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The Optimized Conditions for the in Vitro Micronucleus (MN) Test Procedures Using Chamber Slides

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The Optimized Conditions for the in Vitro Micronucleus (MN) Test Procedures Using Chamber Slides Environ. Mutagen Res., 27: 145-151 (2005) Original Article The optimized conditions for the in vitro micronucleus (MN) test procedures using chamber slides Mika Yamamoto＊, Akira Motegi, Jiro Seki and Youichi Miyamae Toxicology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. 1-6, Kashima 2-chome, Yodogawa-ku, Osaka 532-8514, Japan Summary Optimized conditions for an in vitro micronucleus (MN) test procedure were defined using a chamber slide that enabled the preparation of fine specimens without undergoing complicating pro- cedures using culture dishes. The issues investigated are 1) the effect of slide materials on the adhesion of cells, 2) the number of seeding cells necessary to obtain an adequate number of cells for observation and 3) effects of hypotonic treatment and fixation on the cytoplasmic: nuclear area ratio. In addition, we determined cell viability in each chamber using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay. The results of the investigation were as follows: 1) cell adhesion was best using plastic slides, 2) the optimum number of cells for seeding was 6.6× 103 cells/cm2, 3) the best condition for hypotonic treatment was incubation in 75 mM KCl at 37 ℃ for 5 min, and 4) the best condition for fixation was treatment of cells twice for about 2 min in ice- cold methanol containing 6％ acetic acid. Finally, the result of the MTT assay correlated with the number of viable cells in chamber as determined by the trypan blue dye exclusion assay. An in vitro MN test was conducted under these conditions using the known clastogens, Mitomycin C and dimethylnitrosamine. These clastogens dose-dependently induced a significant increase in the number of micronucleated cells with positive responses at concentrations approxi- mately 10 times lower than those of the chromosomal aberration test. On the other hand, the fre- quency of micronucleated cells in the solvent control was stable and low (0.4 ─ 1.7％). These results indicated that the in vitro MN test has a high level of sensitivity to clastogens. It was concluded that the in vitro MN test using chamber slides is a rapid, simple and sensitive method to detect clastogens. Keywords: in vitro micronucleus test, chamber slide short-term test is required. Introduction Recently, the in vitro micronucleus (MN) test has been The in vitro chromosomal aberration (CA) test is a well- used as a short-term test alternative to the CA test to established, frequently used short-term test to estimate detect the clastogenicity of chemicals (Matsuoka et al., the clastogenicity of chemicals (OECD, 1997). However, it 1993; Miller et al., 1995; Matsushima et al., 1999). The in is not applicable to high throughput screening because vitro MN test is rapid and simple for evaluation, because the slide preparation and metaphase analysis in the CA the measurement of micronucleus is less subjective than test requires considerable skill, experience and time metaphase analysis. Furthermore, the concordance (Matsuoka et al., 1993; Kirsch-Volders, 1997; Fenech, between the result of the in vitro MN and CA tests has 2000). Therefore, the development of a rapid and sensitive been reported to be approximately 90％ (Miller et al., 1997; Matsushima et al., 1999). Therefore, the in vitro MN test is capable of screening larger numbers of drug candi- ＊ E-mail: [email protected] dates (Matsuoka et al., 1993; Miller et al., 1995; Kirsch- Received: March 22, 2005, revised: July 14, 2005, Volders, 1997; Garriott et al., 2002; Matsushima et al., accepted: July 16, 2005 © Japanese Environmental Mutagen Society 1999). However, the slide preparation in the in vitro MN 145 test is usually the same as that of the in vitro CA test, and ferent concentrations. For experiments with metabolic this method still requires complicated, time consuming activation, the fresh medium also contained 16.7％ S9 mix procedures such as the peeling off cells from dishes, cen- (5％ S9 fraction). S9 was prepared by centrifugation trifugation of cell suspensions, and dropping cells onto (9000×g) of liver homogenate from male Sprague-Dawley slides. If complicated slide preparation procedures were rats (Slc:SD) (7 weeks of age) given phenobarbital and not required, the in vitro MN test would be more rapid 5,6-benzoflavone intraperitoneally. The ingredients in and simple. 1 mL of S9 mix were 0.3 mL of S9, 0.2 mL of 40 mM Chamber slides have one to eight small separate areas HEPES buffer solution, 0.1 mL of 50 mM MgCl2, 330 mM (chambers) which can accommodate all procedures for KCl, 50 mM glucose 6-phosphate, 40 mM NADP and 0.1 cell culture from the seeding to the fixation on the slide. mL of distilled water. Cells were treated for 6 h or 24 h by Therefore, if the number of cells obtained by culturing in the method without metabolic activation system and for 6 chamber slides is adequate for the MN assay, the test h by the metabolic activation method. After treatment of 6 capacity for screening of drug candidates would be h or 24 h, the cells were rinsed twice with Hanks’ increased. To obtain evaluative cells which possess clear Balanced Salt Solution and incubated further in 0.8 mL of nuclei surrounded by large cytoplasmic area, it is impor- fresh medium until 48 h after the start of treatment. At the tant to optimize the slide material, the number of seeding end of this incubation, the culture medium was replaced cells, the condition of hypotonic treatment, and the condi- by 75 mM KCl solution for hypotonic treatment. Then tion of fixation in the test procedure. These conditions for cells were fixed every two minutes by two changes of ice- in vitro MN test procedures using chamber slides have cold methanol containing acetic acid and, air-dried, and not been reported. The purpose of this study was to define the chambers were removed from the slides. Each slide the optimized conditions for in vitro MN test procedures was stained with 100 µL acridine orange (3 µg/mL; AO: using chamber slides. Nacalai Tesque, Inc., Kyoto, Japan) and coverslipped. All slides were analyzed at 1,000 magnification using a fluo- Materials and Methods rescence microscope equipped with an excitation filter of 1. Chemicals and solvents 420-490 nm and a long pass barrier filter of 520 nm. The To investigate the test condition for practical treatment, number of micronucleated cells among 1,000 intact inter- cells were treated by Mitomycin C (MMC, CAS No. 50 -07- phase cells was counted. Micronucleus was identified 7, Kyowa Hakko Kogyo Ltd., Tokyo, Japan) or dimethylni- according to the following criteria based on that of P.I. trosamine (DMN, CAS No. 62-75 -9, Nacalai Tesque, Inc., Countryman and J.A. Heddle (Countryman and Heddle, Kyoto, Japan). Both chemicals were dissolved in physio- 1976). logical saline. 1) Micronucleus is surrounded by a nuclear membrane. 2) The diameter of micronucleus varies less than 1/2nd 2. Cell and medium of the diameter of the main nucleus. Chinese hamster lung (CHL/IU) cells obtained from 3) Micronucleus is lying within the cytoplasm, not the National Institute of Health Sciences, Tokyo (Japan) linked or connected to the main nuclei and with were grown in Eagle’s Minimum Essential Medium (E- staining similar to the main nucleus. MEM, GIBCO BRL, Life Technologies, Inc.) supplement- The number of cells with micronuclei in the treated ed with 10％ heat-inactivated (56℃, for 30 min) calf group was compared to that of the solvent control group serum. Cells were grown in a humidified atmosphere of using the Chi-square test. Test chemicals were consid- 95％ air 5％ CO2 at 37℃. The modal chromosome num- ered positive if the number of micronucleated cells ber was 25, and the average generation time was about 15 increased dose-dependently and differed significantly h. from the solvent control in at least 1 treated group (p＜ 0.05). 3. In vitro micronucleus test CHL/IU cells were cultivated in 4-chamber slides made 4. Cell viability determination using the 3-(4,5- of either glass (Lab-Tek, Nunc Inc., Naperville, IL), colla- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro- gen-coated glass (glass slide: Lab-Tek, Nunc Inc., mide (MTT) assay and the trypan blue dye exclusion Naperville, IL; Cellmatrix Type1-A: 3.0 mg/mL, Nitta zer- assay achin Inc., Niigata, Japan), or plastic (permanox: Lab-Tek, After cultivation, the chamber slides were divided into Nunc Inc., Naperville, IL). CHL/IU cells were diluted in two groups for measurement of the number of viable cells culture medium, and 0.8 mL of suspension was seeded in in chamber using two assay methods. For the trypan blue each chamber (1.8 cm2/chamber). After preculture (about dye exclusion assay, cells were rinsed with phosphate 18 h), the culture medium was exchanged with 0.8 mL of buffered solution, detached from each chamber by treat- fresh medium containing solvent or test chemicals at dif- ment with trypsin-EDTA, and suspended in culture medi- 146 Fig. 1 Influence of slide materials on cell morphology (a): glass slide, (b): collagen-coated glass slide, (c): plastic slide, magnification: ×50 um. The cell suspension was mixed with an equal volume optimized the number of seeding cells for observation of of 0.5％ Trypan Blue dye solution, and cells that were not micronuclei using the plastic chamber slide (data not stained were counted as viable cells using a hemacytome- shown). When CHL/IU cells were incubated in chamber ter. For the MTT assay, culture medium was exchanged slides at a density of 2.2×103 ─ 8.8×103 cells/cm2, it was with fresh medium (0.8 mL/chamber) containing 0.25 concluded that a seeding density of 6.6×103 cells/cm2 mg/mL MTT, and the cells were incubated at 37℃ for 4 was optimal for the observation of micronuclei because of h.
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