Animals
Male Crl:CD (SD) rats were purchased from The Jackson Laboratory Japan Inc. (Kanagawa, Japan). The rats were housed together in groups of two or three in each cage with wood chip bedding at 23 ± 3 °C and 30–70% humidity with alternating 12 h intervals of light and dark. Animals were acclimatized for a week or more and treated at 8 weeks of age. All animals were fed F-2 commercial pellets (Funabashi Farm Co. Ltd., Chiba, Japan) and tap water ad libitum throughout the acclimation and experimental period. All experiments were performed in accordance with the guidelines for the care and use of laboratory animals established by the Institutional Animal Care and Use Committee of Yakult Central Institute, and the protocols were approved by this committee.
Chemicals
DEN [55–18-5] (99.9% purity, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), DMH [306–37-6] (100% purity, Tokyo Chemical Industry Co., Ltd.), and KBrO3 [7758-01-2] (≥99.8% purity, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) were dissolved in water for injection (DW; Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) immediately before treatment. Ten% neutral-buffered formalin (Mildform® 10 N, which contains 1 w/w% methanol, FUJIFILM Wako Pure Chemical Corp.), which is routinely used in histopathological studies, was used to fix the cells and tissues used in the present study. Acridine orange (AO; FUJIFILM Wako Pure Chemical Corp.) and/or 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma Aldrich Co. LLC, St. Louis, USA) were used to stain the cells for MN analysis. Potassium hydroxide (KOH; FUJIFILM Wako Pure Chemical Corp.) was used to loosen the cell connections and prepare single-cell suspensions. Tris base and Tris hydrochloride (Sigma Aldrich Co. LLC) were used for preparing a Tris-HCl buffer.
Micronucleus test
Five rats were randomly assigned to either the negative (untreated) control or the treatment group. The dose levels for the treatment group were set at 100 mg/kg body weight for DEN (a single dose), and 90 mg/kg body weight for DMH (a single dose) based on our previous report [12]. The dose level of KBrO3 was set at 120 mg/kg body weight/day (2 doses), showing positive results in the stomach and bone marrow with weak cytotoxicity, based on the results of a 2-week dose-finding study of KBrO3 when rats were administered a combination of DEN, DMH and KBrO3 with the same schedule as the present study. The treatment volume of each chemical was 10 mL/kg body weight. Each formulation was administered to the rats by oral gavage immediately after preparation. The treatment protocol is shown in Fig. 1. Day 1 is designated as the first day of treatment. On the day of necropsy (Day 15), animals were anesthetized with isoflurane and euthanized by exsanguination via the abdominal aorta, and their stomachs, colons, livers, and right femurs were sampled. The MN tests were conducted twice. Experiment 1 (Exp.1) was conducted to compare the performance between the EDTA and FF methods using the GI tract tissues obtained from the same animal; half of the glandular stomach and a portion of the colon were subjected to the EDTA method immediately after dissection from animals, and the remaining portion of these tissues were immersed in 10% neutral-buffered formalin to apply the FF method. Experiment 2 (Exp.2) was conducted to evaluate the aging effect of tissues stored in formalin fixative, comparing tissues from the same animal stored in formalin fixative for approximately 10 days (initial) and 1 year.
EDTA method (GI tract)
Single-cell preparation from fresh GI tract tissues using the EDTA method was performed according to our previous report [10]. The cell suspensions were washed, fixed in 10% neutral-buffered formalin, and stored at 4 °C until analysis. Immediately before microscopic observation, the cell suspensions were mixed with an equal volume of staining solution (250 μg/mL AO–2.5 μg/mL DAPI for the stomach and 500 μg/mL AO–2.5 μg/mL DAPI for the colon) on a glass slide. The cells were observed under a fluorescence microscope (600× magnification) with UV excitation (365 nm).
FF method (GI tract)
The entire stomach and colon were dissected from the rats and immersed in 10% neutral-buffered formalin for a week or more. After washing with DW, the fixed stomach and middle portion (approximately 3 cm long) of the colon were cut open along the greater curvature and longitudinally, respectively, and rinsed with DW. Subsequently, the forestomach was removed, and half of the glandular stomach and opened colon were rinsed with DW again and separately placed into a centrifuge tube containing approximately 10 mL of aqueous solution of KOH. As a result of examining various combinations of the concentrations (0.5, 1, and 4 M) and treatment periods (5, 16, and 24 h) of KOH, suitable cells for MN analysis were obtained when treated with 1 M KOH for 24 h at room temperature, and these treatment conditions were used thereafter. After treatment with KOH, the tissues were rinsed with 0.5 M Tris-HCl buffer (pH 7.5) for neutralization. The epithelial cells of each tissue were scraped with a Cell Scraper S made of silicon rubber (10 mm width, Sumitomo Bakelite Co., Ltd., Tokyo, Japan). The cells were separated by pipetting 10 times with a Pasteur pipette and transferred to a centrifuge tube. The cell suspension was centrifuged at 200×g for 5 min, the supernatant was removed, and the cells were resuspended in Tris-HCl buffer. This step was repeated. Finally, the cells were resuspended in a small amount of the same buffer and stored at 4 °C until analysis. The protocol for observing the cells was the same as that used in the EDTA method except for the concentration of the staining solution: 200 μg/mL AO–15 μg/mL DAPI was used after optimization to distinguish between the nucleus and cytoplasm.
Liver and bone marrow
Slides were prepared from formalin-fixed livers for MN analysis, according to the method reported by Shigano et al. [9] with minor modification in the KOH concentration (1 M), washing buffer (0.5 M Tris-HCl buffer, pH 7.5), staining solution (25 μg/mL DAPI), and excitation wavelength (365 nm) for a fluorescence microscope. The bone marrow (BM) cells were collected by washing the femur cavity with 1 mL of 10% neutral-buffered formalin, stained with 40 μg/mL AO solution, and observed under a fluorescence microscope (600× magnification) with blue excitation (490 nm) [17].
MN analysis
For the stomach, colon, and liver, 2000 cells were analyzed per animal for determining the frequency of the MNed cells. For the BM, 2000 immature erythrocytes (IMEs) from each rat were analyzed for determining the frequency of the MNed IMEs, and more than 500 erythrocytes were analyzed for determining the percentage of IMEs among the total erythrocytes (%IME).
Histological examination
Histological examination of the glandular stomach and colon was performed before and after cell isolation of untreated 8-week-old male rats. For the EDTA method, portions of the glandular stomach and colon were fixed in 10% neutral-buffered formalin and used as histological specimens before cell isolation. The remaining portions were treated with EDTA as described in the section of EDTA method (GI tract), and the tissues after treatment were also fixed in 10% neutral-buffered formalin and used as histological specimens after cell isolation. For the FF method, the entire stomach and colon obtained from another animal were fixed in 10% neutral-buffered formalin. Portions of the glandular stomach and colon were used as histological specimens before cell isolation, and the remaining portions were treated with KOH, as described in the section of FF method (GI tract) and used as histological specimens after cell isolation. Each sample was then embedded in paraffin and sectioned. The sections were stained with hematoxylin–eosin and observed under a light microscope.
Statistical analysis
The statistical significance between the values obtained with the EDTA and FF methods, between the initial values and values 1 year after formalin fixation, and between %IME values of the negative control and treatment groups was analyzed by Student’s t-test (significance level of 0.05) after confirming the homogeneity of variance using BellCurve for Excel version 3.20 (Social Survey Research Information Co., Ltd., Tokyo, Japan). Differences in MNed cell frequency between the negative control and treatment groups were analyzed using Kastenbaum and Bowman’s tables with a significance level of 0.01 [18].