- Email: [email protected]
Micronucleus assays on 5-fluorouracil and 6-mercaptopurine with mouse peripheral blood reticulocytes
131)
MUTGEN
00210
Micronucleus assays on 54uorouracil and 6-mercaptopurine with mouse peripheral blood reticulocytes Akinobu Ohuchida ‘, Akemi Furukawa I, Junichi Yoshida ‘, Minako Watanabe ‘, Fu~ihiko Aruga ‘, Yoshihisa Miwa 3, Kanako Shinkawa 4* and Naohide Kinae ’ ’ 7’uilw Plrortttulnrric.nl Co. Ltd., Drtrg Sufity Lohmmty, __ TV-2 Ebisroto. Hiraishi. Kuwttcciti. Tokttshitnu 771~Oi. ’ Km& Pltcrrttlu~e~ctic~ltI Co. Ltd.. DrtrR S&y Luhorcrtoty. 301, Gctt.wkr. Fujirrlu, Slzkuoku 426. .’ Niltm Biorescarch Ctwter hr. Mujima. Fdwjtt,
Hctsltitm SOI-
atul ’
Uttitwsity of Shimoku, Sdrvul of Food wtd Nutritiwtd Sckttw. 395 Y&a. Slu:rtctka 420 (Juputt l (Accepted 9 August 19Y1)
K~ywrds:
Micronucleus test; 5Fluorouracil;
h-Mercaptopurinr:
Mouse: Peripheral hInod cells
Summary
As part of the Sth collaborative study of the Collaborative Study Group for the Micronucleus Test BCSGMT), the sensitivity and advantages of the micronucleus assay using mouse peripheral blood cells were evaluated using 5fluorouracil (5”FU) and 6-mercaptopurine (6-MP). The peripheral blood cells were collected from a tail vein of CD-I male mice just before and 24-120 h after intraperitoneai injection. At 24-h intervals. The maximum incidence of micronuclcated ret~culocytes (~NRETs) at SO mg/kg S-FU was observed 96 h after injection; at IO0 mg/kg, the peak was delayed to I20 h. and followed severe bone marrow depression, With 6-MP, maximum MNRETs were observed 48 h after treatment at all doses tested. At dose lcvcls higher than 50 mg/kg, severe bone marrow depression iclas observed after maximum MNRETs. Though the appearance patterns of MNRETs and the bone narrow depression were diffcrcnt between 5-FU and 6-MP. the positive response of both chemicals could be dctccted with this assay system as well as with the micronucleus test using femoral bone marrow cells.
As part of the fifth collaborative study on the micronucleus test. a new assay system using mouse peripheral blood reticulocytcs fRETsI was evaluated. Its advantages. and the results of conven-
Correspondence:
Akinohu Ohuchida, Drug Snfcty Labora7%7 Ebisuno, ?iiraishi. tory. Taiho Pharmaceutical Co. Ltd., -_ Kawauchi, Tokushimo 77!-01 (Japitn). * Present address: Finnish Institute of Occupational Health. Department
of Hygiene and Toxicology. Topeliuksenkntu
A. SF-OOZSOHelsinki (Finl~Ind).
-II a
tional micronucleus assays using femoral bone m~~rrow cells, were compared. We carried out micronucleus assays with peripheral blood cells on S-FU and 6-MP. S-FU and 6-MP are analogues of nucleotide bases, pyrimidine and purine, respectively, and are known to inhibit DNA biosynthesis. The assays on S-FU were carried out at two laboratories, Taiho Pharmaceutical Co. Ltd., and Kaken Pharmaceutical Co., Ltd.; those on 6-MP were performed at Nihon Bioresearch Center, Inc., and the University of Shizuoka.
Materinls
Yamamoto and Kikuchi (1981 I, Hara et al. (198% CSGMT (1990), and Ohuchida et al. (1991). The maximum dose in the first experiment with 6-MP was set at 200 mg/kg, and lower dose levels were
and methods
Both 5-FU (CAS No. 5 l-21-X. Lot No. PDK 19901 and h-MP (CA!3 No. 55-44-2. Lot No. KPN2W) were purchased from Wako Pure Chemical Industries (Osaka, Japan). 5-FU was dissolved in physiological saline. and 6-MP was suspended in olive oil. Mitomycin C (CAS No. ?I-07-7. Kyows Hakko Kogyo Co., Ltd., Lot No. t#AlJ, Tokyo. Japan) was used as a positive control to compare the results among four laboratories.
I/2-l/l6 of the maximum dose. In this experiment, severe bone marrow depression was observed at dose levels of more than 100 mg/kg 6-MP, so the second and third experiments were performed at doses of 3.1, 6.2, 12.5, 25, and 50 mg/kg. The dose levels of 5-FU in the two assays were 12.5, 25, 50, and 100 mg/kg. In all experiments. I m&kg MMC was administered as a positive control. Peripheral blood cells were collected just before injection and again i;t 24, 48. 72, and 96 h after treatment using the standardized protocol by CSGMT (1992). In the second cxpetiment with
Six- or seven-week-old male CD- 1 (ICR) SPF mice were purchased from Charles River Japan, Inc. (Japan). The experiments were carried out after more than 1 week of acclimatization. The
5-FU,
animals were randomly distributed into five or six groups and five mice per group were used. Commercial feed and tap water were available ad lihitum throughout imental periods.
the acclimatization
Micronucleus
TABLE
based
and exper-
upon the results
of
assay
I
MEAN FREQUENCIES OF MNRETs IN PERIPHERAL ~\DMINISTRATlON OF 5-FU TO MALE CD-I MICE Chemical
time of 120 h was included
The micronucleus assay with peripheral blood cells was carried out according to the method of Hayashi et al. (19901. The test chemicals were administered once to the ammals intraperitoneally as a solution or a suspension, and the
Selmimr of dose lewls
Dose levels were
a sampling
because we had not found an MNRET time peak in the first experiment.
Dose
Sampling time 01)
(mg/kg)
,)
BLOOD
CELLS
AFTER
A
SINGLE
INTRAPERITONEAL
24
4x
72
Yh
0.0X + 0.04 iI
0.21 It 0. I7
fl.4h + 0.37
0.40 + 0.30
0.20 + 0.3
ft.10; 0.07 0.14 * 0.05 O.Oh& 0.04
0.72 -fI 0. Ih 0.30 i 0.26 0.10+0.10
O.Sh+ 0.48 0.35 f 0.27 0.22 $0. I9
1.OZ rt 0.33 l.hSrt I.07 h
(I.46 + 0.43 3.50 + 1.h7
i).t(O + 0.22
2.58 * O.hO
Toxicity ’ 0.42 -fr0.30
3.20 c
o.II+o*lo
0.40 * 0.32 O.S8 + 0.3
0.1 h + 0.08 0.54 & 0.60
0.12 + 0.12 (1.30 + 0.30
0.0x + O.OKil 0.10 i’
2.34 f 0.75 Toxicity
2.54 f I ss Toxicity
l.XII&O.S4
0.22+0.1s
0.10 * 0. I I
120
Eipr. I Y-FL1
11.5 3.0 50.0 Ift(i
MMC
I .o
0.20 + 0.07
&3r. .? S-FW
MMC
I3
0.10+ 0.00
0. I?. * 0.07
25.0
Il.04 * 0.05
o.o~.l+ 0.00
50.0 Itlo
O.llh f 0.05 O.i~~O.17
1.0
0.02 + 0.04
0.12 + 0.07 0. I2 + 0.07 0.88 i_ 0.3
Expt. I was carried out at Taiho Pharmaceutical Co. Ltd.. and Expt. 2 at Kaken Pharmaceutical ” The frequency (5 1of MNRET, mean&SD.
0.02) 0.04 0.04 f 0.05 l.Ohf I.21 7.(1x*3.13 0.14+0.1.
Co. Ltd.
‘.’ Data for four animals (b) and one animal (cl were used because RETs could not be counted in the other ~ni~is bone marrotv depression. ’
”
RETs could not be counted in all animals because severe bone marrow depression was observed.
due to severe
inje~t~on volume was adjusted to fO rnfikg body weight. Peripheral blood was obtained by piercing the tail and collecting the blood with a mieropipet containing no antj~oa~u~ant. About 5 gtf of blood was pjacled in the center of an acrid& orange (AQI-coated glass slide prepared according to the method of Hayashi et al. (19901 and was covered with a 24 x m mm roverslip.
At 12.5 mgikg 5-FU, peak incidences of MNRET (0.46% in Expt. 0 and 0.40% in Expt. 2) occurred 48 h after injection. When animals were treated with higher doses, the peak response was defayed to 96 h. Higher response peaks (2.5UB in Expt, 1 and 2.54% in Expt. 2) were observed 96 h after treatment with 50 mg/kg 5-FU. The highest dose, 100 mg/kg, produced severe bone marrow depression from 48 h to 96 h. The incidence of MNRETs 120 h after treatment with 100 mg/kg was ‘?,fS%, the maximum 5-FU-induced incidence in Q& study
Rwults
Mi~~~~~leus tests wi& muuse ~~r~phera~ RETs were carried out independently at two iaboratories. The results with SFU- and 6-MP* treated mice are shown in Tables 1 and 2, respectiveiy. Results were similar in the two taboraturies, indicating reproducibility.
TABLE 2 MEAN FREQUENCIES QF MNRETs IN PERIPHERAL ADMINIST~AT~DN QF 6MP X-0 MALE CD-l MICE Chemical
fir. I 6”MP
MMC J?X@*z 6-MP
MMC Expr. 3 6&P
MMC
BLOOD
CELLS
AFTER
A SYNCLE INTRAPERITONEAL
Dose
Sampling time (h)
(mg/ kg)
0
24
48
72
96
0.14ctO.06 a 0.14~0.11 0.2Xf0.15
3.14+ 1.04 3.30 f 1.oo 2.34* 1.10 t,24*Ct.ft3 0.74 _t D.60 4.74 + 2.34
2.26 + 1.77 1.40 rf:0.2Y 2.43 + 1.4(i Toxicity h Toxicity 0.56 * 0.29
I.08 ?0.81 ILSHfO.19 I .40 * 0.50
f%[email protected] ff.t2+0.13 0.85 + 0.40 F 0.40+ n.32 LfO J U.3O-3_O.Zi
12.5 25.0 50.0 IO@
~.~~~O_~~
O.46+1)*11 0.34+O.II O-34+ 0.20 0.2610.15
ml
u.06
il.2h-L.O~IS
Fk 0.06
1.0
o.nrr;* 0.08
0.98 * 0.46
;I 6.3 12.5 25.0
#.I8 * o&S 0.04 * 0.M 0.10~0.12 3.06 & 0.06
&36*0X
50.0 I.@
0.@8rl:a.08 0.04 & OXB
@*#8f tr.ax 0.10+0.1u 0.20 + 0.1 Ei 0.22 + 0.08 &22+O*13 I.t4-l0+40
3.1 6_3 12.5 25.0
0.1&*0.14 0.10*0X XXI * 0. it’ 0.14&O.lr6
0.284li.f5 0.38 If O”22 0.64 + 0.43 0.40 rl_0. I9
0.54 * 2.06 2 2&l i 2.90 f
50.0 I .o
0.14fO.li c)lf8 + #.tX
0.24+0.18 I.I4,0.67
2X12* 0.93 2.90 5 0.89
0.56” 0.27 1.48rtO.43 1.54-eO.61 f.“?&B.47 4.42 + Ff.44 0.62 U&l 6.45 1.32
(3.21;4 0.11 $1-96f G.45 I .h6 + 0.48 1.60 + 0.80 2.30+ 0.27 ’ lI4urtu.19
TOXi&!
Toxicity 0.52-,0.19
K?x f 0.27 0.52 i 0.26 0,511f rt.27 0.68 * 0.51 0.90+ a.30 c [email protected]
,Expts. f and 3 were carried out at Nihon Sioresenrch Center, Inc., and Expt. 2 at the University of Shizuoka. 8 The frequency (%I of MNRETs, mean f SD. Ir RETs could not be counted in all animals due to severe bone marrow depression. c&e Data &am four a&r& (E), one &mat (rtf, and three animals fe) were used because RI33 cOUt& Eatk countedin tfteother animalsdue $0severe!bone marrow depression.
prourcol. The maximum incidences of MNRETs (3.W~ in Espt. I. I.70c; in Expt. 2. and 2.YOS: in Espt. 3) were observed 48 h after treatment with 29 or 50 mg/kg (I-MP. The incidence of MNRETs at doses higher than SO mg/kg decreased clearly at all sampling times (Expt. 1 in Table 2). The incidcncc also decreased at all doses mote than 4s h after the treatment. RETs could not be observed 72 and 06 h after treatmcnt with doses higher than SO mg/kg, due to suppression of proliferation of erythropoietic cells.
The incidence of spontaneously induced MNRETs was determined in all animals just before injection. The average frequency varied from 0.067% (Expt. 2) to 0.143% (Espt. 3). and there were thought to be few differences among these five experiments. MMC. the positive control, clearly increased the frequency of MNRETs with values varying from 1.80% to 4.7496, yielding positive results in all experiments.
Discussion Recently, micronucleus tests with mice have been widely used to detect potential clastogens or spindle poisons. Some chemicals, such as metabolic inhibitors (S-FU, 6-MP. etc.), delay the peak incidcncc of micronucleated polychromatic erythrocytes (MNPCEs) to more than 48 h iHara et al., 1089; Chuchida et al., lY91). Hayashi et al. (1984b) reported that the optimum sampling times and the effective dose ranges differed for many chemicals and it was therefore important to perform the micronucleus test under optimal conditions. Hayashi et al. (lY84b) recommended the adoption of a pilot test, that is the multi-sampling at multi-dose levels method, in order to select optimal conditions for the full-scale tests. In the micronucleus assay with peripheral blood cells, it is possible to make preparations at various sampling times from the same individual. This study showed that this assay system could detect in vivo clastogenicity by chemicals that delay peak micronucleus formation, such as 5-FU and 6-MP, and supported the opinion of Hayashi et al. ( 1990) that this system could be used as an alternative to the bone marrow micronucleus assay.
In bone marrow, the peak incidence of MNPCEs, which was induced by SO mg/kg S-FU, appeared 72 h after a single dosing (CSGMT, 1990: Ohuchida et al., lY911, while in peripheral blood the maximum response of MNRETs by S-FU at the same dose was obtained 96 h after treatment (Table I). The maximum frequencies of MNPCEs in bone marrow and MNRETs in peripheral blood cells at their optimal sampling times were almost identical, 3.13% and 2.500/c, respectively. h-MP induced MNPCEs with a peak response 48 h after a single intraperitoneal dose (Havashi et al.. 1984a; Hara et al., l989), and induced MNRETs which peaked 48 h after the dose (Tablc 2). The maximum frequencies of MNRETs and MNPCEs were different at their optimal sampling times, that is, the frequencies of MNRETs were 1.70-X30% in our results, and those of MNPCEs were about 6.0% (Hayashi et al., 1984a) or 6.98% (Hara et al., 1989). Hayashi et al. (1990) reported that the frequencies of MNRETs and MNPCEs induced by mitomycin C (MMC) were almost identical, though the induction of MNRETs by MMC was delayed for about 12 h compared to that of MNPCEs. Our results showed that the incidences of MNRETs by 5-FU and 6-MP were a little lower than those of MNPCEs reported. However, both 5-FU and 6-MP could be detected as clastogens by both micronucleus assays, with peripheral blood cells as well as with femoral bone marrow cells. There appear to be few differences in sensitivity between the two assay systems. 5-FU and 6-MP are analogues of purine and pyrimidine bases, respectively, and are known to inhibit DNA biosynthesis. 5-FU is converted to some active forms, S-FU-riboside, 5-FU-ribosephosphate, 5-FU-dcoxyribose-monophosphate, and so on. 6-MP is converted to an active form, h-MP-ribose-phosphate (6-MPRP), by hypoxanthine-guanine phosphoribosyl transfcrase. 6MPRP inhibits the pathw;y of adenylic acid biosynthesis. The metabolic pattern and metabolites are different for the above two chemicals, but both are thought to attack DNA directly or indirectly and, ultimately, to cause chromosomal aberrations. The peak responses of MNRETs were induced by higher doses of 5-FU after re-
covcry from bone marrow depression, while at higher doses of A-MP, the peak response of MNRETs was observed before the depression of bone marrow. The differences in the appearance pattern of MNRETs and in the bone marrow depression for the two chemicals are thought to be due to differences in their metabolic patterns and their metabolites. This study was carried out by four laboratories independently, two each testing S-FU and 6-MP. Similar results were obtained by all laboratories, although there were slight differences among the peak MNRET values. The present results suggest that this new assay system using peripheral blood cells and AO-coated slides produces an improved micronucleus test. References CSGMT (Collaborative Study Group for the Micronucleus Test) (1990~ Single versus multiple dosing in the micronucleus test: the summary of the fourth collaborative study by CSGMT/JEMS MMS. Mutation Res.. 233, 205-271. CSGMT f IVY11 Micronucleus test with mouse peripheral blood
crythrocptcs hy acridinc ctrange wpravi~;rl qsining: The lummox report of the 5th ctdllrhorative \tudy hy CSGMT/J~~~S.MMS. Mut~ti(~n RIB.. 27X. X3-Y>i. Ham. T.. T. Makita. N. tfttriya. S. Ozawa. M. Ohha. _I. Naitcr anrf T. Shibuya (IYXut Micronucleus test with h-mercaptopurine monnhydratc administered orally. Mutation Rcs.. 3. 340-352.
intraperittmeally
and
Hayashi, M.. T. Sofuni and M. lshidatc Jr. (lW4t1) Kinetic!, of micronuclru!+ formation in relation to chrnmosomal aherrations in mou%r hone marrou. 137.
Mutation
Res.. 117. l7Y-
Hayashi. M.. T. Sofuni and M. I%hidate Jr. (IYXJhb A pilot experiment for micronucleus Tess. The multi-sampling at multi-dose Ievcls method. Mutation Res.. 141. 165- IhY. Hayashi. M.. T. Morita. Y. Kodama. T. Sofuni and M. Lhidate Jr. 1lYYO) The micr~~nucl~us assay with mouse peripheral hltd rcticubqtrs using acridinr ~)~n~~-c(~~t~~ dides. MuI~ltil~n Res.. 34% 3-31). Ohuchida. A.. A. Furukawa. Y. Umrno. H. Tamura. K. Iwakuru. T. Ham. 1-i. Ishihara. M. Katoh. T. Shihuya and M. I layashi (IYYI)The effects of cdl cycle in hone marrow cells and !i-lluorouracil content in hone marrow on the delaying of the appearance of micronucleated polychromatic rrythrocytes hy S-fluorouracil, in preparation. Yamamoto. K.I.. and Y, Kikuchi (1081) Studies on micronucloi time response and on the effects of multiple trt’atments of mutagens cm induction of micronuclei. Mutation Rrs.. YO. 163-173.
MUTGEN
00210
Micronucleus assays on 54uorouracil and 6-mercaptopurine with mouse peripheral blood reticulocytes Akinobu Ohuchida ‘, Akemi Furukawa I, Junichi Yoshida ‘, Minako Watanabe ‘, Fu~ihiko Aruga ‘, Yoshihisa Miwa 3, Kanako Shinkawa 4* and Naohide Kinae ’ ’ 7’uilw Plrortttulnrric.nl Co. Ltd., Drtrg Sufity Lohmmty, __ TV-2 Ebisroto. Hiraishi. Kuwttcciti. Tokttshitnu 771~Oi. ’ Km& Pltcrrttlu~e~ctic~ltI Co. Ltd.. DrtrR S&y Luhorcrtoty. 301, Gctt.wkr. Fujirrlu, Slzkuoku 426. .’ Niltm Biorescarch Ctwter hr. Mujima. Fdwjtt,
Hctsltitm SOI-
atul ’
Uttitwsity of Shimoku, Sdrvul of Food wtd Nutritiwtd Sckttw. 395 Y&a. Slu:rtctka 420 (Juputt l (Accepted 9 August 19Y1)
K~ywrds:
Micronucleus test; 5Fluorouracil;
h-Mercaptopurinr:
Mouse: Peripheral hInod cells
Summary
As part of the Sth collaborative study of the Collaborative Study Group for the Micronucleus Test BCSGMT), the sensitivity and advantages of the micronucleus assay using mouse peripheral blood cells were evaluated using 5fluorouracil (5”FU) and 6-mercaptopurine (6-MP). The peripheral blood cells were collected from a tail vein of CD-I male mice just before and 24-120 h after intraperitoneai injection. At 24-h intervals. The maximum incidence of micronuclcated ret~culocytes (~NRETs) at SO mg/kg S-FU was observed 96 h after injection; at IO0 mg/kg, the peak was delayed to I20 h. and followed severe bone marrow depression, With 6-MP, maximum MNRETs were observed 48 h after treatment at all doses tested. At dose lcvcls higher than 50 mg/kg, severe bone marrow depression iclas observed after maximum MNRETs. Though the appearance patterns of MNRETs and the bone narrow depression were diffcrcnt between 5-FU and 6-MP. the positive response of both chemicals could be dctccted with this assay system as well as with the micronucleus test using femoral bone marrow cells.
As part of the fifth collaborative study on the micronucleus test. a new assay system using mouse peripheral blood reticulocytcs fRETsI was evaluated. Its advantages. and the results of conven-
Correspondence:
Akinohu Ohuchida, Drug Snfcty Labora7%7 Ebisuno, ?iiraishi. tory. Taiho Pharmaceutical Co. Ltd., -_ Kawauchi, Tokushimo 77!-01 (Japitn). * Present address: Finnish Institute of Occupational Health. Department
of Hygiene and Toxicology. Topeliuksenkntu
A. SF-OOZSOHelsinki (Finl~Ind).
-II a
tional micronucleus assays using femoral bone m~~rrow cells, were compared. We carried out micronucleus assays with peripheral blood cells on S-FU and 6-MP. S-FU and 6-MP are analogues of nucleotide bases, pyrimidine and purine, respectively, and are known to inhibit DNA biosynthesis. The assays on S-FU were carried out at two laboratories, Taiho Pharmaceutical Co. Ltd., and Kaken Pharmaceutical Co., Ltd.; those on 6-MP were performed at Nihon Bioresearch Center, Inc., and the University of Shizuoka.
Materinls
Yamamoto and Kikuchi (1981 I, Hara et al. (198% CSGMT (1990), and Ohuchida et al. (1991). The maximum dose in the first experiment with 6-MP was set at 200 mg/kg, and lower dose levels were
and methods
Both 5-FU (CAS No. 5 l-21-X. Lot No. PDK 19901 and h-MP (CA!3 No. 55-44-2. Lot No. KPN2W) were purchased from Wako Pure Chemical Industries (Osaka, Japan). 5-FU was dissolved in physiological saline. and 6-MP was suspended in olive oil. Mitomycin C (CAS No. ?I-07-7. Kyows Hakko Kogyo Co., Ltd., Lot No. t#AlJ, Tokyo. Japan) was used as a positive control to compare the results among four laboratories.
I/2-l/l6 of the maximum dose. In this experiment, severe bone marrow depression was observed at dose levels of more than 100 mg/kg 6-MP, so the second and third experiments were performed at doses of 3.1, 6.2, 12.5, 25, and 50 mg/kg. The dose levels of 5-FU in the two assays were 12.5, 25, 50, and 100 mg/kg. In all experiments. I m&kg MMC was administered as a positive control. Peripheral blood cells were collected just before injection and again i;t 24, 48. 72, and 96 h after treatment using the standardized protocol by CSGMT (1992). In the second cxpetiment with
Six- or seven-week-old male CD- 1 (ICR) SPF mice were purchased from Charles River Japan, Inc. (Japan). The experiments were carried out after more than 1 week of acclimatization. The
5-FU,
animals were randomly distributed into five or six groups and five mice per group were used. Commercial feed and tap water were available ad lihitum throughout imental periods.
the acclimatization
Micronucleus
TABLE
based
and exper-
upon the results
of
assay
I
MEAN FREQUENCIES OF MNRETs IN PERIPHERAL ~\DMINISTRATlON OF 5-FU TO MALE CD-I MICE Chemical
time of 120 h was included
The micronucleus assay with peripheral blood cells was carried out according to the method of Hayashi et al. (19901. The test chemicals were administered once to the ammals intraperitoneally as a solution or a suspension, and the
Selmimr of dose lewls
Dose levels were
a sampling
because we had not found an MNRET time peak in the first experiment.
Dose
Sampling time 01)
(mg/kg)
,)
BLOOD
CELLS
AFTER
A
SINGLE
INTRAPERITONEAL
24
4x
72
Yh
0.0X + 0.04 iI
0.21 It 0. I7
fl.4h + 0.37
0.40 + 0.30
0.20 + 0.3
ft.10; 0.07 0.14 * 0.05 O.Oh& 0.04
0.72 -fI 0. Ih 0.30 i 0.26 0.10+0.10
O.Sh+ 0.48 0.35 f 0.27 0.22 $0. I9
1.OZ rt 0.33 l.hSrt I.07 h
(I.46 + 0.43 3.50 + 1.h7
i).t(O + 0.22
2.58 * O.hO
Toxicity ’ 0.42 -fr0.30
3.20 c
o.II+o*lo
0.40 * 0.32 O.S8 + 0.3
0.1 h + 0.08 0.54 & 0.60
0.12 + 0.12 (1.30 + 0.30
0.0x + O.OKil 0.10 i’
2.34 f 0.75 Toxicity
2.54 f I ss Toxicity
l.XII&O.S4
0.22+0.1s
0.10 * 0. I I
120
Eipr. I Y-FL1
11.5 3.0 50.0 Ift(i
MMC
I .o
0.20 + 0.07
&3r. .? S-FW
MMC
I3
0.10+ 0.00
0. I?. * 0.07
25.0
Il.04 * 0.05
o.o~.l+ 0.00
50.0 Itlo
O.llh f 0.05 O.i~~O.17
1.0
0.02 + 0.04
0.12 + 0.07 0. I2 + 0.07 0.88 i_ 0.3
Expt. I was carried out at Taiho Pharmaceutical Co. Ltd.. and Expt. 2 at Kaken Pharmaceutical ” The frequency (5 1of MNRET, mean&SD.
0.02) 0.04 0.04 f 0.05 l.Ohf I.21 7.(1x*3.13 0.14+0.1.
Co. Ltd.
‘.’ Data for four animals (b) and one animal (cl were used because RETs could not be counted in the other ~ni~is bone marrotv depression. ’
”
RETs could not be counted in all animals because severe bone marrow depression was observed.
due to severe
inje~t~on volume was adjusted to fO rnfikg body weight. Peripheral blood was obtained by piercing the tail and collecting the blood with a mieropipet containing no antj~oa~u~ant. About 5 gtf of blood was pjacled in the center of an acrid& orange (AQI-coated glass slide prepared according to the method of Hayashi et al. (19901 and was covered with a 24 x m mm roverslip.
At 12.5 mgikg 5-FU, peak incidences of MNRET (0.46% in Expt. 0 and 0.40% in Expt. 2) occurred 48 h after injection. When animals were treated with higher doses, the peak response was defayed to 96 h. Higher response peaks (2.5UB in Expt, 1 and 2.54% in Expt. 2) were observed 96 h after treatment with 50 mg/kg 5-FU. The highest dose, 100 mg/kg, produced severe bone marrow depression from 48 h to 96 h. The incidence of MNRETs 120 h after treatment with 100 mg/kg was ‘?,fS%, the maximum 5-FU-induced incidence in Q& study
Rwults
Mi~~~~~leus tests wi& muuse ~~r~phera~ RETs were carried out independently at two iaboratories. The results with SFU- and 6-MP* treated mice are shown in Tables 1 and 2, respectiveiy. Results were similar in the two taboraturies, indicating reproducibility.
TABLE 2 MEAN FREQUENCIES QF MNRETs IN PERIPHERAL ADMINIST~AT~DN QF 6MP X-0 MALE CD-l MICE Chemical
fir. I 6”MP
MMC J?X@*z 6-MP
MMC Expr. 3 6&P
MMC
BLOOD
CELLS
AFTER
A SYNCLE INTRAPERITONEAL
Dose
Sampling time (h)
(mg/ kg)
0
24
48
72
96
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,Expts. f and 3 were carried out at Nihon Sioresenrch Center, Inc., and Expt. 2 at the University of Shizuoka. 8 The frequency (%I of MNRETs, mean f SD. Ir RETs could not be counted in all animals due to severe bone marrow depression. c&e Data &am four a&r& (E), one &mat (rtf, and three animals fe) were used because RI33 cOUt& Eatk countedin tfteother animalsdue $0severe!bone marrow depression.
prourcol. The maximum incidences of MNRETs (3.W~ in Espt. I. I.70c; in Expt. 2. and 2.YOS: in Espt. 3) were observed 48 h after treatment with 29 or 50 mg/kg (I-MP. The incidence of MNRETs at doses higher than SO mg/kg decreased clearly at all sampling times (Expt. 1 in Table 2). The incidcncc also decreased at all doses mote than 4s h after the treatment. RETs could not be observed 72 and 06 h after treatmcnt with doses higher than SO mg/kg, due to suppression of proliferation of erythropoietic cells.
The incidence of spontaneously induced MNRETs was determined in all animals just before injection. The average frequency varied from 0.067% (Expt. 2) to 0.143% (Espt. 3). and there were thought to be few differences among these five experiments. MMC. the positive control, clearly increased the frequency of MNRETs with values varying from 1.80% to 4.7496, yielding positive results in all experiments.
Discussion Recently, micronucleus tests with mice have been widely used to detect potential clastogens or spindle poisons. Some chemicals, such as metabolic inhibitors (S-FU, 6-MP. etc.), delay the peak incidcncc of micronucleated polychromatic erythrocytes (MNPCEs) to more than 48 h iHara et al., 1089; Chuchida et al., lY91). Hayashi et al. (1984b) reported that the optimum sampling times and the effective dose ranges differed for many chemicals and it was therefore important to perform the micronucleus test under optimal conditions. Hayashi et al. (lY84b) recommended the adoption of a pilot test, that is the multi-sampling at multi-dose levels method, in order to select optimal conditions for the full-scale tests. In the micronucleus assay with peripheral blood cells, it is possible to make preparations at various sampling times from the same individual. This study showed that this assay system could detect in vivo clastogenicity by chemicals that delay peak micronucleus formation, such as 5-FU and 6-MP, and supported the opinion of Hayashi et al. ( 1990) that this system could be used as an alternative to the bone marrow micronucleus assay.
In bone marrow, the peak incidence of MNPCEs, which was induced by SO mg/kg S-FU, appeared 72 h after a single dosing (CSGMT, 1990: Ohuchida et al., lY911, while in peripheral blood the maximum response of MNRETs by S-FU at the same dose was obtained 96 h after treatment (Table I). The maximum frequencies of MNPCEs in bone marrow and MNRETs in peripheral blood cells at their optimal sampling times were almost identical, 3.13% and 2.500/c, respectively. h-MP induced MNPCEs with a peak response 48 h after a single intraperitoneal dose (Havashi et al.. 1984a; Hara et al., l989), and induced MNRETs which peaked 48 h after the dose (Tablc 2). The maximum frequencies of MNRETs and MNPCEs were different at their optimal sampling times, that is, the frequencies of MNRETs were 1.70-X30% in our results, and those of MNPCEs were about 6.0% (Hayashi et al., 1984a) or 6.98% (Hara et al., 1989). Hayashi et al. (1990) reported that the frequencies of MNRETs and MNPCEs induced by mitomycin C (MMC) were almost identical, though the induction of MNRETs by MMC was delayed for about 12 h compared to that of MNPCEs. Our results showed that the incidences of MNRETs by 5-FU and 6-MP were a little lower than those of MNPCEs reported. However, both 5-FU and 6-MP could be detected as clastogens by both micronucleus assays, with peripheral blood cells as well as with femoral bone marrow cells. There appear to be few differences in sensitivity between the two assay systems. 5-FU and 6-MP are analogues of purine and pyrimidine bases, respectively, and are known to inhibit DNA biosynthesis. 5-FU is converted to some active forms, S-FU-riboside, 5-FU-ribosephosphate, 5-FU-dcoxyribose-monophosphate, and so on. 6-MP is converted to an active form, h-MP-ribose-phosphate (6-MPRP), by hypoxanthine-guanine phosphoribosyl transfcrase. 6MPRP inhibits the pathw;y of adenylic acid biosynthesis. The metabolic pattern and metabolites are different for the above two chemicals, but both are thought to attack DNA directly or indirectly and, ultimately, to cause chromosomal aberrations. The peak responses of MNRETs were induced by higher doses of 5-FU after re-
covcry from bone marrow depression, while at higher doses of A-MP, the peak response of MNRETs was observed before the depression of bone marrow. The differences in the appearance pattern of MNRETs and in the bone marrow depression for the two chemicals are thought to be due to differences in their metabolic patterns and their metabolites. This study was carried out by four laboratories independently, two each testing S-FU and 6-MP. Similar results were obtained by all laboratories, although there were slight differences among the peak MNRET values. The present results suggest that this new assay system using peripheral blood cells and AO-coated slides produces an improved micronucleus test. References CSGMT (Collaborative Study Group for the Micronucleus Test) (1990~ Single versus multiple dosing in the micronucleus test: the summary of the fourth collaborative study by CSGMT/JEMS MMS. Mutation Res.. 233, 205-271. CSGMT f IVY11 Micronucleus test with mouse peripheral blood
crythrocptcs hy acridinc ctrange wpravi~;rl qsining: The lummox report of the 5th ctdllrhorative \tudy hy CSGMT/J~~~S.MMS. Mut~ti(~n RIB.. 27X. X3-Y>i. Ham. T.. T. Makita. N. tfttriya. S. Ozawa. M. Ohha. _I. Naitcr anrf T. Shibuya (IYXut Micronucleus test with h-mercaptopurine monnhydratc administered orally. Mutation Rcs.. 3. 340-352.
intraperittmeally
and
Hayashi, M.. T. Sofuni and M. lshidatc Jr. (lW4t1) Kinetic!, of micronuclru!+ formation in relation to chrnmosomal aherrations in mou%r hone marrou. 137.
Mutation
Res.. 117. l7Y-
Hayashi. M.. T. Sofuni and M. I%hidate Jr. (IYXJhb A pilot experiment for micronucleus Tess. The multi-sampling at multi-dose Ievcls method. Mutation Res.. 141. 165- IhY. Hayashi. M.. T. Morita. Y. Kodama. T. Sofuni and M. Lhidate Jr. 1lYYO) The micr~~nucl~us assay with mouse peripheral hltd rcticubqtrs using acridinr ~)~n~~-c(~~t~~ dides. MuI~ltil~n Res.. 34% 3-31). Ohuchida. A.. A. Furukawa. Y. Umrno. H. Tamura. K. Iwakuru. T. Ham. 1-i. Ishihara. M. Katoh. T. Shihuya and M. I layashi (IYYI)The effects of cdl cycle in hone marrow cells and !i-lluorouracil content in hone marrow on the delaying of the appearance of micronucleated polychromatic rrythrocytes hy S-fluorouracil, in preparation. Yamamoto. K.I.. and Y, Kikuchi (1081) Studies on micronucloi time response and on the effects of multiple trt’atments of mutagens cm induction of micronuclei. Mutation Rrs.. YO. 163-173.