- Email: [email protected]
JEMS · MMS
MUTGEN
002t1l
Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary report of the 5th collaborative study by CSGMT/JEMS - MMS The Collaborative
Study Group for the Micronucleus fAcceptcd 9 Allgus
Kqww-A:
Micronucleus;
I’)01
Test
1
Periphcrai blood: Reticulocyte: Acridine orange: Supravital staining
Summary The main goal of the Collaborative Study Group for the Micronucleus Test (CSGMT) was to validate a new method for the micronucleus test, recently introduced b/ Hayashi et al. (lc)YOl. using mouse peripheral blood cells stained supravitally with acridine orange (AO). The micronucleus tests were performed on CD-l mice using 23 chemicals with various modes of action. As a rule, one chemical was studied by two participants. Peripheral blood sampled from the same animal was examined 0. 24.48, and 72 h (or longer) after treatment. The frequencies of micronucleated peripheral reticulocytes (MNRETs) were recorded based on observation of 1000 reticulocytcs per mouse. All chemicals induced MNRETs dose-dependently. Interlaboratory differences in the induction of MNRETs were in an acceptable range for most chemicals tested. Although differences wcrc observed with some chemicals, there were no discrepancies iit qualitative judgment. Most chemicals gave the greatest response 48 h after treatment, which was less variable than in the bone marrow assay (grcatcst response, 24-48 hl. These results suggest that the peripheral blood assay using the A0 supravital staining technique generates reproducible and reliable data to evaluate the clastogcnicity of chemicals. This makes the peripheral blood micronucleus assay an attractive alternative to the conventional bone marrow assay.
The Collaborative Study Group for the Micronucleus Test (CSGMT) is a subgroup of the Mammalian Mutagenesis Study Group, a suborganizatton of the l%vironmental Mutagen Society of Japan (JEMS * MMS). CSGMT has investigated the influence of a number of factors involved in the micronucleus test, namely, sex of
Correspondence:
Makoto
Mutagenesis, National
Hayashi. Division of Genetics and
Institute of Hygienic Scicnccs. I-IX 1.
Kamiyoga, Setagaya-ku, Tokyo iSX (Japan).
the test animals (CSGMT, 1986). strain (CSGMT. lY8X). administration route (Hayashi et al.. IYXY). and dosing times (CSGMT. 1900). Results from those collaborative studies provided basal data for the micronucleus test in bone marrow cells. Micronuclei are also detectable in smeared peripheral etythrocytes (MacGregor et al., IYtiO). Because the fraction of young erythrocytcs in the peripheral blood is small, which makes micronuclcus evaluation very laborious. and because objective discrimination between young and mature erythrocytes is not easy. the peripheral blood
T:\BLE
I
PARTICIPANTS
IN THE 5TH COLLABORATIVE
STUDY
Chemical studied %’ 7-AAF
N. Asano *: Nitto Denko Corporation T. Hagiwara: Sankyo Co.. Ltd.
&a-C
K. Iwakura. H. Tamura and A. Matsumoto: Nippon Shinyaku Co.. Ltd. S. Ajimi, S. Ogura, K. &kimoto and Y. Kajiwara: Chemicals Inspection
BEN
Y. Hatakeyama. H. Atai and S. Suzuki: Central Institute E. Nakajima: Toyobo Co.. Ltd.
B[a]P
H. Shimada *. S. Itoh. C. Hattori. Y. Matsuura and S. Tada: Developmental Research P~d~aceutical Co.. Ltd. H. Suzuki and C. Watan~I~: Research Center of Taisho Pharmaceutical Co.. Ltd.
COL
M. Nakajim~~. T. Kitazawa, M. Fujiwara. K. Suzuki and Y. Ukai: B&safety Pesticides K. Miyahana and K. Inour: Nihon Noyaku. Co.. Ltd.
CYP
Y. Hatanaka, Y. Kitagawa and Y. Toyoda: Suntory Co., Ltd. T. Kawatn. N. Ando, Y. KaWabata. M. lwai and H. Arimura: Green Cross Co.. Ltd.
DMBA
M. Hayashi *, T. Suzuki. Y. Kodama, A.O. Asita. A. Matsuoka and T. Sofuni: National Sciences K. Tamai, M. Kurita, H. Ohtsuki and T. Hiwatashi: Health Soienccs Research Institute
DMN
s. Sat0 * and M. Taketomi: Japan Tobacco. Inc. T. Morita *: Nippon Glaxo. Ltd.
EMS
K. Kondo: Shionogi and Co., Ltd. S. Gzawa: Kissei Pha~a~eutic~I Co.. Ltd.
ENU
S. Hitotsumachi and Y. Kimura: Takeda Chemical industries. Ltd. M. Katoh, N. Ishihara, T. Ham and T. Shibuya: Food and Drug Safety Center
5-FU
A. Ohuchida and A. Furukawa: Taiho Pharmaceutical Co., Ltd. J. Yoshida and M. Watanabe: Kaken Pharmaceutical Co., Ltd.
K.BrG,
T. Morita *: Nippon Glaxo Ltd. M. Uejima. T. Kuwahara, S. Asanami and K. Shimono: Otsuka Pharmaceutical
for Experimental
and Testing Institute.
Japan
Animals
Research
bbOKitOfy
Center,
of Daiichi
Foods. Drugs and
Institute
of Hygienic
Factory. Inc.
K $30,
T. Awogi *: Otsuka Pharmaceutical Co., Ltd. K. Murata: Kumiai Chemical Industry Co.. Ltd.
MMC
M. Hara and S. Nakagawa: Sumitomo Chemical Co.. Ltd. E. Fujioka, E. Ayukawa and T. Izushi: Fuji Photo Film Co., Ltd.
MMS
c. Sugiyama. H. Kt~bayashi and M. Mori: Shiseido Toxicological Analytical Research Y. Miyamae. Y. Fujino and K. Ohara: Fujisawa Pharmaceutical Co., Ltd.
MNNG
M. Kuramochi. H. Seki and T. Tazawa: Biomedical Laboratories, S. Sasaki and Y. Sakai: Ono Pharmaceutical Co., Ltd.
h-MP
F. Aruga and Y. Miwa: Nihon Bioresearch Center, Inc. K. Shinkawa and N. Kinae: University of Shizuoka
MTX
Y. Kasahara. Y. Nakai. C. Miura. K. Yagi, K. Hirabayashi and T. Makita: Trijin Ltd. A. Wakata * and K. Yuno: Yamanouchi Pharmaceutical Co.. Ltd.
PCZ
F. Romagna: Sandoz Pharma Ltd. H. Matsumura, M. Watanabe, T. Kato, Y.F. Sasaki * and Y. Shirasu: institute K. Ohmori and H. Yamada: Pfizer Pharmaceutical, Inc.
Center
Inc.
of Environmental
Toxicology
x5 TABLE I (continued) Chemical studied “
Investigators
PFf EN
N. Higashikuni. T. Nakamura and S. Sutou *: Itoham Central Research lnstitute T. Baba, K. Tsurekawa and T. Sera: Daicel Chemical Industries
TEM
E. Yamamura, H. Hirono and M. Takeuchi: Yoshitomi Pharmaceutical Industries. Ltd. M. Kojima and S. Aoki: The Biological Research Center for the Protection of Environment
URET
M. Kishi: Kanagawa Prefectural
VINCI
Y. Kondo, S. Nito and F. Ariyuki: Tanabe Seiyaku Cc Ltd. S. Honda, M. Hayashi, Y. Shinagawa and S. Sato: Toyama Institute of Health
Public Health Laboratories
” For the full names of the chemicals see Table 2. * Organizer.
method has not been widely used. The clastogenicity of chemicals is thus routinely assayed in bone marrow cells stained with Giemsa or acridine orange (AO). Recently, Hayashi et al. (1990) reported a new method for the preparation and observation of micronuclei in peripheral blood reticulocytes using AO-coated glass slides. This method greatly facilitates discrimination between young and mature erythrocytes. The purpose of this 5th collaborative study is to validate the new method using 23 clastogens and spindle poisons with different modes of action; 46 laboratories participated (Table 1). Detailed results on each chemical are reported individually. This summary report presents the overall design, results, and discussion.
Materials and methods
The 23 chemicals used in the present study are listed in Table 2 with their abbreviations, CAS registry numbers, sources, and lot numbers. The chemicals were classified into eight groups according to their mode of action, as shown in Table 2. Each chemical was evaluated by two laboratories independently, with two exceptions: procarbazine was studied by three laboratories and urethane was evaluated by only one laboratory. As a rule, the same chemical lots were used in the paired laboratories. All laboratories used the same lot of mitomycin C (1 mg/kgI as the positive reference chemical,
Animals
CD-I male mice (Charies River Japan or Germany, Inc.) were used by all participants. Approximately 7-week-old mice were purchased, acclimatized for a week or more, and treated at S-10 weeks of age. They were given commercial pellets (not specified) and water ad libitum throughout the acclimatization and experimental periods.
AO-coated slides were prepared at each Iaboratory according to Hayashi et a1. (1990). Briefly, 10 ~1 of 1 mg/ml A0 aqueous solution was spread homogeneously on a warmed glass slide. 5 ~1 of peripheral blood, collected by piercing the ventral tail, was placed without any anticoagulant on the center of an AO-coated glass slide and covered immediately with a 24 x 40 mm coverslip. AO supravitai~y stained reticulocytes were examined by fluorescence microscope with a blue excitation and a yellow barrier fiiter. The frequencies of micronucleated peripheral reticulocytes (MNRETs) were recorded based on the observation of 1000 reticulocytes per mouse. Reticulocytes were restricted to types I, II, and III (Fig. 1) of the classification by Vander et al. (1963). The i?~~niFn~~required protocol
The minimum requirements for each participating laboratory proposed by the organizing committee were as follows.
I‘.ABl.I:
2
LlSl‘ OF CtlEXlICXLS
TESTED Ahhreviation
C‘hemicai tested
source .’
Lot No.
75Y-73-Y
A s W N N
CWOShOYLT 3XFOXXZ TLP7US MOAh4SO VOAl700 VOH4.39 I VOBl7hl 07717TV X4505
CAS No.
CYP DMN I-c!-so-o
Ethyl methanesulfonatr ~-Ethyl-,V-nitrosourea
EMS ENU
Methyl methancsulfonate .V-Methyl-N ‘-nitro-:V-nitrohoguanidine Triethylcnemelamine
MMS MNNG TEM
66-27-3 70-25-7 Sl-IX-3
A P
Ara-C 5-FU h-MP MTX
l-17-04-4 5l-‘I-X 55-44-2 59-0.5-2
W W W
‘40 l2Y PDKIYYO KPN2934 ECQl303
‘-AAF PHEN
53-96-3
W W
AWFh303 KPL453
R[a]P DMBA
50-32-X
s7-07-h
T W
AXO! SAN I X74
MMC
50-07-7
K
h4YAIJ
N
NS
.-lnmtrrtic cmOw
2-Acetylaminofluorene Phenacetin
62-34-2
Polwyc-lie orormrtic- h~dr0curh0n.s
Benzo[lr]pyrene 7.12-Dimethylhenz[o]anthracrne
Inor.wrric c~iwinic~ctis
Potassium hrom:.Le Potuss .n chromate(VI)
KBrO, K $30,
775%01-2 77XY-00-h
W W
SAR3708 KPG72Y7
Spitz& poisur2.s Colchicinr Vincristine sulfate
COL VINC
h-l-Xh-X ZOhX-?X-2
W S
SANIhlY hYF-OS36
BEN PCZ URET
71-43-Z 3%70- 1 5I -70-h
W NR S
KPE73YX TO161 h9F-0633
~li.~~~cll~tttt~oi~s chcvnicab
Benzene Procarbazine Urethane
hydrochloride
” A: Aldrich Chemical Co.. Inc.. Milwaukee. WI (U.S.A.); K: Kyowa Hakko Kogyo Co.. Tokyo (Japan): N: Nacalai Tesque Inc.. Kyoto (Japan): NR: Nippon Roche K.K., Tohyo (Japan); NS: Nippon Shinyaku Co., Ltd.. Kyoto (Japan): S: Sigma Chemical Co.. St. Louis. MO (U.S.A.): T: Tokyo Chemical Industry Co.. Ltd.. Tokyo (Japan): P: Polysciences Inc.. Washington. PA (U.S.A.): W: Wako Pure Chemical Industries. Ltd.. Osaka (Japan).
Erythroblast Fig. I. Schematic presentation
Nucleus expulsion
Reticulocyte
of red fluorescing reticular structure in each type of reticulocyte. II. and 111reticulocytes per mouse were observed.
MZUWZ erythmcyte In the present study, lOOI type I,
72
Fig. 2. Experiments
were performed
frequencies of MNRETs
independently
at two different
laboratories
using peripheral
after treatment with (11)CYP: (b) DMN: (c) EMS: (d) ENU: (e) MMS: (f)
blood of CD-I
mice. Mean
MNNG: (g)TEM; (h) am-C: (i)
S-FU: Q) (I-Ml’: (Ii) MTX: (I) 7-AAF: (m) PHEN: (n) B[a]P: (01 DMBA; (p) MMC: (q) KBrO,: (r) K,CrO,: (5) COL: (1) VINC: BEN; (v) PCZ: (w) URET. The experiments in (VI were performed indcpcndently at three laboratories.
(~1
I.
n
00
M td R E T s
f%I
9,
-2
r
0
MNR E T
MN R E T
s
s
(Xl
(%I
Q, 0 M
s; N
R
E
T
3
(Xl
M
N
R
E
M N R E
T
I
3
s
(%I
I%)
100
,d
50
50 p'
Fig.2 (continued).
4 b9
-3 m -1
800 ,a’
Fig.
2 !rontinucd).
80G ,d
Yl
212 iL%’
60 pa
2.0 (d
Fig. ?. (canhwi).
60
Fig. 2 (continued).
(Ii Each dose group consisted of at least five male mice. (21 At leas? three dose groups and a positive control group were used. (3) The doses were chosen based on the data reported by CSGMT ( 1986, 1989. 1990) or determined experimentally. (4) Mice were treated once intraperitoncally or by gavage. (5) The peripheral blood was examined at 0, 24, 48, and 72 h (or later) after treatment. Blood was sampled at 48 h after mitomycin C (MM0 treatment in the positive control group. In addition, optional experiments, e.g., comparison between peripheral blood and bone marrow, use of rats instead of mice, and multiple treatments, were designed freely by the participants. Before starting the collaborative study, a workshop was held for the participants where details of the experimental methods and standardization of the classification of reticulocytes were discussed. Results
The results of the micronucleus tests are presented in tabular form in each report. Here, the overall results are shown in Fig. 2. All chemicals tested induced MNRETs dose-dependently. The majority of chemicals showed a maximum response 48 h after treatment. Although laboratory differences wcrc seen with some chemicals, most chemicals tested showed minor or no differences between laboratories.
All alkylating agents studied, i.e., CYP (Fig. 2a), DMN (2b), EMS (2~). ENU (2d), MMS (2ej, MNNG (2fj, and TEM (2g), induced MNRETs dose-dependently with good agreement between laboratories. Almost all cases showed similar time-response curves with a single peak at 48 h after treatment. At both laooratories DMN gave a weak but positive response and peaked at 48 h at the only effective dose, 10 mg/kg. At one laboratory MMS showed a maximum response 36 h after treatment while it was 48 h at the paired laboratory.
Ara-C (Fig. 2h) and h-MP (2jj induced MNRETs strongly and dose-dependently at both laboratories in like manner. These two base analogues evoked time-response curves with one peak at 48 h, which is comparable to the alkylating agents. MNRET frequencies induced by S-FU, however, did not follow the time-response curves obtained by the above two chemicals (2i). The maximum response times tended to be dose-related. MTX also showed a positive response at both laboratories (2kj. Peak MNRET frequencies were observed at 72 h in both laboratories.
The frequencies of MNRETs increased after treatment with 2-AAF or PHEN dose-depcndently (Fig. 21. 2mj. Time response curves peaked 48 h after treatment with 2-AAF at all dose levels at both laboratories. PHEN also induced its maximum response at 4X h. Poiyqclic Iryilrocurhotis B[a]P induced MNRETs dose-dependently peaking at 4X h after treatment (Fig. 3n). DMBA also increased frequencies of MNRETs dose-dependently, with the maximum responses being observed at or later than 4X h after treatment (201.
Fig. 2p shows time-response curves for each MMC dose level. The results of the two laboratories were comparable, and the maximum responses were observed 48 h after treatment.
Both K&O, and K,CrO, induced MNRETs dose-dependently with the peak usually observed at 48 h (Fig. 2r, s). There was a hint in these data that the time response was dose-dependent.
COL and VINC showed positive responses (Fig. 2s 0. These two spindle poisons also induced peak responses 48 h after treatment, and were thus comparable to other chemicals. This contrasts with the bone marrow method, in which the maximum response was usually observed ear-
The masimum frequency of MNRETs was ohsc~cd -18 h after treatment with BEN (Fig. 3) in fhc two laboratories. PCZ was studied at three laboratories. each of which ohtainCd positive. dose-rclntcd responses with peak freyucncies of MNRETs obscrvcd at 4X h (3). URET, which W:ISassayed at one I:+oratory. induced MNRETs dose-dcpendcntly wittl peak frequencies also obsened IS h after treatment (2~).
The spontaneous frequencies of MNRETs wt‘re evaluated on peripheral blood collcctcd from 1179 mice immediately before trcatmcnt with each chemical throughout the present collahorative study. MNRET frequencies ranged between 0 and 7 per 1OW RETs. and the mean + SD was I.15 5 1.24 per 101)ORETs (Fig. 3). This distribution fit the binomial distribution surprisingly well (R(WOi3~. IOWA. P = 0.7 by x2 test). ft indicates that vari~~tions in the data are due to chance, which is an advantage for evaluating the clasto-
No. MNRETs Fig. 3. Distribution
of MNRET
/ 1000
RETs
frcqurncies in mice immcdi-
at&y before trc:mnrnt. Da13 from ail l~l~or~t(~ri~~ were combined. for a total of 117’1 CD-1 mice. Bars show the tthsrrv~d frcyuaxk
and circles the cxpcctcd values from the thectretical hinomi;li distrihutittn 6(0.001~, 1000).
genicity of the tusted chemicals. The reproducibility and reliability of the new simple method are shown by the stable distribution of MNRETs in the negative control. Some participants performed experiments including solvent control groups, i.e., mice were treated with physiological saline or olive oil alone. Time-response curves arc shown in Fig. 4, and no positive response was observed at any time after treatment. This is good evidence to support the experimental designs that sample blood at 0 h instead of using a solvent control group. It also indicates that stress from the expcrimcntal procedures did not influence the background frequency of MNRETs, cvcn after S daily samplings.
In this collaborative study, all participants were requested to examine the MNRETs from mice treated with I mg/kg MMC (same loti 48 h after treatment as the reference control. The individual numbers of MNRETs per 1000 RETs scored at each laboratory arc piotted in Fig. 5. MNRET frcquencics were distributed widely bctwccn 2 and X9 per 1000 KETs. and frequencies at each laboratory (mean of:SD) ranged between 16.X L5.81 and 51.2 + 13.0 per 1000 RETs. One of the possible factors causing such laboratory differcnces is different definitions of RETs among microscopists. Although two mice showed values within the negative control range, possibly bccause of improper dosing, all other mice showed significantly increased frequencies of MNRETs,
0
. (2)
. (2)
Laboratories Fig. 5. Pistrih!6nn
of the numher of MNRETs
mice had been tread
i.p. with
4X h after
1 mg/kg MMC.
with one mouse showing an exceptionally high frequency of MNRETs. Excluding these three outliers, other points were distributed randomly around the global mean, 33.7 MNRETs/lOOO RETs. Optional studies Detailed data are presented in individual papers in this issue but are summarized very brieily here. CYP (Hatanaka et al., 19921, DMN (Sat0 et al., 19921, ara-C (Iwakura et al., 1992), 2-AAF (Asano et al., 1992), DMBA (Suzuki et al., 19921, and URET (Kishi et al., 1992) were studied with both peripheral blood and bone marrow erythrocytes, and comparable responses were obtained. On the other hand, although VINC, MMS, and PHEN gave positive responses in peripheral blood, a lower percentage of micronucleated cells was observed in these young erythrocytes than in\ the bone marrow test. CYP (Hayashi et al., 1992), B[a]P (Shimada elt al., 19921, MMC (Hayashi et al., 1992), and BEr\l (Hatakeyama et al., 1992) were used for a comparison between mice and rats. CYP, MMC, and BEN induced MNRETs comparably in rat and mouse peripheral blood. Only a slight increase of MNRETs was observed among rat peripheral reticulocytes after treatment with B[alP. In the
rat, the effect of splenectomy was studied with MMC in the induction of MNRETs. Splenectomy was found to be effective in increasing the number of MNRETs observed especially at the high dose level, but with large deviations. The effects of repeated treatments were studied with MTX (Kasahara et al., 1992), 2-AAF (Asano et al., 19921, PHEN (Higashikuni et al., 19921, and MMC (Hara et al., 1992). With PHEN and MMC, repeated treatments induced higher MNRET frequencies. One experiment with MTX showed an enhancement of MNRET induction after triple treatments but the other experiments did not show significant differences or single treatment was more effective. The ratio of type I, II, III, and IV RETs to total RETs was studied after treatment with ara-C by Iwakura et al. (1992). They concluded that the proportion of type I RETs to total storable RETs was potentially a good way to assess the cytotoxicity of the test chemical, as is the ratio of PCEs to total erythrocytes in the conventional bone marrow method. Discussion The micronr&eus assay with peripheral blood There are two main uses for the peripheral blood micronucleus test. One is to evaluate the acute clastogenic effects of test chemicals (MacGregor et al.. 1980, 1990; Tice et al., 1987, 1990), and the other is to assess the chronic chromosome-damaging activity of chemical substances (Schlegel and MacGregor, 1982: Barale et al., 1985; Choy et al., 1985; Jauhar et al., 1988; Rithidech et al., 1988; MacGregor et al., 1990). The former is done by scoring restrictively young erythrocytes, and the latter is done by scoring mature erythrocytes. The aim of the 5th collaborative study presented here was to validate the new A0 supravital staining method described by Hayashi et al. (1990) using known clastogenic chemicals with various modes of action. The micronucleus assay using peripheral blood has also received attention in the field of regulatory sciences. The Gene-Tox Program organized by the U.S. Environmental Protection Agency regarded it as a variation of the conventional bone marrow micronucleus assay (Mavournin et
al.. 1~~0). In the manual for the Japanese guidclinch (J;~pan MHW. 1YYI) for drug toxicity studich. it is htatcd that ‘c\vn those data obtained by studies using tissues other tl‘,rn the bone marrow (e.g.. the peripheral blood) and those obtained by a testing method involving differentiation and counting of micronuclei by some mechanical means may 31~0 be acceptable as a subject of evaluation if they are justified from the scientific viewpoint’. Thereforc, at the present moment, it is important to validate the method using peripheral blood as the material for the micronucleus iMay.
The transition time from bone marrow MNPCEs to peripheral blood MNRETs was reported to be about 24 h by MacGregor et al. (Ic)W, iW0). The same transition time was shown in the present study with several chemica!s. According to more precise time-course studies. however. the transition time was shown to be about 12 h with MMC and ara-C (Hayashi et al., 1YYO: lwakura et al., 19%). This difference might simply depend on the study protocol, i.e., the former results were obtained in the study sampling with a 24-h interval. The advantages of the micronucleus test using peripheral blood instead of bone maT;‘ow crythrocytes were discussed by MacGregor et al. (1980). They pointed out the following. (1) Repeated samples are easily obtained from the same animal without killing it. (2) Sample preparation is simpler. (3) It is simpler to score peripheral blood smears than conventional bone marrow smears. (4) The micronuclei are easily distinguished because they are the only darkly staining features on the slide. (5) The cell population is uniform enough to apply automated scoring of micronucleated cells. They also reported that the test using peripheral blood was at least as sensitive as the bone marrow method.
The A0 supravital staining method has additional advantages to those stated by MacGregor et al. ( 1980). ( 1) A more accurate identification of micronuclei can be made (Hayashi et al., 1983). (2) Classification of young erythrocytes is less subjective because supravital staining identifies reticulocytes. (3) More precise classification of
RETs is possible by yuantificaticjp of the amount of red fluorescing reticular material. Therefore. rat peripheral blood can also be used by restricting the scoring population to very young reticulocytes (types I and II). (4) Very stable background frequencies of MNRETs have been shown in the present study. (5) Sample preparation, only involving placing blood on the AO-coated glass slide and putting a cover slip on it, is :;mpler than smear preparation. Disadvantages of the A0 supravital staining method are: (1) slide preparations cannot be stored for long; (2) preselection of good quality regions is frequently necessary; (3) the method to assess the inhibition of crythropoiesis caused by the test chemical is still under development.
Although the new method for the micronucleus test using peripheral blood is considered to have several advantages as mentioned above, its validity has not been shown. To validate this new method, the following should be confirmed. (1) The new method is sensitive enough to detect the clastogenic activity of known clastogens with different modes of action. (2) The frequency of MNRETs in peripheral blood reflects that of MNPCEs in the bone marrow. (3) The data are reproducible. (1) All clastogenic chemicals with different modes of action tested here gave a positive response. These chemicals included MNNG and DMN, which gave contradicting results in the conventional bone marrow assay. COL and VINC, which are spindle poisons, also induced MNRETs dose-dependently. (2) In the present collaborative study, CYP, DMN, and BEN gave comparable responses in peripheral MNRETs and bone marrow MNPCEs. However, VINC, MMS, and PHEN induced higher frequencies of MNPCEs than MNRETs. It is difficult to precisely compare the frequencies of micronucleated young erythrocytes in the peripheral blood and in the bone marrow, because the populations might be different according to the criteria for RETs and PCEs. In the case of VINC, cells containing large micronuclei may have been eliminated before they could enter the peripheral blood, because several large-micro-
nucleated young crythrocytes were observed in the bone marrow, but not in the peripheral blood
(data not shown). In the hone marrow studies, the majority of chemicals studied by CSGMT induced maximum MNPCE frequencies 24-48 h after treatment. For example, COL (Asano et al., 19X9)and VINC (Ohuchida et al., 1989) induced maximum MNPCEs at 24 h, and 6-MP (Hara et al., 19891, B[a]P (Awogi and Sato, 19X9), and DMBA (Mizuhashi et al., 1989) did so at 48 h. On the other hand, all those chemicals, and the others tested here, gave positive, but not necessarily maximum responses 48 h after treatment. This is quite useful for determining sampling times for unknown chemicals, because peak responses seem iu be cunfined to a narrower period, around 48 h after treatment. (3) The reproducibility of results by this new method was illustrated in the present collaborative study. There were almost no qualitative differences between laboratories at which the same chemicals were studied independently, although the absolute values of the frequencies of MNRETs varied between laboratories, These differences may not be critical; data from the conventional bone marrow method also varied between laboratories and even between experiments at the same laboratory. Conclusion
In conclusion, our validation study for the A0 supravital staining method for rodent peripheral blood reticulocytes clearly showed its usefulness for the evaIuation of the clastogenjcity of test chemicals. We also confirmed the usefulness of peripheral blood reticulocytes for the rodent micronucleus assay. Therefore, this method offers an alternative to the conventional bone marrow micronucleus assay. Because it is possible to obtain time-response data concomitantly with the dose-response relationship, more reliable information about micronucleus induction could be obtained than with the conventional bone marrow assay. Moreover, since samples at 0 h can serve as the negative control, the number of animals, at least for the negative control group, can be reduced without any reduction in scnsitiv-
ity. The fact that most chemicals gave peak responses at 4X h could sirnplj~ the test method by more narrowly defining and reducing sampling times. The negative aspects of the A0 supravital staining technique are few. Acknowledgement
We are indebted to Dr. Miriam Bloom for reviewing all the manuscripts of this collaborative study. References A~no.
N.. and T. Hagiwara
(1’1921 The mouse peripheral
hl<~~~dmi~~~~u~ieus test of 2-acetylaminc,nuorcne with acridine OI: .lge supravital staining method, Mutation Ruk.. 178. 153-157. Asano. N.. T. Morita and Y. Watanabe
(IYXY) Micronucleus
test with colchicine given hy intraperitc>nral injection and oral gavage. Mutation Res.. 213. 3Y I -3Y4. Awogi. T.. and T. .%to (3989) Micronucleus test with henzo[ a]pyrenc using a single peroral administration and intraperitoneal injection in males of the MS/Ae and CD-I mouse strains, Mutation Res.. 113. 353-356. Barale. R.. F. Giorgrlli. L. Mi~li~~r~. R. Ciranni. D. Casini. D. Zucconi and N. Loprieno (1985) Benzene induces micmnuclei in circulating crythrocytos of chronically treated mice. Mutation Res., 144. lY3-lYh. Choy. W.N., J.T. MacGregor. M.D. Shclhy and R.R. Maronpot (10x5) Induction of micronuclei hy benzene in BbUF, mice: retrospective analysis of peripheral blood smears from the NTP csrcinogencsis hioashay. Mutation Res.. 14.3. 5%50. CSGMT (Colla,llahorativeStudy Group
for the Micronucleus
Test) (1’186) Sex difference in the micronucleus lest. Mutation Res.. 172. 151-163. CSGMT (lY8X) Strain difference
in the micronucleus test.
Mut~t~~)n Res., 314. 307-316. CSGMT f IYYO) Single versus multiple dosing in the micronucleus test: The sumrn~~ of the fourth collaborative study by CSGMT/JEMS.MMS. Mutation Res.. 234. X5-22 Iiara, T., T. Makita. N. Horiya. S. Ozawa, M. Ohha. J. Naito and 1‘. Shihuya (IYXY) Micronucleus test with h-mrrcaptopurine monohydrutc administered orally. Mutation
intrapcritoneally
and
Res.. 233. 349-352.
Hatab;y:ina. Y.. H. Atai. S. Suzuki and E. Nakajima (IYY?) Effects trf benzene in ;I micrcmuclcu> tust on peripheral blood utilizing
acridine
orange-coated
slides. Mutation
Res.. 27X. 19% IYS. IIatanaka. Y.. Y. Kitagawa, Y. Topda. T. Kawata. N. Ando. Y. Kawubata. M. Iw;li and H. Arimura (It)911 Micronucleus test with cycl~ph~sphumide using mouse peripheral blood retir*b!ctcytes. Mutation Rrs.. 27X. YY- 101. Httyashi. M., ‘I. Sofuni and M. Ishidate Jr. (ICI831 An applica-
&+;~&I. M.. S. Sutctu. IT Shims&. S. Sate. Y.F. Sasaki and A. Waknta IIWW Difference between intr~p~ritoneal and oral savage appl~cati~~n in the micronucleus test. The 3rd eoilabocuivr study hy CSGMT/JEMS~ MMS. Mutation Rcs.. 27.3, 329-3-J-t. Hayitshi. M.. T. Morita. Y. Kodama. T. Sofuni and M. lshidate Jr. (1V-M)) The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides, Mutation Res.. 215. 2-15-240. tlayashi. hl.. Y. Kodama. T. Awogi. T. Suzuki. A.O. Asita and T. Sofuni ( lY92) The micronucleus assay using peripheral blood reticulocytes from mitomycin C- and cyctophosphamide-treated rats. Mutation Res.. 2%. X9-213. Higashikuni N.. T. Nakamura. T. Baba and S. Sutou flYY21 The mi~r~~nucleus test of ph~na~etin with reticulocytes of mice, Mutati~~n Res.. 27% IS9-164. iwakura. K.. H. Tamu*a. A. ~l~tt~~rnc~t~~. S. Ajimi, S. Ogura, K. Kakimota and T. Matsumoto (1992) The micronucleus assay with peripheral blood retictdocytes by acridine OFange supravital staining with I-B-o-arabinofuranosylcytosine. Mutation Res., 27X. 131-137. Japan MHW (1991) Guidelines for Toxicity Studies of Drugs Manual. Is: edn. for 1990, editorial supervision by New Drugs Division, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare. Yakuji Nippo, Tokyo. pp. 55-59. Jauhar, P.P., P.R. Hmika. J.T. MaCciFegOF, C.M. Wehr. M.D. Shelby. S.A. Murphy and B-H. Margolin (198X) 1,3Butadiene: induction of micronucleated erythrocytes in the peripheral blood of BhC3F, mice exposed by inhalation for 13 weeks. Mutation Res.. 209, 171-176. Kasahara, Y.. A. Wakata. Y. Nakai, K. Yuno, D. Miura, K. Yagi. K. Hirabayashi and T. Makita (1992) Use of mouse peripheral blood reticulocytes for micronucleus test with methotrexate. Mutation Res.. 278, 145-151. Kishi. M.. Y. Horiguchi. S. Watanabe and M. Hayashi (lYY2) Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with ethyl carhamate. Mutation Res., 278, 205-208. MacGregor. J.T.. CM. Wehr and D.H. Gould (1980) Clastogen-induced micronuclei in peripheral blood erythroeytes: the basis of an improved micronucleus test, Environ. Mutagen.. 2.509-514. MacGregor, J.T., C.M. Wehr, P.R. Henika and M.D. Shelby ftYY0) The in vivo erythrocyte micronucleus test: measurement at steady state increases assay efficiency and permits
integration with toxicity studies. Fund. Appl. Toxicoi.. 14. 513-522. Mav~~urnin, K.H., D.H. Blakey. M.C. Cimino, M.F. Salamone and J.A. Heddle (1990) 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. Mizuhashi, F.. K. Murata, T. Kitagaki. T. Nishitomi, A. Hashimoto and A. Kido (1989) Administration-route-related difference in the micronucleus test with 7,12-dimethylbenz[a]anthracene. Mutation Res., 223, 357-360. Ohuchida. A., A. Furukawa. Y. Kondo. T. Ono and S. Nito (1989) Micronucleus test with vincristine administered by intraperitoneal injection and oral gavage, Mutation Res., 223. 395-398. Rithidech. K.. W.W. Au, V.M.S. Ramanujam. E.B. Whorton Jr. and MS. Legator f198XI Persistence of micronuclei in peripheral hlood normochromalie erythrocytes of subchronically benzene-treated male mice, Environ. Mol. Mutagen.. 12, 319-329. Sato. S.. M. Taketomi and T. Morita (1992) Simplified mouse peripheral reticuiocyte micronucleus test with dimethylnitrosamine. Mutation Res.. 278. 103-107. Schlegel, R. and J.T. MacGregor (1982) The persi-trnce of micronuclei in peripheral blood erythrocytcs: detection of chronic chromosome breakage in mice, Mutation Res.. 104, 367-369. Shimada, I-I., H. Suzuki, S. Itoh. C. Hattori. Y. Matsuura. S. Tada and C. Watanabe (1992) The micronucleus test of ben2~~]pyrene with mouse and rat peripheral blood reticulocytes, Mu~tion Res., 278, 165-168. Suzuki, T.. T. Hiwatashi. K. Tamai. Y. KOddm. A.O. Asita. A. Matsuoka. T. Sofuni, M. Kurita. H. Ohtsuki and M. Hayashi (1992) Micronucleus induction in mouse peripheral reticulocytes by 7,12-dimethylbenz[a]anthracene. Mutation Res., 27X. 169-173. Tice, R.R.. C.A. Luke and M.D. Shelby (1987) Methyl isocyanate: an evaluation of m vivo cytogenetic activity. Environ. Mutagen.. 9, 37-Z. Tice, R.R.. G.L. Erexson. C.J. Hillard. J.L. Huston. R.M. Boehm, D. Gulati and M.D. Shelby (1990) Effect of treatment protocol and sample time on the frequencies of micronu~ieated polychronIati~ erythrocytes in mouse bone marrow and peripheral blood. Mutagenesis, 5, 3 23-321. Vander. J.B., C.A. Harris and S.R. Ellis (1963) Reticulocyte counts by means of fluorescence microscopy, J. Lab. Clin. Med., 62. l32- 140.
002t1l
Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary report of the 5th collaborative study by CSGMT/JEMS - MMS The Collaborative
Study Group for the Micronucleus fAcceptcd 9 Allgus
Kqww-A:
Micronucleus;
I’)01
Test
1
Periphcrai blood: Reticulocyte: Acridine orange: Supravital staining
Summary The main goal of the Collaborative Study Group for the Micronucleus Test (CSGMT) was to validate a new method for the micronucleus test, recently introduced b/ Hayashi et al. (lc)YOl. using mouse peripheral blood cells stained supravitally with acridine orange (AO). The micronucleus tests were performed on CD-l mice using 23 chemicals with various modes of action. As a rule, one chemical was studied by two participants. Peripheral blood sampled from the same animal was examined 0. 24.48, and 72 h (or longer) after treatment. The frequencies of micronucleated peripheral reticulocytes (MNRETs) were recorded based on observation of 1000 reticulocytcs per mouse. All chemicals induced MNRETs dose-dependently. Interlaboratory differences in the induction of MNRETs were in an acceptable range for most chemicals tested. Although differences wcrc observed with some chemicals, there were no discrepancies iit qualitative judgment. Most chemicals gave the greatest response 48 h after treatment, which was less variable than in the bone marrow assay (grcatcst response, 24-48 hl. These results suggest that the peripheral blood assay using the A0 supravital staining technique generates reproducible and reliable data to evaluate the clastogcnicity of chemicals. This makes the peripheral blood micronucleus assay an attractive alternative to the conventional bone marrow assay.
The Collaborative Study Group for the Micronucleus Test (CSGMT) is a subgroup of the Mammalian Mutagenesis Study Group, a suborganizatton of the l%vironmental Mutagen Society of Japan (JEMS * MMS). CSGMT has investigated the influence of a number of factors involved in the micronucleus test, namely, sex of
Correspondence:
Makoto
Mutagenesis, National
Hayashi. Division of Genetics and
Institute of Hygienic Scicnccs. I-IX 1.
Kamiyoga, Setagaya-ku, Tokyo iSX (Japan).
the test animals (CSGMT, 1986). strain (CSGMT. lY8X). administration route (Hayashi et al.. IYXY). and dosing times (CSGMT. 1900). Results from those collaborative studies provided basal data for the micronucleus test in bone marrow cells. Micronuclei are also detectable in smeared peripheral etythrocytes (MacGregor et al., IYtiO). Because the fraction of young erythrocytcs in the peripheral blood is small, which makes micronuclcus evaluation very laborious. and because objective discrimination between young and mature erythrocytes is not easy. the peripheral blood
T:\BLE
I
PARTICIPANTS
IN THE 5TH COLLABORATIVE
STUDY
Chemical studied %’ 7-AAF
N. Asano *: Nitto Denko Corporation T. Hagiwara: Sankyo Co.. Ltd.
&a-C
K. Iwakura. H. Tamura and A. Matsumoto: Nippon Shinyaku Co.. Ltd. S. Ajimi, S. Ogura, K. &kimoto and Y. Kajiwara: Chemicals Inspection
BEN
Y. Hatakeyama. H. Atai and S. Suzuki: Central Institute E. Nakajima: Toyobo Co.. Ltd.
B[a]P
H. Shimada *. S. Itoh. C. Hattori. Y. Matsuura and S. Tada: Developmental Research P~d~aceutical Co.. Ltd. H. Suzuki and C. Watan~I~: Research Center of Taisho Pharmaceutical Co.. Ltd.
COL
M. Nakajim~~. T. Kitazawa, M. Fujiwara. K. Suzuki and Y. Ukai: B&safety Pesticides K. Miyahana and K. Inour: Nihon Noyaku. Co.. Ltd.
CYP
Y. Hatanaka, Y. Kitagawa and Y. Toyoda: Suntory Co., Ltd. T. Kawatn. N. Ando, Y. KaWabata. M. lwai and H. Arimura: Green Cross Co.. Ltd.
DMBA
M. Hayashi *, T. Suzuki. Y. Kodama, A.O. Asita. A. Matsuoka and T. Sofuni: National Sciences K. Tamai, M. Kurita, H. Ohtsuki and T. Hiwatashi: Health Soienccs Research Institute
DMN
s. Sat0 * and M. Taketomi: Japan Tobacco. Inc. T. Morita *: Nippon Glaxo. Ltd.
EMS
K. Kondo: Shionogi and Co., Ltd. S. Gzawa: Kissei Pha~a~eutic~I Co.. Ltd.
ENU
S. Hitotsumachi and Y. Kimura: Takeda Chemical industries. Ltd. M. Katoh, N. Ishihara, T. Ham and T. Shibuya: Food and Drug Safety Center
5-FU
A. Ohuchida and A. Furukawa: Taiho Pharmaceutical Co., Ltd. J. Yoshida and M. Watanabe: Kaken Pharmaceutical Co., Ltd.
K.BrG,
T. Morita *: Nippon Glaxo Ltd. M. Uejima. T. Kuwahara, S. Asanami and K. Shimono: Otsuka Pharmaceutical
for Experimental
and Testing Institute.
Japan
Animals
Research
bbOKitOfy
Center,
of Daiichi
Foods. Drugs and
Institute
of Hygienic
Factory. Inc.
K $30,
T. Awogi *: Otsuka Pharmaceutical Co., Ltd. K. Murata: Kumiai Chemical Industry Co.. Ltd.
MMC
M. Hara and S. Nakagawa: Sumitomo Chemical Co.. Ltd. E. Fujioka, E. Ayukawa and T. Izushi: Fuji Photo Film Co., Ltd.
MMS
c. Sugiyama. H. Kt~bayashi and M. Mori: Shiseido Toxicological Analytical Research Y. Miyamae. Y. Fujino and K. Ohara: Fujisawa Pharmaceutical Co., Ltd.
MNNG
M. Kuramochi. H. Seki and T. Tazawa: Biomedical Laboratories, S. Sasaki and Y. Sakai: Ono Pharmaceutical Co., Ltd.
h-MP
F. Aruga and Y. Miwa: Nihon Bioresearch Center, Inc. K. Shinkawa and N. Kinae: University of Shizuoka
MTX
Y. Kasahara. Y. Nakai. C. Miura. K. Yagi, K. Hirabayashi and T. Makita: Trijin Ltd. A. Wakata * and K. Yuno: Yamanouchi Pharmaceutical Co.. Ltd.
PCZ
F. Romagna: Sandoz Pharma Ltd. H. Matsumura, M. Watanabe, T. Kato, Y.F. Sasaki * and Y. Shirasu: institute K. Ohmori and H. Yamada: Pfizer Pharmaceutical, Inc.
Center
Inc.
of Environmental
Toxicology
x5 TABLE I (continued) Chemical studied “
Investigators
PFf EN
N. Higashikuni. T. Nakamura and S. Sutou *: Itoham Central Research lnstitute T. Baba, K. Tsurekawa and T. Sera: Daicel Chemical Industries
TEM
E. Yamamura, H. Hirono and M. Takeuchi: Yoshitomi Pharmaceutical Industries. Ltd. M. Kojima and S. Aoki: The Biological Research Center for the Protection of Environment
URET
M. Kishi: Kanagawa Prefectural
VINCI
Y. Kondo, S. Nito and F. Ariyuki: Tanabe Seiyaku Cc Ltd. S. Honda, M. Hayashi, Y. Shinagawa and S. Sato: Toyama Institute of Health
Public Health Laboratories
” For the full names of the chemicals see Table 2. * Organizer.
method has not been widely used. The clastogenicity of chemicals is thus routinely assayed in bone marrow cells stained with Giemsa or acridine orange (AO). Recently, Hayashi et al. (1990) reported a new method for the preparation and observation of micronuclei in peripheral blood reticulocytes using AO-coated glass slides. This method greatly facilitates discrimination between young and mature erythrocytes. The purpose of this 5th collaborative study is to validate the new method using 23 clastogens and spindle poisons with different modes of action; 46 laboratories participated (Table 1). Detailed results on each chemical are reported individually. This summary report presents the overall design, results, and discussion.
Materials and methods
The 23 chemicals used in the present study are listed in Table 2 with their abbreviations, CAS registry numbers, sources, and lot numbers. The chemicals were classified into eight groups according to their mode of action, as shown in Table 2. Each chemical was evaluated by two laboratories independently, with two exceptions: procarbazine was studied by three laboratories and urethane was evaluated by only one laboratory. As a rule, the same chemical lots were used in the paired laboratories. All laboratories used the same lot of mitomycin C (1 mg/kgI as the positive reference chemical,
Animals
CD-I male mice (Charies River Japan or Germany, Inc.) were used by all participants. Approximately 7-week-old mice were purchased, acclimatized for a week or more, and treated at S-10 weeks of age. They were given commercial pellets (not specified) and water ad libitum throughout the acclimatization and experimental periods.
AO-coated slides were prepared at each Iaboratory according to Hayashi et a1. (1990). Briefly, 10 ~1 of 1 mg/ml A0 aqueous solution was spread homogeneously on a warmed glass slide. 5 ~1 of peripheral blood, collected by piercing the ventral tail, was placed without any anticoagulant on the center of an AO-coated glass slide and covered immediately with a 24 x 40 mm coverslip. AO supravitai~y stained reticulocytes were examined by fluorescence microscope with a blue excitation and a yellow barrier fiiter. The frequencies of micronucleated peripheral reticulocytes (MNRETs) were recorded based on the observation of 1000 reticulocytes per mouse. Reticulocytes were restricted to types I, II, and III (Fig. 1) of the classification by Vander et al. (1963). The i?~~niFn~~required protocol
The minimum requirements for each participating laboratory proposed by the organizing committee were as follows.
I‘.ABl.I:
2
LlSl‘ OF CtlEXlICXLS
TESTED Ahhreviation
C‘hemicai tested
source .’
Lot No.
75Y-73-Y
A s W N N
CWOShOYLT 3XFOXXZ TLP7US MOAh4SO VOAl700 VOH4.39 I VOBl7hl 07717TV X4505
CAS No.
CYP DMN I-c!-so-o
Ethyl methanesulfonatr ~-Ethyl-,V-nitrosourea
EMS ENU
Methyl methancsulfonate .V-Methyl-N ‘-nitro-:V-nitrohoguanidine Triethylcnemelamine
MMS MNNG TEM
66-27-3 70-25-7 Sl-IX-3
A P
Ara-C 5-FU h-MP MTX
l-17-04-4 5l-‘I-X 55-44-2 59-0.5-2
W W W
‘40 l2Y PDKIYYO KPN2934 ECQl303
‘-AAF PHEN
53-96-3
W W
AWFh303 KPL453
R[a]P DMBA
50-32-X
s7-07-h
T W
AXO! SAN I X74
MMC
50-07-7
K
h4YAIJ
N
NS
.-lnmtrrtic cmOw
2-Acetylaminofluorene Phenacetin
62-34-2
Polwyc-lie orormrtic- h~dr0curh0n.s
Benzo[lr]pyrene 7.12-Dimethylhenz[o]anthracrne
Inor.wrric c~iwinic~ctis
Potassium hrom:.Le Potuss .n chromate(VI)
KBrO, K $30,
775%01-2 77XY-00-h
W W
SAR3708 KPG72Y7
Spitz& poisur2.s Colchicinr Vincristine sulfate
COL VINC
h-l-Xh-X ZOhX-?X-2
W S
SANIhlY hYF-OS36
BEN PCZ URET
71-43-Z 3%70- 1 5I -70-h
W NR S
KPE73YX TO161 h9F-0633
~li.~~~cll~tttt~oi~s chcvnicab
Benzene Procarbazine Urethane
hydrochloride
” A: Aldrich Chemical Co.. Inc.. Milwaukee. WI (U.S.A.); K: Kyowa Hakko Kogyo Co.. Tokyo (Japan): N: Nacalai Tesque Inc.. Kyoto (Japan): NR: Nippon Roche K.K., Tohyo (Japan); NS: Nippon Shinyaku Co., Ltd.. Kyoto (Japan): S: Sigma Chemical Co.. St. Louis. MO (U.S.A.): T: Tokyo Chemical Industry Co.. Ltd.. Tokyo (Japan): P: Polysciences Inc.. Washington. PA (U.S.A.): W: Wako Pure Chemical Industries. Ltd.. Osaka (Japan).
Erythroblast Fig. I. Schematic presentation
Nucleus expulsion
Reticulocyte
of red fluorescing reticular structure in each type of reticulocyte. II. and 111reticulocytes per mouse were observed.
MZUWZ erythmcyte In the present study, lOOI type I,
72
Fig. 2. Experiments
were performed
frequencies of MNRETs
independently
at two different
laboratories
using peripheral
after treatment with (11)CYP: (b) DMN: (c) EMS: (d) ENU: (e) MMS: (f)
blood of CD-I
mice. Mean
MNNG: (g)TEM; (h) am-C: (i)
S-FU: Q) (I-Ml’: (Ii) MTX: (I) 7-AAF: (m) PHEN: (n) B[a]P: (01 DMBA; (p) MMC: (q) KBrO,: (r) K,CrO,: (5) COL: (1) VINC: BEN; (v) PCZ: (w) URET. The experiments in (VI were performed indcpcndently at three laboratories.
(~1
I.
n
00
M td R E T s
f%I
9,
-2
r
0
MNR E T
MN R E T
s
s
(Xl
(%I
Q, 0 M
s; N
R
E
T
3
(Xl
M
N
R
E
M N R E
T
I
3
s
(%I
I%)
100
,d
50
50 p'
Fig.2 (continued).
4 b9
-3 m -1
800 ,a’
Fig.
2 !rontinucd).
80G ,d
Yl
212 iL%’
60 pa
2.0 (d
Fig. ?. (canhwi).
60
Fig. 2 (continued).
(Ii Each dose group consisted of at least five male mice. (21 At leas? three dose groups and a positive control group were used. (3) The doses were chosen based on the data reported by CSGMT ( 1986, 1989. 1990) or determined experimentally. (4) Mice were treated once intraperitoncally or by gavage. (5) The peripheral blood was examined at 0, 24, 48, and 72 h (or later) after treatment. Blood was sampled at 48 h after mitomycin C (MM0 treatment in the positive control group. In addition, optional experiments, e.g., comparison between peripheral blood and bone marrow, use of rats instead of mice, and multiple treatments, were designed freely by the participants. Before starting the collaborative study, a workshop was held for the participants where details of the experimental methods and standardization of the classification of reticulocytes were discussed. Results
The results of the micronucleus tests are presented in tabular form in each report. Here, the overall results are shown in Fig. 2. All chemicals tested induced MNRETs dose-dependently. The majority of chemicals showed a maximum response 48 h after treatment. Although laboratory differences wcrc seen with some chemicals, most chemicals tested showed minor or no differences between laboratories.
All alkylating agents studied, i.e., CYP (Fig. 2a), DMN (2b), EMS (2~). ENU (2d), MMS (2ej, MNNG (2fj, and TEM (2g), induced MNRETs dose-dependently with good agreement between laboratories. Almost all cases showed similar time-response curves with a single peak at 48 h after treatment. At both laooratories DMN gave a weak but positive response and peaked at 48 h at the only effective dose, 10 mg/kg. At one laboratory MMS showed a maximum response 36 h after treatment while it was 48 h at the paired laboratory.
Ara-C (Fig. 2h) and h-MP (2jj induced MNRETs strongly and dose-dependently at both laboratories in like manner. These two base analogues evoked time-response curves with one peak at 48 h, which is comparable to the alkylating agents. MNRET frequencies induced by S-FU, however, did not follow the time-response curves obtained by the above two chemicals (2i). The maximum response times tended to be dose-related. MTX also showed a positive response at both laboratories (2kj. Peak MNRET frequencies were observed at 72 h in both laboratories.
The frequencies of MNRETs increased after treatment with 2-AAF or PHEN dose-depcndently (Fig. 21. 2mj. Time response curves peaked 48 h after treatment with 2-AAF at all dose levels at both laboratories. PHEN also induced its maximum response at 4X h. Poiyqclic Iryilrocurhotis B[a]P induced MNRETs dose-dependently peaking at 4X h after treatment (Fig. 3n). DMBA also increased frequencies of MNRETs dose-dependently, with the maximum responses being observed at or later than 4X h after treatment (201.
Fig. 2p shows time-response curves for each MMC dose level. The results of the two laboratories were comparable, and the maximum responses were observed 48 h after treatment.
Both K&O, and K,CrO, induced MNRETs dose-dependently with the peak usually observed at 48 h (Fig. 2r, s). There was a hint in these data that the time response was dose-dependent.
COL and VINC showed positive responses (Fig. 2s 0. These two spindle poisons also induced peak responses 48 h after treatment, and were thus comparable to other chemicals. This contrasts with the bone marrow method, in which the maximum response was usually observed ear-
The masimum frequency of MNRETs was ohsc~cd -18 h after treatment with BEN (Fig. 3) in fhc two laboratories. PCZ was studied at three laboratories. each of which ohtainCd positive. dose-rclntcd responses with peak freyucncies of MNRETs obscrvcd at 4X h (3). URET, which W:ISassayed at one I:+oratory. induced MNRETs dose-dcpendcntly wittl peak frequencies also obsened IS h after treatment (2~).
The spontaneous frequencies of MNRETs wt‘re evaluated on peripheral blood collcctcd from 1179 mice immediately before trcatmcnt with each chemical throughout the present collahorative study. MNRET frequencies ranged between 0 and 7 per 1OW RETs. and the mean + SD was I.15 5 1.24 per 101)ORETs (Fig. 3). This distribution fit the binomial distribution surprisingly well (R(WOi3~. IOWA. P = 0.7 by x2 test). ft indicates that vari~~tions in the data are due to chance, which is an advantage for evaluating the clasto-
No. MNRETs Fig. 3. Distribution
of MNRET
/ 1000
RETs
frcqurncies in mice immcdi-
at&y before trc:mnrnt. Da13 from ail l~l~or~t(~ri~~ were combined. for a total of 117’1 CD-1 mice. Bars show the tthsrrv~d frcyuaxk
and circles the cxpcctcd values from the thectretical hinomi;li distrihutittn 6(0.001~, 1000).
genicity of the tusted chemicals. The reproducibility and reliability of the new simple method are shown by the stable distribution of MNRETs in the negative control. Some participants performed experiments including solvent control groups, i.e., mice were treated with physiological saline or olive oil alone. Time-response curves arc shown in Fig. 4, and no positive response was observed at any time after treatment. This is good evidence to support the experimental designs that sample blood at 0 h instead of using a solvent control group. It also indicates that stress from the expcrimcntal procedures did not influence the background frequency of MNRETs, cvcn after S daily samplings.
In this collaborative study, all participants were requested to examine the MNRETs from mice treated with I mg/kg MMC (same loti 48 h after treatment as the reference control. The individual numbers of MNRETs per 1000 RETs scored at each laboratory arc piotted in Fig. 5. MNRET frcquencics were distributed widely bctwccn 2 and X9 per 1000 KETs. and frequencies at each laboratory (mean of:SD) ranged between 16.X L5.81 and 51.2 + 13.0 per 1000 RETs. One of the possible factors causing such laboratory differcnces is different definitions of RETs among microscopists. Although two mice showed values within the negative control range, possibly bccause of improper dosing, all other mice showed significantly increased frequencies of MNRETs,
0
. (2)
. (2)
Laboratories Fig. 5. Pistrih!6nn
of the numher of MNRETs
mice had been tread
i.p. with
4X h after
1 mg/kg MMC.
with one mouse showing an exceptionally high frequency of MNRETs. Excluding these three outliers, other points were distributed randomly around the global mean, 33.7 MNRETs/lOOO RETs. Optional studies Detailed data are presented in individual papers in this issue but are summarized very brieily here. CYP (Hatanaka et al., 19921, DMN (Sat0 et al., 19921, ara-C (Iwakura et al., 1992), 2-AAF (Asano et al., 1992), DMBA (Suzuki et al., 19921, and URET (Kishi et al., 1992) were studied with both peripheral blood and bone marrow erythrocytes, and comparable responses were obtained. On the other hand, although VINC, MMS, and PHEN gave positive responses in peripheral blood, a lower percentage of micronucleated cells was observed in these young erythrocytes than in\ the bone marrow test. CYP (Hayashi et al., 1992), B[a]P (Shimada elt al., 19921, MMC (Hayashi et al., 1992), and BEr\l (Hatakeyama et al., 1992) were used for a comparison between mice and rats. CYP, MMC, and BEN induced MNRETs comparably in rat and mouse peripheral blood. Only a slight increase of MNRETs was observed among rat peripheral reticulocytes after treatment with B[alP. In the
rat, the effect of splenectomy was studied with MMC in the induction of MNRETs. Splenectomy was found to be effective in increasing the number of MNRETs observed especially at the high dose level, but with large deviations. The effects of repeated treatments were studied with MTX (Kasahara et al., 1992), 2-AAF (Asano et al., 19921, PHEN (Higashikuni et al., 19921, and MMC (Hara et al., 1992). With PHEN and MMC, repeated treatments induced higher MNRET frequencies. One experiment with MTX showed an enhancement of MNRET induction after triple treatments but the other experiments did not show significant differences or single treatment was more effective. The ratio of type I, II, III, and IV RETs to total RETs was studied after treatment with ara-C by Iwakura et al. (1992). They concluded that the proportion of type I RETs to total storable RETs was potentially a good way to assess the cytotoxicity of the test chemical, as is the ratio of PCEs to total erythrocytes in the conventional bone marrow method. Discussion The micronr&eus assay with peripheral blood There are two main uses for the peripheral blood micronucleus test. One is to evaluate the acute clastogenic effects of test chemicals (MacGregor et al.. 1980, 1990; Tice et al., 1987, 1990), and the other is to assess the chronic chromosome-damaging activity of chemical substances (Schlegel and MacGregor, 1982: Barale et al., 1985; Choy et al., 1985; Jauhar et al., 1988; Rithidech et al., 1988; MacGregor et al., 1990). The former is done by scoring restrictively young erythrocytes, and the latter is done by scoring mature erythrocytes. The aim of the 5th collaborative study presented here was to validate the new A0 supravital staining method described by Hayashi et al. (1990) using known clastogenic chemicals with various modes of action. The micronucleus assay using peripheral blood has also received attention in the field of regulatory sciences. The Gene-Tox Program organized by the U.S. Environmental Protection Agency regarded it as a variation of the conventional bone marrow micronucleus assay (Mavournin et
al.. 1~~0). In the manual for the Japanese guidclinch (J;~pan MHW. 1YYI) for drug toxicity studich. it is htatcd that ‘c\vn those data obtained by studies using tissues other tl‘,rn the bone marrow (e.g.. the peripheral blood) and those obtained by a testing method involving differentiation and counting of micronuclei by some mechanical means may 31~0 be acceptable as a subject of evaluation if they are justified from the scientific viewpoint’. Thereforc, at the present moment, it is important to validate the method using peripheral blood as the material for the micronucleus iMay.
The transition time from bone marrow MNPCEs to peripheral blood MNRETs was reported to be about 24 h by MacGregor et al. (Ic)W, iW0). The same transition time was shown in the present study with several chemica!s. According to more precise time-course studies. however. the transition time was shown to be about 12 h with MMC and ara-C (Hayashi et al., 1YYO: lwakura et al., 19%). This difference might simply depend on the study protocol, i.e., the former results were obtained in the study sampling with a 24-h interval. The advantages of the micronucleus test using peripheral blood instead of bone maT;‘ow crythrocytes were discussed by MacGregor et al. (1980). They pointed out the following. (1) Repeated samples are easily obtained from the same animal without killing it. (2) Sample preparation is simpler. (3) It is simpler to score peripheral blood smears than conventional bone marrow smears. (4) The micronuclei are easily distinguished because they are the only darkly staining features on the slide. (5) The cell population is uniform enough to apply automated scoring of micronucleated cells. They also reported that the test using peripheral blood was at least as sensitive as the bone marrow method.
The A0 supravital staining method has additional advantages to those stated by MacGregor et al. ( 1980). ( 1) A more accurate identification of micronuclei can be made (Hayashi et al., 1983). (2) Classification of young erythrocytes is less subjective because supravital staining identifies reticulocytes. (3) More precise classification of
RETs is possible by yuantificaticjp of the amount of red fluorescing reticular material. Therefore. rat peripheral blood can also be used by restricting the scoring population to very young reticulocytes (types I and II). (4) Very stable background frequencies of MNRETs have been shown in the present study. (5) Sample preparation, only involving placing blood on the AO-coated glass slide and putting a cover slip on it, is :;mpler than smear preparation. Disadvantages of the A0 supravital staining method are: (1) slide preparations cannot be stored for long; (2) preselection of good quality regions is frequently necessary; (3) the method to assess the inhibition of crythropoiesis caused by the test chemical is still under development.
Although the new method for the micronucleus test using peripheral blood is considered to have several advantages as mentioned above, its validity has not been shown. To validate this new method, the following should be confirmed. (1) The new method is sensitive enough to detect the clastogenic activity of known clastogens with different modes of action. (2) The frequency of MNRETs in peripheral blood reflects that of MNPCEs in the bone marrow. (3) The data are reproducible. (1) All clastogenic chemicals with different modes of action tested here gave a positive response. These chemicals included MNNG and DMN, which gave contradicting results in the conventional bone marrow assay. COL and VINC, which are spindle poisons, also induced MNRETs dose-dependently. (2) In the present collaborative study, CYP, DMN, and BEN gave comparable responses in peripheral MNRETs and bone marrow MNPCEs. However, VINC, MMS, and PHEN induced higher frequencies of MNPCEs than MNRETs. It is difficult to precisely compare the frequencies of micronucleated young erythrocytes in the peripheral blood and in the bone marrow, because the populations might be different according to the criteria for RETs and PCEs. In the case of VINC, cells containing large micronuclei may have been eliminated before they could enter the peripheral blood, because several large-micro-
nucleated young crythrocytes were observed in the bone marrow, but not in the peripheral blood
(data not shown). In the hone marrow studies, the majority of chemicals studied by CSGMT induced maximum MNPCE frequencies 24-48 h after treatment. For example, COL (Asano et al., 19X9)and VINC (Ohuchida et al., 1989) induced maximum MNPCEs at 24 h, and 6-MP (Hara et al., 19891, B[a]P (Awogi and Sato, 19X9), and DMBA (Mizuhashi et al., 1989) did so at 48 h. On the other hand, all those chemicals, and the others tested here, gave positive, but not necessarily maximum responses 48 h after treatment. This is quite useful for determining sampling times for unknown chemicals, because peak responses seem iu be cunfined to a narrower period, around 48 h after treatment. (3) The reproducibility of results by this new method was illustrated in the present collaborative study. There were almost no qualitative differences between laboratories at which the same chemicals were studied independently, although the absolute values of the frequencies of MNRETs varied between laboratories, These differences may not be critical; data from the conventional bone marrow method also varied between laboratories and even between experiments at the same laboratory. Conclusion
In conclusion, our validation study for the A0 supravital staining method for rodent peripheral blood reticulocytes clearly showed its usefulness for the evaIuation of the clastogenjcity of test chemicals. We also confirmed the usefulness of peripheral blood reticulocytes for the rodent micronucleus assay. Therefore, this method offers an alternative to the conventional bone marrow micronucleus assay. Because it is possible to obtain time-response data concomitantly with the dose-response relationship, more reliable information about micronucleus induction could be obtained than with the conventional bone marrow assay. Moreover, since samples at 0 h can serve as the negative control, the number of animals, at least for the negative control group, can be reduced without any reduction in scnsitiv-
ity. The fact that most chemicals gave peak responses at 4X h could sirnplj~ the test method by more narrowly defining and reducing sampling times. The negative aspects of the A0 supravital staining technique are few. Acknowledgement
We are indebted to Dr. Miriam Bloom for reviewing all the manuscripts of this collaborative study. References A~no.
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