Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides

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Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides Koji Kondo

Kcyvordx

Micronucleus test; Ethyl methanesulfonate:

’

and Sigenari Ozawa ’

Peripheral blood reticulocytes; Acridine orange

Summary

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-l mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.

The studies on the micronucleus test with ethyl methanesulfonate (EMS) described here were conducted as part of the 5th collaborative study organized by the Collaborative Study Group for the Micronucleus Test (CSGMT). The study was designed to validate a new method for the micronucleus test with mouse peripheral blood reticulocytes stained supravitally using acridine orange (AOJ-coated $ides (Hayashi et al., 1990). The scope and sunimaly of this study are reported in an accomp;mying article (CSGMT. 1992). The studies with EMS were performed independently at Kissei Pharmaceutical Co., Ltd.

Correspondence: ries.

Shionogi

(Japan).

Koji Kondo. Shionogi Research Laboratoand

Co.,

Ltd..

Fukushima-ku.

Osaka

553

(Expt. 1) and at Shionogi & Co., Ltd. (Expt. 2). EMS is an alkylating agent and is known to be a micronucleus inducer (Matter and Grauwiler, 1974; CSGMT, 1986, 1990; Kondo et al.. 1989). Materials

and methods

Male 7-week-old CD-l mice were purchased from Charles River Japan, Inc. (Atsugi. Japan). They were given commercial food pellets and water ad libitum throughout the l-week acclimatization period and the experiments. Body weights (mean f SD) at the start of the experiment were 34.6 + 2.88 g for Expt. 1 and 37.5 I 1.42 g for Expt. 2. Five animals were randomly allocated to each group. EMS (Nacalai Tesque, Inc., Kyoto. Japan, Lot No. MOA6540) was dissolved in physiological saline and was administered once to mice in-

tr~q~~rit0ncrtlly at dose kvds of l(R). 200, 300 nlg,/kg ibpts. I and 3, and 400 mg/kg (Expt. 2) in a volume of 111ml/kg, based on the results of previous collaborative studies by CSGMT (CSGMT, l986; Kondo et al., 1989). Another group of mice in both experiments rcccived mitomycin C (MMC, Kyowa Hakko Kogyo Co.. Ltd.. Tokyo, Japan, Lot No. 649AIJI intraperitoneally at a dose of 1 mg/kg as the common reference compound in the present collaborrtivc study (CSGMT, 1992). Details of the method used hcrc are given in the summary report (CSGMT, 1992). Briefly, the prcp~~rati[~n of A~-cited slides and the collection of peripheral blood from the tail by piercing and without anticoagulant were done according to the method of Hayashi et al. (1990). Peripheral blood from each mouse was examined at 0. 21, 48, 72 h (Expts. 1 and 2) and 96 h (Expt. 2) after treatment with EMS or MMC. The frequencies of micronucleated peripheral blood reticulocytes (MNRETsI were estimated by observing 1000 reticulocytes per mouse on coded slides using fluorescence microscopy within 2 days after sampling. Results and discussion The results of the two experiments with the new method using peripheral blood are shown in

Table 1. Both experiments showed the same induction pattern of MNRETs, that is, EMS dosedependently induced a significant number of MNRETs (p < 0.051, with the peak frequency at 4X h after treatment. Frequencies of MNRETs decreased rapidly to the control (0 h) level thereafter. At a dose of 100 mg/kg frequencies of MNRETs stayed at the control level at all sampling times in both experiments. At each EMS dose the frequencies of micronuclei in peripheral blood reticulocytes at a sampling time of 48 h appeared to be at the same level as those in bone marrow poiychromatic erythrocytes (PCEsI at 24-h sampling times reported in previous collaborative studies (CSGMT, 1986, 1990; Kondo et al., 1989). Kondo et al. (1989) reported that the maximum frequency of micronuclei in bone marrow PCEs was observed 24 h after treatment with EMS. The present data indicate that the peak frequency (at the optimal sampling time) of micronuclei in peripheral blood reticulocytes was delayed by 24 h compared to that in bone marrow PCEs. This time lag in the appearance of micronuclei for blood reticulocytes compared to bone marrow PCEs after MMC treatment has previously been observed by Hayashi et al. (1990). It was also found after treatment with other compounds used in this coIIaborative study (CSGMT, 1992). Mitomycin C, the common reference com-

TABLE I RESULTS OF THE MICRONUCLEUS Ci~mpound

TEST WITH EMS USING MOUSE PERIPHERAL

Dose (mg/kg)

Number of mice

&?f. f EMS EMS EMS Mitomycin C

100 200 300

I

&?I. 7 EMS EMS EMS EMS Mit~~mycin C

100 ‘00 3011 400

I

Frequencies

BLOOD

RETICULOCYTES

of MNRETs (? mean + SD) *‘,at sampling time of

0h

24 h

48h

72 h

46 h

5 5 5

0.22 * 0.1 I 0.14 + 0.0s 0.24 + 0. I 1

0.1 h 5 I).13 03 + 0.1 I 0.42 * 0.1 1

022i0.15

5

o.lX+o.lh

1.22+0.31

0.24 + 0.11 0.98 + O.hD I .h2 + 0.64 1.78 + 0.72

0.26+0.li 0.20+0.10 0.52 + 0.2’

NT NT NT NT

5 5 s s

0.22 * 0.13 O.lXlt:O.13 0.16 f 0.09 0. IO + 0.07

!I.22 * 0. I I 0.30 * 0.10 0.28 & 0.16 0.‘2*0.14

0.22+o.lh O.Sh + 0.05 1.8X*0.61 x06+ 1.73

5

0.20+o. I3

1.OZ+ fl.hZ

4.56It 1.28

0.22+o.lh 0.2~0+0.10 0.28 + 0.19 0.44+0.15 ft.76+0.17

N’T 0.14 * 0.09 0.16tD.05 0.22 * 0.13 0.24 & 0.09

.’ Frcquencirs of MNRETs were ewluatcd NT. not tested.

based on l(W)0reticulocytes

per mouse.

Ill

pound in the present collaborative study, showed a similar time-response pattern in the induction of micronuclei in both experiments, agreeing with Hayashi et al.3 (1990) results, i.e., the peak frequency of MNRETs was observed at the 48 h sampling time. These results with EMS and MMC using the new method show that, although some interlaboratory differences were observed, reproducible induction patterns of micronuclei can be obtained. Furthermore, EMS could induce substantial numbers of micronuclei in peripheral blood reticulocytes, as it had in bone marrow PCEs. It is concluded from the present studies that, for EMS at least, this new method can be an alternative to the conventional bone marrow micronucleus test.

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