan as well as in other countries. The experimental protocol of the test, however, varies slightly from guideline to guideline in details such as number of animals, sex, dosing and sampling time. The optimum response may vary according to the chemical concerned, so that Hayashi et al. (1984) have recommended that a pilot preliminary experiment be carded out for each test substance before the start of full-scale experimentation. Collaborative Japanese studies on the micronucleus test in mice have been in progress since 1985 as part of the project of the
Correspondence: Dr. H. Shimada,Research Institute of Daiichi PharmaceuticalCo., Ltd.,Tokyo(Japan).
Environmental Mutagen Society of Japan. The first study, conducted to compare sex differences, indicated that males seem to be more sensitive than females (except in the case of ethyl methanesulfonate), but no chemicals that were tested were positive only in males (Collaborative Study Group, 1986). The second study was intended to compare the strain differences, and indicated that no strain (except a mutagen-sensitive MS/Ae strain) appeared to be superior in response to the chemicals tested (Collaborative Study Group, 1988). The third study compared the route of administration and indicated that intraperitoneal injection seemed to be more sensitive than oral administration (except in the case of benzene) (Collaborative Study Group, 1989a). The fourth study was intended to compare sampling times and multiple-dosing effects. That final study is, coincidentally, related to the present study. The present study, as a part of this cooperative effort,
0165-1161/90/$03.50 © 1990 ElsevierScianc~PublishersB.V.(BiomedicalDivision)
180 c o m p a r e d the m u l t i p l e dosing effects of benzo[ a]p y r e n e (B[a]P) after oral a d m i n i s t r a t i o n in the m i c r o n u c l e u s test. Materials
and methods
Test chemical B[a]P ( C A S N o . 50-32-8) of 98% p u r i t y was p u r c h a s e d f r o m W a k o Pure C h e m i c a l Inc., Osaka. It was s u s p e n d e d in olive oil ( K o z a k a i S e i y a k u Co. Ltd., T o k y o ) b e f o r e use. T h e q u a l i t y o f the olive oil was verified a c c o r d i n g to the J a p a n e s e Pharmacopeia. Animals M a l e CD-1 mice of 8 - 1 0 weeks old were p u r c h a s e d f r o m Charles River J a p a n Inc., Atsugi. T h e y were h o u s e d in m e t a l m e s h cages in an a i r - c o n d i t i o n e d r o o m a n d given f o o d a n d w a t e r a d lib±turn. Dose levels F o r e s t i m a t i o n o f the 4 - d a y m e d i a n lethal d o s e
( M L D 4 ) for single, d o u b l e a n d triple dosings, g r o u p s of 7 mice each were given oral doses of 10 m l / k g of 500, 707, 1000, 1414 or 2000 m g / k g . T h e highest d o s e was l i m i t e d to 2000 m g / k g , b e c a u s e of the d i f f i c u l t y f o u n d in a d m i n i s t e r i n g the s u s p e n s i o n o w i n g to its high viscosity. N o a n i m a l s d i e d at a n y d o s e tested. T w o a n i m a l s f r o m the t r e a t e d g r o u p were killed 24 h after the last a d m i n i s t r a t i o n a n d their b o n e m a r r o w was e x a m i n e d for the p i l o t a s s a y for the m i c r o n u c l e u s test. A s a result o f these p r e l i m i n a r y tests, the doses of B[a]P to b e used for the m i c r o n u c l e u s test were set at 500, 1000 a n d 2000 m g / k g . G r o u p s t r e a t e d with 250 m g / k g were also a d d e d as a s u p p l e m e n t a r y e x p e r i m e n t , in o r d e r to f i n d the m a x i m a l r e s p o n s e o f m i c r o n u c l e u s i n d u c t i o n , since the d o s e of 500 m g / k g s h o w e d the highest r e s p o n s e of a n y d o s e regimen.
Micronucleus test B[a]P was a d m i n i s t e r e d o r a l l y to g r o u p s of 5 mice at single, d o u b l e a n d triple doses of 500,
TABLE 1 INCIDENCE OF MPE IN THE BONE MARROW OF MICE TREATED WITH BENZO[a]PYRENE Number of doses
Chemical
Single
Olive oil B[a]P
CP Double
Triple
Single Double Triple
Olive oil B[a]P
Olive oil B[a]P
B[a]P B[a]P B[a]P Olive oil
Dose (mg/kg)
Number of mice
MN/1000 PE based on 2000 PE assessed per animal PE (%) Individual animal data Group mean ± S.D. Group mean ± SD (range)
500 1000 2000 40
5 5 5 5 5
0.5, 0.5, 2, 1.5, 1.5 16, 16, 21, 16, 16.5 11, 18.5, 14, 8, 11 13.5, 16, 12.5, 7.5, 9 16, 38, 34, 33, 24.5
1.2±0.67 17.1±2.19"* 12.5±3.97"* 11.7±3.44 ** 29.1±8.82"*
56.44 + 2.95 (52.45-60.4) 58.46 + 3.98 (54.8 -63.95) 59.25 -1-4.34 (52.85-64.75) 60.73 + 1.70 (59.3 -63.5) 58.184-4.18 (52.35-63.9)
500 1000 2000
5 5 5 5
2, 1, 1, 2.5, 2 27, 20, 10.5, 22.5, 24 22, 14.5, 17.5, 13, 14 15.5, 14, 20, 12.5, 23.5
1.7±0.67 20.8±6.29** 16.2±3.65"* 17.1±4.55"*
57.08 + 1.65 (55.25-59.45) 49.90 + 5.70 (42.65-55.3) * 49.89 + 1.65 (47.6 -51.65) * 49.64+ 3.76 (46.6 -55.85) *
500 1000 2000
5 5 5 5
2, 0.5, 1, 2, 3 12, 12, 11.5, 18.5, 11 11.5, 13.5, 17.5, 10.5, 8 6, 11.5, 6.5, 9.5, 7.5
1.7±0.97 13.0±3.10"* 12.2±3.56"* 8.2±2.28**
54.66 + 1.48 (52.9 -56.25) 48.75 + 4.94 (40.1 -52.2) * 48.99+4.99 (42.75-53.0) * 48.79+ 1.27 (46.8 -50.1) *
5 5 5 3
9.5, 5.5, 11, 7, 14.5 15, 15.5, 18.5, 20, 14.5 17.5, 12.5, 11, 8.5, 15.5 2.5, 0.5, 1.5
9.5-t-3.52 ** 16.7 + 2.41 * * 13.0+3.57 ** 1.5+1.00
54.74 ± 3.89 (51.90-61.35) 48.02 + 3.47 (43.50-52.5) 47.70 + 4.11 (36.4 -51.4) 53.82 + 4.85 (48.85-58.55)
250 250 250
* Significant difference from control (p < 0.05). * * Significant difference from control (p < 0.01).
181
1000 or 2000 mg/kg. Cyclophosphamide (Nacalai Tesque, Inc., Kyoto) were used as the positive control, and was dissolved in physiological saline and injected intraperitoneaUy. A group of 5 animals treated with olive oil alone served as a negative control group. Animals were killed by cervical dislocation 24 h after the last treatment, and the bone marrow of the femur was flushed into a test tube with fetal bovine serum. Smear preparations were made and stained with acridine orange (Merck Ltd., Darmstadt, F.R.G.) according to the technique described by Hayashi et al. (1983). Briefly, the preparations were stained with 0.007% acridine orange for 3 min, rinsed with the phosphate buffer (pH 6.8) for 10-15 min, and mounted with the same buffer. A fluorescent microscope (Olympus AHBRFL with BG-12 excitation filter and O-515 barrier filter) was used for observation. For each animal, 2000 polychromatic erythrocytes (PE) were observed and the incidence of the PE with micronuclei (MPE) was recorded. The ratio of PE to erythrocytes was also counted. The Kastenbaum and Bowman method and Wilcoxon's rank-sum test were used for statistical analysis of micronuclei and the PE ratio, respectively. Results and discussion
The results are summarized in Table 1. The incidence of MPE in the vehicle control was in the range of 0.05-0.3%. At all doses of B[a]P, the incidence of MPE was significantly higher than in the negative control, for all (single-, double- and triple-dose) regimens. The positive response, however, showed no dose response and the maximal response was obtained in the group treated with
500 mg/kg with any dosing regimen. The positive response seemed to be enhanced by double dosing but reduced by triple dosing. As regards B[a]P, it can be concluded from the present experiment that the double-dose regimen shows a more effective response than the singleand triple-dose regimens, when the bone marrow samples are prepared 24 h after the last treatment. Our findings support the simphfied protocol introduced by Garriott et al. (1988) and recent results obtained by the Japanese Collaborative Study Group for the Micronucleus Test (1989b) in mice. The question still remains, however, whether or not the same conclusion can be drawn when B[a]P is injected intraperitoneally instead. References Collaborative Study Group for the Micronucleus Test (1986) Sex difference in the micronucleus test, Mutation Res., 172, 151-163. Collaborative Study Group for the Micronucleus Test (1988) Strain difference in the micronucleus test, Mutation Res., 204, 307-316. Collaborative Study Group for the Micronucleus Test (1989a) Difference between intraperitoneal and oral gavage application in the micronucleus test, Mutation Res., 223, 329344. Collaborative Study Group for the Micronucleus Test (1989b) Single versus multiple dosing in the micronuclens test, in preparation (see summary herein, Sutou et al.). Garriott, M.L., C.E. Piper and A.J. Kokkino (1988) A simphfiecl protocol for the mouse bone marrow micronucleus assay, J. Appl. Toxicol., 8, 141-144. Hayashi, M., T. Sofuni and M. Ishidate, Jr. (1983) An application of acridine orange fluorescent staining to the micronucleus test, Mutation Res., 120, 241-247. Hayashi, M., T. Sofuni and M. Ishidate, Jr. (1984) A pilot experiment for the micronucleus test: the multi-sampling at multi-dose levels method, Mutation Res., 141, 165-169.
179
Elsevier MUTREV 02912
Multiple-dosing effects of benzo[ a]pyrene in the mouse bone marrow micronucleus test H i r o y a s u S h i m a d a 1, S a c h i k o S a t a k e 1, S a t o r u I t o h 1, C h i h a l ' u H a t t o r i M a k o t o H a y a s h i 2 a n d M o t o i I s h i d a t e , Jr. 2
1,
Research Institute of Daiichi Pharmaceutical Co., Tokyo (Japan) and 2 National Institute of Hygienic Sciences, Tokyo (Japan)
(Accepted25 January1990)
Keywords: Benzo[a]pyrene;Micronucleustest; Multipledosing
Summary Multiple-dosing effects of benzo[a]pyrene (B[a]P) in the micronucleus test were studied using CD-1 male mice. Mice were treated orally once, twice or 3 times with 250, 500, 1000 or 2000 mg/kg, at 24-h intervals. Bone marrow cells were sampled 24 h after the last administration. The present study indicated that the incidence of polychromatic erythrocytes with micronuclei significantly increased more in the group of animals that received B[a]P twice than in those receiving it one or 3 times. The dose of 500 mg/kg B[a]P yielded the greatest response of any dose regimen.
The micronucleus test has been widely used as a sensitive in vivo genotoxicity test and has been recommended for use in regulatory guidelines for the safety evaluation of new drugs or industrial chemicals in Japan as well as in other countries. The experimental protocol of the test, however, varies slightly from guideline to guideline in details such as number of animals, sex, dosing and sampling time. The optimum response may vary according to the chemical concerned, so that Hayashi et al. (1984) have recommended that a pilot preliminary experiment be carded out for each test substance before the start of full-scale experimentation. Collaborative Japanese studies on the micronucleus test in mice have been in progress since 1985 as part of the project of the
Correspondence: Dr. H. Shimada,Research Institute of Daiichi PharmaceuticalCo., Ltd.,Tokyo(Japan).
Environmental Mutagen Society of Japan. The first study, conducted to compare sex differences, indicated that males seem to be more sensitive than females (except in the case of ethyl methanesulfonate), but no chemicals that were tested were positive only in males (Collaborative Study Group, 1986). The second study was intended to compare the strain differences, and indicated that no strain (except a mutagen-sensitive MS/Ae strain) appeared to be superior in response to the chemicals tested (Collaborative Study Group, 1988). The third study compared the route of administration and indicated that intraperitoneal injection seemed to be more sensitive than oral administration (except in the case of benzene) (Collaborative Study Group, 1989a). The fourth study was intended to compare sampling times and multiple-dosing effects. That final study is, coincidentally, related to the present study. The present study, as a part of this cooperative effort,
0165-1161/90/$03.50 © 1990 ElsevierScianc~PublishersB.V.(BiomedicalDivision)
180 c o m p a r e d the m u l t i p l e dosing effects of benzo[ a]p y r e n e (B[a]P) after oral a d m i n i s t r a t i o n in the m i c r o n u c l e u s test. Materials
and methods
Test chemical B[a]P ( C A S N o . 50-32-8) of 98% p u r i t y was p u r c h a s e d f r o m W a k o Pure C h e m i c a l Inc., Osaka. It was s u s p e n d e d in olive oil ( K o z a k a i S e i y a k u Co. Ltd., T o k y o ) b e f o r e use. T h e q u a l i t y o f the olive oil was verified a c c o r d i n g to the J a p a n e s e Pharmacopeia. Animals M a l e CD-1 mice of 8 - 1 0 weeks old were p u r c h a s e d f r o m Charles River J a p a n Inc., Atsugi. T h e y were h o u s e d in m e t a l m e s h cages in an a i r - c o n d i t i o n e d r o o m a n d given f o o d a n d w a t e r a d lib±turn. Dose levels F o r e s t i m a t i o n o f the 4 - d a y m e d i a n lethal d o s e
( M L D 4 ) for single, d o u b l e a n d triple dosings, g r o u p s of 7 mice each were given oral doses of 10 m l / k g of 500, 707, 1000, 1414 or 2000 m g / k g . T h e highest d o s e was l i m i t e d to 2000 m g / k g , b e c a u s e of the d i f f i c u l t y f o u n d in a d m i n i s t e r i n g the s u s p e n s i o n o w i n g to its high viscosity. N o a n i m a l s d i e d at a n y d o s e tested. T w o a n i m a l s f r o m the t r e a t e d g r o u p were killed 24 h after the last a d m i n i s t r a t i o n a n d their b o n e m a r r o w was e x a m i n e d for the p i l o t a s s a y for the m i c r o n u c l e u s test. A s a result o f these p r e l i m i n a r y tests, the doses of B[a]P to b e used for the m i c r o n u c l e u s test were set at 500, 1000 a n d 2000 m g / k g . G r o u p s t r e a t e d with 250 m g / k g were also a d d e d as a s u p p l e m e n t a r y e x p e r i m e n t , in o r d e r to f i n d the m a x i m a l r e s p o n s e o f m i c r o n u c l e u s i n d u c t i o n , since the d o s e of 500 m g / k g s h o w e d the highest r e s p o n s e of a n y d o s e regimen.
Micronucleus test B[a]P was a d m i n i s t e r e d o r a l l y to g r o u p s of 5 mice at single, d o u b l e a n d triple doses of 500,
TABLE 1 INCIDENCE OF MPE IN THE BONE MARROW OF MICE TREATED WITH BENZO[a]PYRENE Number of doses
Chemical
Single
Olive oil B[a]P
CP Double
Triple
Single Double Triple
Olive oil B[a]P
Olive oil B[a]P
B[a]P B[a]P B[a]P Olive oil
Dose (mg/kg)
Number of mice
MN/1000 PE based on 2000 PE assessed per animal PE (%) Individual animal data Group mean ± S.D. Group mean ± SD (range)
500 1000 2000 40
5 5 5 5 5
0.5, 0.5, 2, 1.5, 1.5 16, 16, 21, 16, 16.5 11, 18.5, 14, 8, 11 13.5, 16, 12.5, 7.5, 9 16, 38, 34, 33, 24.5
1.2±0.67 17.1±2.19"* 12.5±3.97"* 11.7±3.44 ** 29.1±8.82"*
56.44 + 2.95 (52.45-60.4) 58.46 + 3.98 (54.8 -63.95) 59.25 -1-4.34 (52.85-64.75) 60.73 + 1.70 (59.3 -63.5) 58.184-4.18 (52.35-63.9)
500 1000 2000
5 5 5 5
2, 1, 1, 2.5, 2 27, 20, 10.5, 22.5, 24 22, 14.5, 17.5, 13, 14 15.5, 14, 20, 12.5, 23.5
1.7±0.67 20.8±6.29** 16.2±3.65"* 17.1±4.55"*
57.08 + 1.65 (55.25-59.45) 49.90 + 5.70 (42.65-55.3) * 49.89 + 1.65 (47.6 -51.65) * 49.64+ 3.76 (46.6 -55.85) *
500 1000 2000
5 5 5 5
2, 0.5, 1, 2, 3 12, 12, 11.5, 18.5, 11 11.5, 13.5, 17.5, 10.5, 8 6, 11.5, 6.5, 9.5, 7.5
1.7±0.97 13.0±3.10"* 12.2±3.56"* 8.2±2.28**
54.66 + 1.48 (52.9 -56.25) 48.75 + 4.94 (40.1 -52.2) * 48.99+4.99 (42.75-53.0) * 48.79+ 1.27 (46.8 -50.1) *
5 5 5 3
9.5, 5.5, 11, 7, 14.5 15, 15.5, 18.5, 20, 14.5 17.5, 12.5, 11, 8.5, 15.5 2.5, 0.5, 1.5
9.5-t-3.52 ** 16.7 + 2.41 * * 13.0+3.57 ** 1.5+1.00
54.74 ± 3.89 (51.90-61.35) 48.02 + 3.47 (43.50-52.5) 47.70 + 4.11 (36.4 -51.4) 53.82 + 4.85 (48.85-58.55)
250 250 250
* Significant difference from control (p < 0.05). * * Significant difference from control (p < 0.01).
181
1000 or 2000 mg/kg. Cyclophosphamide (Nacalai Tesque, Inc., Kyoto) were used as the positive control, and was dissolved in physiological saline and injected intraperitoneaUy. A group of 5 animals treated with olive oil alone served as a negative control group. Animals were killed by cervical dislocation 24 h after the last treatment, and the bone marrow of the femur was flushed into a test tube with fetal bovine serum. Smear preparations were made and stained with acridine orange (Merck Ltd., Darmstadt, F.R.G.) according to the technique described by Hayashi et al. (1983). Briefly, the preparations were stained with 0.007% acridine orange for 3 min, rinsed with the phosphate buffer (pH 6.8) for 10-15 min, and mounted with the same buffer. A fluorescent microscope (Olympus AHBRFL with BG-12 excitation filter and O-515 barrier filter) was used for observation. For each animal, 2000 polychromatic erythrocytes (PE) were observed and the incidence of the PE with micronuclei (MPE) was recorded. The ratio of PE to erythrocytes was also counted. The Kastenbaum and Bowman method and Wilcoxon's rank-sum test were used for statistical analysis of micronuclei and the PE ratio, respectively. Results and discussion
The results are summarized in Table 1. The incidence of MPE in the vehicle control was in the range of 0.05-0.3%. At all doses of B[a]P, the incidence of MPE was significantly higher than in the negative control, for all (single-, double- and triple-dose) regimens. The positive response, however, showed no dose response and the maximal response was obtained in the group treated with
500 mg/kg with any dosing regimen. The positive response seemed to be enhanced by double dosing but reduced by triple dosing. As regards B[a]P, it can be concluded from the present experiment that the double-dose regimen shows a more effective response than the singleand triple-dose regimens, when the bone marrow samples are prepared 24 h after the last treatment. Our findings support the simphfied protocol introduced by Garriott et al. (1988) and recent results obtained by the Japanese Collaborative Study Group for the Micronucleus Test (1989b) in mice. The question still remains, however, whether or not the same conclusion can be drawn when B[a]P is injected intraperitoneally instead. References Collaborative Study Group for the Micronucleus Test (1986) Sex difference in the micronucleus test, Mutation Res., 172, 151-163. Collaborative Study Group for the Micronucleus Test (1988) Strain difference in the micronucleus test, Mutation Res., 204, 307-316. Collaborative Study Group for the Micronucleus Test (1989a) Difference between intraperitoneal and oral gavage application in the micronucleus test, Mutation Res., 223, 329344. Collaborative Study Group for the Micronucleus Test (1989b) Single versus multiple dosing in the micronuclens test, in preparation (see summary herein, Sutou et al.). Garriott, M.L., C.E. Piper and A.J. Kokkino (1988) A simphfiecl protocol for the mouse bone marrow micronucleus assay, J. Appl. Toxicol., 8, 141-144. Hayashi, M., T. Sofuni and M. Ishidate, Jr. (1983) An application of acridine orange fluorescent staining to the micronucleus test, Mutation Res., 120, 241-247. Hayashi, M., T. Sofuni and M. Ishidate, Jr. (1984) A pilot experiment for the micronucleus test: the multi-sampling at multi-dose levels method, Mutation Res., 141, 165-169.