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Evaluation of a 4-day repeated-dose micronucleus test in rat glandular stomach and colon using aneugens and non-genotoxic non-carcinogens

Evaluation of a 4-day repeated-dose micronucleus test in rat glandular stomach and colon using... Background: We previously developed a rodent gastrointestinal (GI) tract micronucleus (MN) test using the glan- dular stomach and/or colon, and evaluated this test method using several genotoxic carcinogens (clastogens) and genotoxic non-carcinogens; we demonstrated that this test method could detect genotoxic stomach and/or colon carcinogens with target organ specificity. In the present study, we further evaluated the sensitivity and specificity of the MN test for the rat glandular stomach and colon using three aneugens (colchicine, vinblastine sulfate, and doc- etaxel hydrate) and two non-genotoxic non-carcinogens (sodium chloride and sucrose). Results: Male Crl:CD (SD) rats were administered test compounds through clinical administration route (orally or intravenously) for four consecutive days and then examined for the micronucleated cell frequencies in the glandular stomach and colon. We observed that all three aneugens significantly and dose-dependently increased the micro - nucleated cell frequencies in the stomach and colon. In contrast, neither of the two non-genotoxic non-carcinogens increased the micronucleated cell frequency in these tissues. Notably, an increase in cell proliferation was observed in the glandular stomach of rats administered a stomach toxicant, sodium chloride, but this increase did not affect the induction of micronuclei in the gastric cells. Conclusions: In the present study, it was demonstrated that the glandular stomach and colon MN tests could detect aneugens as positive and could adequately evaluate non-genotoxic non-carcinogens as negative, including a chemi- cal that enhances cell proliferation. These results provide important evidence supporting good performance of the rat glandular stomach and colon MN tests with a 4-day treatment regimen. Keywords: Micronucleus test, Gastrointestinal tract, Glandular stomach, Colon, 4-day treatment regimen, Aneugen showing a positive result in an in  vitro genotoxicity test Introduction gives a negative result in the erythrocyte MN test, it is The erythrocyte micronucleus (MN) test using rodent necessary to conduct follow-up evaluation with a second bone marrow/peripheral blood is one of the stand- in  vivo test using appropriate target tissues and/or end- ard genotoxicity tests in regulatory use and is valuable points based on factors such as exposure, metabolism, for evaluating the clastogenicity and aneugenicity of and distribution in the drug development [1]. Regarding chemicals in  vivo [1–3]. However, when a test chemical evaluation of orally administered substances, the gas- trointestinal (GI) tract is useful being a direct contact *Correspondence: emiko-okada@yakult.co.jp site for the substances. In particular, the stomach is con- Yakult Central Institute, Yakult Honsha Co., Ltd., 5-11 Izumi, Kunitachi-shi, Tokyo 186-8650, Japan sidered to be important because of its high exposure to © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Okada et al. Genes and Environment (2022) 44:12 Page 2 of 11 orally administered substances. The colon is also useful inter-laboratory reproducibility was confirmed using for evaluating the genotoxicity of substances that reach negative- and positive-control substances with a 4-day the intestine via intestinal contents and/or bloodstream treatment regimen [19]. Furthermore, the glandular as well as substances that are metabolized by intesti- stomach and colon MN tests showed good performance nal microbiota. For genotoxicity tests targeting the GI even with the long-term RD regimen of 14 and/or 28 days tract, the comet assay to detect DNA damage and the [19, 20]. This evidence suggests that the GI tract MN test transgenic rodent gene mutation assay to detect gene could be incorporated into a 28-day RD toxicity test, sim- mutations have already been established and listed in ilar to the erythrocyte MN test. Based on these findings, the OECD Test Guidelines [4, 5]; however, the GI tract the GI tract MN test was concluded to be a promising MN test to detect chromosome damage has not yet been method to examine the clastogenicity of test chemicals in standardized for regulatory use. the stomach and/or colon of rats in the 6th and 7th Inter- Micronuclei are formed when cells with aberrations national Workshop on Genotoxicity Testing (IWGT) [21, of chromosomes and/or mitotic apparatus divide into 22]. However, it was also mentioned that further data two daughter cells. The GI tract epithelium that has would be needed to identify the sensitivity and specific - high cell proliferative activity is a suitable tissue for ity of this test, in particular, validation of sensitivity using analysis of micronucleated (MNed) cells. GI tract MN aneugens and specificity using both genotoxic and non- tests using mice or rats have been developed by several genotoxic non-carcinogens. In response to these require- researchers [6–13]. They have shown that the GI tract ments, we further evaluated the specificity of the test MN test could detect genotoxic stomach and/or colon using two genotoxic non-carcinogens, amaranth (AM) carcinogens with target organ specificity and that the and quercetin dihydrate (QN) in a 4- and/or 28-day treat- MNed cells in the stomach and colon increased rela- ment regimen, and two non-genotoxic non-carcinogens, tively early after the administration of genotoxic agents NaCl and sucrose (SUC) in a 28-day treatment regimen (mainly clastogens). In our previous study using rats, [15, 20]. However, aneugens and non-genotoxic non-car- the maximum frequency of MNed cells was observed cinogens have not been evaluated with a 4-day treatment at 48–72 h (glandular stomach) and 96 h (colon) after regimen that allows setting higher doses. a single oral administration of genotoxic-carcinogens To obtain additional data to validate the performance [13]. These time-response patterns of MN induction of the GI tract MN test in the present study, we con- were considered to be ascribed to the relatively short ducted rat glandular stomach, colon, and erythrocyte turnover time (approximately 2–4 days) of the GI tract MN tests using three aneugens—colchicine (COL), vin- epithelium [14]. blastine sulfate (VBS), and docetaxel hydrate (DOC)— Based on such time-response patterns of MN induc- and two non-genotoxic non-carcinogens—NaCl (also tion, we developed a rat GI tract MN test combined stomach toxicant) and SUC. These tests were per - with the bone marrow MN test with a 4-day treatment formed with a 4-day treatment regimen using higher regimen [15]. The advantage of this regimen is that MN doses than those in a long-term treatment study. In the induction in cells of the glandular stomach, colon, and present study with aneugens, MN induction in hepato- bone marrow can be simultaneously evaluated in the cytes was preliminarily evaluated. same rat. The test showed good performance using seven genotoxic carcinogens (clastogens) and two geno- toxic non-carcinogens, and the results of the GI tract Materials and methods MN test correlated well with their carcinogenicity to the Animals stomach and/or colon. Notably, N-methyl-N′-nitro-N- Male Crl:CD (SD) rats were purchased from Charles nitrosoguanidine (MNNG) and N-methyl-N-nitrosoure- River Laboratories Japan, Inc. (Kanagawa, Japan), accli- thane (NMUT), genotoxic-stomach-carcinogens, which mated for a week, and used for experiments at an age of are known to be rapidly degraded by contact with high 8 weeks. Either two or three rats were housed in a cage concentrations of thiols in the gastric mucosa after oral with wood-chip bedding, under constant temperature administration [16–18], were positive only in the glandu- (20 ± 3 °C) and humidity (50 ± 20%) with alternating lar stomach, but not in the colon and bone marrow. As 12 h intervals of light and dark throughout the acclima- part of a collaborative study of the repeated-dose (RD) tion and experimental periods. The rats were provided liver MN test in the Mammalian Mutagenicity Study with food and water ad  libitum. All experiments were (MMS) group, a subgroup of the Japanese Environmen- performed according to the guidelines for the care and tal Mutagen and Genome Society (JEMS), a collaborative use of laboratory animals of the Institutional Animal trial of the GI tract MN test among six laboratories was Care and Use Committee of Yakult Central Institute, conducted. After the technical transfer of the method, and the protocols were approved by the committee. Ok ada et al. Genes and Environment (2022) 44:12 Page 3 of 11 Chemicals of 10 mL/kg body weight/day. Each animal in the test We used five test chemicals (COL, VBS, DOC, NaCl and chemical groups was administered once a day for 4 days SUC) for evaluation of the performance of the GI tract (days 1–4), at three doses, via oral gavage (po) or intra- MN test. The rationale for selecting of these chemicals is venous (iv) consistent with their administration routes as follows; COL, VBS, and DOC were selected as aneu- for humans (Table  1). The maximum dose of aneugens gens with different mechanisms of action and routes of was determined based on the toxicity data (for example, administration. COL and VBS exert their action by inhi- decrease in body weight) in a preliminary experiment. bition of tubulin polymerization [23, 24], while DOC acts For non-genotoxic non-carcinogens, the maximum mainly through inhibition of tubulin depolymerization dose was set at 2 g/kg body weight/day (the limit dose in [25]. Furthermore, route of administration for COL is ICH/OECD guidelines [1, 2]) for NaCl and 10 g/kg body oral, while that of VBS and DOC is intravenous injection weight/day (5-fold of the limit dose) for SUC; these doses [24, 26–28]. NaCl and SUC were selected as non-geno- correspond to 2/3 and 1/3 of the L D values [29], respec- toxic non-carcinogens. Additionally, NaCl is a stomach tively. The negative control animals were treated with the toxicant. Vehicles were used as negative controls and vehicle, water for injection (DW, po) or saline (iv), once a three clastogens, 1,2-dimethylhydrazine dihydrochloride day for 4 days. The positive control animals were treated (DMH), N-nitroso-N-methylurea (MNU), and mitomy- via oral gavage with DMH on day 1 and MNU on days 3 cin C (MMC), were used as positive controls. Details and 4 (P1 regimen). This regimen was demonstrated in regarding the test chemicals and positive control chemi- our preliminary test to increase the frequency of MNed cals (source, lot number, purity, and vehicle) are given in cells in the liver as well as in the glandular stomach, Table  1. Each chemical was dissolved in its vehicle and colon, and bone marrow [15], but not in the peripheral administered to rats immediately after preparation. blood. Thus, the positive control animals for the periph - eral blood MN test were intravenously treated with Dose levels and treatment MMC for 4 days (P2 regimen). Each treatment group consisted of five randomly selected During the treatment period including the nec- rats. All administrations were performed in a volume ropsy day, the animals were weighed, and their general Table 1 Chemical information Chemicals Abbreviation CAS No. Source Lot No. Purity Vehicle Dosage Route (mg/10 mL/ kg body weight/day) Aneugens Colchicine COL 64-86-8 FUJIFILM Wako Pure CTP2407 > 95% DW 4, 8, 16 po Chemical Vinblastine sulfate VBS 143-67-9 Nippon Kayaku Y30400 (PF) Saline 0.125, 0.25, 0.5 iv (Exal for Inj. 10 mg) b) Docetaxel hydrate DOC 148408-66-6 Sanofi 4D023J (PF) Saline 1, 2, 4 iv (TAXOTERE 20 mg for I.V. Infusion) Non-genotoxic non-carcinogens Sodium chloride NaCl 7647-14-5 FUJIFILM Wako Pure KPH4443 ≥99.5% DW 500, 1000, 2000 po Chemical Sucrose SUC 57-50-1 Tokyo Chemical Industry E3PLM > 99% DW 1000, 5000, 10000 po Positive control (clastogens) 1,2-Dimethylhydrazine DMH 306-37-6 Tokyo Chemical Industry VU5VG-RD 100% DW 90 po dihydrochloride a) c) N-Nitroso-N-methylurea MNU 684-93-5 Sigma-Aldrich 100 M1436 45% DW 20 po Mitomycin C MMC 50-07-7 Kyowa Hakko Kirin 569ACH (PF) Saline 1 iv (MITOMYCIN Injection 2 mg) DW Water for injection, PF Pharmaceutical formulation The remaining 55% contains stabilizer (water and acetic acid combined) As amount of docetaxel As amount of MNU Okada et al. Genes and Environment (2022) 44:12 Page 4 of 11 condition was observed once a day. On day 3, 24 h after blood samples were washed and suspended in 20 μL the second administration, approximately 0.1 mL of buffer solution. The suspensions (10 μL) were mixed with blood was collected from the tail vein for peripheral 40 μL of labeling solution containing RNase solution, rat blood MN test. Twenty-four hours after the final dosing, anti-CD71, and platelet antibody, and kept in the dark the rats were anesthetized with isoflurane and eutha - for 30 min on ice and then for 30 min at room tempera- nized by exsanguination via abdominal aorta, and their ture. The samples were stained with DNA stain solution stomachs, colons, livers, and right femurs were obtained. containing propidium iodide (PI) and immediately used for flow cytometric analysis; flow cytometric analysis Glandular stomach, colon, liver, and bone marrow MN tests acquired approximately 10,000 to 20,000 CD71-positive MN tests using the glandular stomach, colon, liver, and erythrocytes per animal to determine the frequency of bone marrow were performed according to our previous PI/CD71 double-positive erythrocytes (MNed IMEs) and reports of the MMS/JEMS collaborative study [19, 30]. percentage of CD71-positive erythrocytes among total Briefly, the glandular stomach and colon were everted erythrocytes (%IMEs). Before the analysis, a biological separately on a glass rod and incubated in a solution con- standard sample, malaria-infected erythrocytes, was used taining 1 mmol/L ethylenediaminetetraacetic acid diso- to set up and calibrate the instrument. dium salt (EDTA) and 2 mmol/L dithiothreitol for the stomach and 1 mmol/L EDTA for the colon to isolate the Ki‑67 immunohistochemistry epithelium. The liver (left lateral lobe, approximately 1 g) In the experiment using non-genotoxic non-carcinogens, was sliced and incubated in a flask containing 100 units/ cell proliferation in the glandular stomach and colon was mL of collagenase (Collagenase Yakult-S; Yakult Phar- assessed using Ki-67-positive cells as the marker. We maceutical Industry, Tokyo, Japan) to isolate hepato- considered that this assay was unsuitable for aneugens cytes. Bone marrow cells were collected by washing the known to induce G1, G2, and/or M arrest and, therefore, femur cavity with 1 mL of 10% neutral-buffered forma - did not perform the assay. lin. These cell suspensions were washed, fixed with 10% Ki-67 analysis was performed according to our previ- neutral-buffered formalin, and stored at 4 °C until further ous report [20]. Briefly, a part of the glandular stomach analysis. (containing the fundus) and colon (middle region, 1 cm) Immediately before observation, the cell suspensions were fixed in 10% neutral-buffered formalin, embedded were mixed with staining solution (acridine orange in paraffin, and cut into 4 μm sections. The sections were (AO)/4′,6-diamidino-2-phenylindole dihydrochloride deparaffinized and placed in antigen retrieval solution mixture for the stomach, colon, and liver; AO solution (Target Retrieval Solution; Agilent Technologies Inc., for the bone marrow) on glass slides. Scoring was car- Santa Clara, CA, USA) at 100 °C. Endogenous peroxidase ried out under blinded conditions using a fluorescence activity was inhibited by incubation with 3% H O . The 2 2 microscope (magnification: 600× for the stomach, colon, sections were incubated with monoclonal mouse anti-rat and bone marrow; 400× for the liver) with UV excitation Ki-67 antigen (clone MIB-5; Agilent Technologies Inc.) (365 nm) for the stomach, colon, and liver, and with blue followed by biotinylated rabbit anti-mouse immuno- excitation (490 nm) for the bone marrow. Two thousand globulin (Agilent Technologies Inc.), and subsequently cells or immature erythrocytes (IMEs) were scored per with streptavidin/horseradish peroxidase (Agilent Tech- tissue per animal to determine the frequency of MNed nologies Inc.). Staining was developed with diaminoben- cells. Additionally, 1000 erythrocytes from each rat were zidine (Agilent Technologies Inc.) and the sections were analyzed to determine the percentage of IMEs among counterstained with hematoxylin. Scoring was performed total erythrocytes (%IME). using a light microscope (600×). Thirty glands of the gastric fundus and 30 crypts of Peripheral blood MN test the colon were observed to determine the number of Ki- Peripheral blood MN test was performed using the Rat 67-positive cells per gland and crypt. A cell was scored MicroFlow Plus Micronucleus Analysis Kit (Litron Labo- positive for Ki-67 when the nucleus of the cell was dis- ratories, Rochester, MN, USA) and a flow cytometer (BD tinctively brown. ™ ™ FACSVerse flow cytometer with BD FACSuite soft- ware, Becton, Dickinson and Company, Franklin Lakes, Statistical analyses NJ, USA) following manufacturer’s instructions with Differences in the MNed cell frequency between the slight modification. Briefly, peripheral blood (80 μL) from test chemical groups or positive control group and the each animal was mixed with 250 μL anticoagulant/dilu- negative control group were analyzed statistically using ent, fixed in ultra-cold methanol, and stored at − 80 °C Kastenbaum and Bowman’s tables with an upper-tailed until further analysis. On the day of analysis, the fixed significance level of 0.05. When the frequency of MNed Ok ada et al. Genes and Environment (2022) 44:12 Page 5 of 11 cells increased, the Cochran-Armitage test for a dose- one of them died on the day of necropsy (day 5). In the related trend was also performed, with a one-sided sig- NaCl and SUC treatment groups, no changes in the mean nificance level of 0.05. body weight and general condition were observed com- The other data were analyzed for statistical signifi - pared to the negative control group. cance using two- or multiple-comparison test. Briefly, the statistical significance between two groups was Glandular stomach, colon, liver, and erythrocyte MN tests determined using Student’s t-test for homogenous data Aneugens (COL, VBS, and DOC) or Aspin-Welch test for non-homogenous data, whereas Two animals administered 16 mg/kg COL exhibited the statistical significance between multiple groups was severe toxicity (death and/or diarrhea); therefore, we determined using Dunnett’s test for homogenous data judged this dosage as inappropriate for genotoxicity eval- or Steel test for non-homogenous data, with a two-sided uation and used the samples from animals administered 4 significance level of 0.05. The variance homogeneity of and 8 mg/kg COL for MN analysis. In the glandular stom- two groups or multiple groups was examined using the ach and colon of the animals administered COL orally, F-test or Bartlett’s test, respectively, with a two-sided sig- statistically significant and dose-dependent increases in nificance level of 0.05. the MNed cell frequencies were observed at doses of 4 All analyses were performed using the SAS, version 9.4 and 8 mg/kg compared to those of the respective nega- (SAS Institute Inc., Cary, NC, USA). tive controls (Fig.  2A(a) and B(a)). In the bone marrow, no significant increase in the MNed IME frequency was Results observed at any dose; however, %IME decreased sig- Body weight and general condition nificantly at 8 mg/kg, providing evidence for the bone A significant decrease in the mean body weight was marrow exposure (Fig. 2C). In the liver, although the fre- observed in the treatment groups at middle and high quency of MNed cells increased in both the 4 and 8 mg/ doses of COL, high dose of VBS, and all doses of DOC kg groups, a statistically significant increase was observed compared to that of the respective negative control only in the 4 mg/kg group; this increase in frequency for groups (Fig.  1). No change in the general condition was both groups was not dose-dependent (Fig. 2E(a)). observed in these groups except COL 16 mg/kg body In the animals administered VBS and DOC intra- weight/day (hereafter mg/kg) treatment group; in this venously for 4 days, statistically significant and dose- group, diarrhea was observed in two rats after day 3 and dependent increases in the MNed cell frequencies were Fig. 1 Changes in body weight of different treatment groups: COL (A), VBS (B), and DOC (C). The data are expressed as the mean ± SD. ○: Negative (vehicle) control group, ■: low-dose group (4 mg/kg/day (A), 0.125 mg/kg/day (B), 1 mg/kg/day (C)), ▲: middle-dose group (8 mg/kg/day (A), 0.25 mg/kg/day (B), 2 mg/kg/day (C)), ♦: high-dose group (16 mg/kg/day (A), 0.5 mg/kg/day (B), 4 mg/kg/day (C)). Statistical significance: *p < 0.05, **p < 0.01 (Dunnett’s test) compared to the negative control (See figure on next page.) Fig. 2 Micronucleus test in the glandular stomach, colon, bone marrow, peripheral blood, and liver with aneugens. The test was performed in rats administered COL (po), VBS (iv), and DOC (iv) for 4 days. The sampling points were as follows: 24 h after 4 daily administration (stomach, colon, bone marrow, and liver), and 24 h after the second administration (peripheral blood). Each bar represents the frequency of MNed cells or MNed immature erythrocytes (IMEs) (mean ± SD). Each closed circle represents %IME (mean ± SD). The horizontal axis represents chemical and/or dosage (mg/kg/ day). N, Negative (vehicle) control; P, positive control (P1: MNU and DMH treatment, P2: MMC treatment); TOX, data were excluded due to severe # § ‡‡ toxicity; ND, not done. Statistical significance: **p < 0.01 (Kastenbaum & Bowman test), p < 0.05 (Dunnett’s test), p < 0.05 (Steel test), and p < 0.01 (Aspin-Welch test) compared to the negative control Okada et al. Genes and Environment (2022) 44:12 Page 6 of 11 Fig. 2 (See legend on previous page.) Ok ada et al. Genes and Environment (2022) 44:12 Page 7 of 11 observed in the glandular stomach and colon (Fig. 2A(b) peripheral blood but also in the glandular stomach and and B(b)). In the bone marrow, cell proliferation was colon (Fig. 2). markedly inhibited (% IME values were less than 10% of the negative control values) at all dosages after 4-day Discussion treatment; hence, the MNed cell frequencies could not We assessed the performance of the GI tract MN test be analyzed. Instead, MN induction was analyzed using for aneugens (COL, VBS, and DOC) and non-genotoxic peripheral blood collected on the day after the sec- non-carcinogens (NaCl and SUC) with a 4-day treatment ond treatment and a statistically significant and dose- regimen. The results of MN tests, in  vitro genotoxicity dependent increase in the frequency of MNed IMEs was tests, and carcinogenicity studies are shown in Table 2 on observed (Fig.  2D). The %IMEs decreased significantly several chemicals that were used in this study and past at all doses for each chemical. In the liver, statistically studies [15, 18, 30–51]. significant and dose-dependent increases in MNed cell COL, an alkaloid product of Colchicum autumnale, frequencies were observed at all doses of both VBS and inhibits tubulin polymerization, and it is used as an oral DOC after 4-day treatment. medicine to treat gout and other inflammations [26, 27]. VBS, a vinca alkaloid, and DOC, a taxoid, are known to inhibit tubulin polymerization and depolymerization, Non‑genotoxic non‑carcinogens (NaCl and SUC) respectively, and are used as anti-cancer drugs admin- In the NaCl and SUC treatment groups, no significant istered intravenously [24, 25, 28]. After administering increase in MNed cell frequency was observed in any of these aneugens to rats according to their clinical admin- the three tissues (Fig.  3). In the NaCl treatment group, istration route, the frequencies of MNed cells in the the number of Ki-67-positive cells significantly increased glandular stomach and colon significantly increased in a only in the glandular stomach at doses of 1 and 2 mg/kg, dose-dependent manner for all aneugens. It is considered while in the SUC treatment group, no change in cell pro- that intravenously administered VBS and DOC reach the liferation was observed in any of the three tissues. stomach and colon via the bloodstream and then induce MN in the cells of these organs. Being administered intra- Positive control gastrically, COL was in contact with the stomach. After In all of the P1 positive control groups (DMH + MNU absorption from the small intestine, a proportion of the treatment groups) set for the glandular stomach, colon, unchanged drug possibly reaches the stomach and colon bone marrow, and liver MN tests, statistically significant via the blood stream, and the major proportion reaches increases in MNed cell frequencies were observed in all the colon from the luminal side due to the involvement target tissues (Figs.  2, 3). In addition, in the P2 positive of P-glycoprotein in the small intestine and liver [27]. In control group (MMC treatment group) set for the periph- the present study, it was revealed that the GI tract MN eral blood MN test, statistically significant increase in test could detect aneugens in the glandular stomach the MNed cell frequencies were observed not only in the and colon of rats when administered via clinical route Fig. 3 Micronucleus test in the glandular stomach, colon, and bone marrow with non-genotoxic non-carcinogens. The test was performed in rats administered NaCl or SUC for 4 days. Each bar represents the frequency of MNed cells or MNed immature erythrocytes (IMEs) (mean ± SD). Each closed circle represents the number of Ki-67 positive cells (per gland or crypt) or %IME (mean ± SD). The horizontal axis represents chemical and/or dosage (mg/kg/day). N, Negative (vehicle) control; P1, positive control (DMH and MNU treatment). Statistical significance: **p < 0.01 (Kastenbaum & ## †† Bowman test), p < 0.01 (Dunnett’s test), and p < 0.01 (Student’s t-test) compared to the negative control Okada et al. Genes and Environment (2022) 44:12 Page 8 of 11 Table 2 Summary of the results of 4-day repeated-dose micronucleus test Classes Chemicals Route Micronucleus test with a 4‑ day treatment In vitro genotoxicity test Carcinogenicity in regimen rodent (main target) [Ref.] Glandular Colon Bone marrow Ref. Ames [Ref.] Chromosomal stomach aberration [Ref.] Aneugens COL po + + – Present study – [31] + (poly) [31] No data a) VBS iv + + Tox (PB; +) Present study – [31] + (poly) [31, 32] No data a) DOC iv + + Tox (PB; +) Present study – [33] + (poly) [33] No data Clastogens MNU po + + + [15] + [34] + [34] + (Stomach) [35, 36] 4NQO po + – + [15] + [31] + [31] + (Stomach) [37] MNNG po + – – [15] + [34] + [34] + (Stomach) [35, 38] NMUT po + – – [15] + [39] + [39] + (Stomach) [18] DMH po – + + [15] + [34] + [34] + (Colon) [35, 40] PhIP po – + + [15] + [31] + [31] + (Colon) [35, 41] KBrO po + Eq + [15, 30] + [42] + [42] + (Kidney) [35, 42] MMC iv + + + Present study + [31, 34] + [31, 34] + (Peritoneum) [35] Genotoxic AM po – – – [15] – [43] + [39] – [44, 45] non-carcinogens QN po – – – [15] + [43] + [43] – [46–48] Non-genotoxic NaCl po – – – Present study – [34] – [34], + [49] – [34, 35] non-carcinogens SUC po – – – Present study – [50] – [51], + [49] – [35] +, Positive; −, negative; Eq Equivocal, Tox Toxic, PB Peripheral blood, Ref. References MN induction in erythrocytes was analyzed using peripheral blood on the day after the second administration COL Colchicine, VBS Vinblastine sulfate, DOC Docetaxel hydrate, MNU N-nitroso-N-methylurea, 4NQO 4-nitroquinoline-1-oxide, MNNG N-methyl-N′-nitro-N- nitrosoguanidine, NMUT N-methyl-N-nitrosourethane, DMH 1,2-dimethylhydrazine dihydrochloride, PhIP 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride, KBrO Potassium bromate, MMC Mitomycin C, AM Amaranth, QN Quercetin dihydrate, NaCl Sodium chloride, SUC Sucrose (intravenous and oral administration). To date, there are present study, and dose-dependent MN induction (posi- few reports of MN induction by aneugens in GI tract tive response) was confirmed for VBS and DOC. For cells, particularly in the stomach epithelium. Therefore, COL, although the frequency of MNed cells increased the present results provide important evidence showing in both the 4 and 8 mg/kg groups, a statistically signifi - good sensitivity of the GI tract MN test to aneugens. cant increase was observed only in the 4 mg/kg group; In erythrocytes, on the other hand, MN induction this increase in frequency for both groups was not dose- could not be detected even when COL was administered dependent. The MN test using the liver, the primary tis - up to the maximum tolerated dose. Cammerer et  al. sue involved in metabolism, as well as the GI tract MN [52] reported a delayed increase in MNed erythrocytes test are considered important for in  vivo genotoxicity (peak at 96 h after the final dosing) in rats orally admin - evaluation. Therefore, various methods such as the par - istered 6 and 8 mg/kg COL for five consecutive days. In tial hepatectomy (PH) method, the juvenile/young rat addition, in the present study, since severe cytotoxicity method, and the RD method have been developed [22, was observed in the bone marrow of rats administered 53, 54]. Sensitivity verification using clastogens has been VBS or DOC for 4 days at all dosages, MNed erythro- carried out using these methods but that using aneugens cyte induction could not be assessed in the bone marrow has been mainly performed using the PH method. Cur- (data not shown). Instead, MN induction was detected in rently, the MMS group is evaluating the sensitivity of the the peripheral blood on the day after the second adminis- liver MN test with the 28-day RD method using aneu- tration. Therefore, the 4-day treatment regimen allows to gens (Shigano M et  al., manuscript in preparation). The collect peripheral blood during the dosing period or col- RD method has the potential to detect MN induction lect bone marrow cells after the recovery period to evalu- in cells in several tissues including the liver, bone mar- ate MNed erythrocyte induction by compounds such as row, and GI tract. However, the cell division cycles dif- aneugens that cause severe bone marrow toxicity and/or fer substantially among these tissues, and in the case of delay of MN induction. aneugens that induce mitotic inhibition and severe cyto- The MNed hepatocyte frequencies in rats treated toxicity, it is important to determine the optimal dos- with aneugens were preliminarily monitored in the age, dosing period, and sampling time for MN analyses Ok ada et al. Genes and Environment (2022) 44:12 Page 9 of 11 in the respective tissues. In the present study, a signifi - of genotoxicity (clastogenicity and aneugenicity) of test cant increase in MNed cell frequency was observed in chemicals. the liver in addition to the GI tract and peripheral blood in rats administered aneugens with a 4-day treatment Conclusions regimen. These results suggest that it may be possible to To further clarify the sensitivity and specificity of the rat detect the aneugenic potential of chemicals in the liver GI tract MN test, we conducted additional studies using using this 4-day treatment regimen. However, further three aneugens and two non-genotoxic non-carcinogens research is required to elucidate the performance of this with a 4-day treatment regimen. The study demonstrated short-term administration method. that the glandular stomach and colon MN tests could To investigate the specificity of the GI tract MN test detect aneugens as positive and could adequately evalu- with a 4-day treatment regimen, NaCl and SUC were ate non-genotoxic non-carcinogens as negative, includ- selected as non-genotoxic non-carcinogens. They did not ing a chemical that enhances cell proliferation. These induce MNed cells in the GI tract and bone marrow of results provide important evidence supporting the good rats when administered up to 2 g/kg of NaCl (2/3 of the performance of the rat glandular stomach and colon MN LD value) and 10 g/kg of SUC (1/3 of the LD value) 50 50 tests with a 4-day treatment regimen. [29]. No treatment-related changes were observed in the clinical signs or body weight gain. NaCl is a stomach Abbreviations toxicant that promotes the proliferation of the glandular MN: Micronucleus; GI: Gastrointestinal; RD: Repeated-dose; COL: Colchicine; VBS: stomach epithelium [55] and exerts tumor-promoting Vinblastine sulfate; DOC: Docetaxel hydrate; SUC: Sucrose; DMH: 1,2-dimethylhy- drazine dihydrochloride; MNU: N-nitroso-N-methylurea; MMC: Mitomycin C. activity [56] when orally administered to rats. SUC is known to enhance the proliferation of the colonic epithe- Acknowledgments lium of rats administered orally [57]. As an index of cell We would like to thank Dr. S. Hamada (Bozo Research Center Inc., Tokyo, Japan), the principal investigator of the MMS/JEMS collaborative study (the proliferation in the GI tract, we analyzed proliferating repeated-dose liver micronucleus study), and the MMS/JEMS members for cells in tissue sections using immunohistochemistry of advice on the study experiments. We are grateful to Drs. T. Kobayashi and S. Ki-67, a nuclear protein expressed in the cell cycle phase Kado for their encouragement throughout the study. We would like to thank Editage (www. edita ge. com) for English language editing. other than the G0 phase [58]. In the present study, the Ki- 67-positive cell counts significantly increased only in the Authors’ contributions gastric epithelium of the rats treated with NaCl, indicat- EO, YF, KN, and WO conducted micronucleus assays and performed a comprehensive evaluation. EO created the tables and figures and wrote the ing that cell proliferation was enhanced, but not in the manuscript. All authors read and approved the final manuscript. cells of all three tissues evaluated including the colon in the rats treated with SUC. It has been reported that an Funding Not applicable. increase in erythropoiesis that is not due to chemical- induced genotoxic damage causes an increase in the Availability of data and materials number of spontaneous MNed erythrocytes, resulting in All data generated or analyzed during this study are included in this published article. false-positive results in the routine erythrocyte MN test [59]. Under the conditions of the present experiment, Declarations however, it was demonstrated that both NaCl and SUC did not increase MNed cell frequencies in the glandular Ethics approval and consent to participate stomach and colon, regardless of whether cell prolifera- Animal experiments were approved by the Institutional Animal Care and Use Committee of Yakult Central Institute before the experiments were conducted. tion was enhanced. These results show that non-geno - toxic non-carcinogens including chemicals that enhance Consent for publication cell proliferation could be correctly evaluated as negative Not applicable. in the glandular stomach and colon MN tests. Competing interests In our previous study, the rat glandular stomach and The authors declare that they have no competing interests. colon MN tests with a 4-day treatment regimen were Received: 23 December 2021 Accepted: 27 March 2022 performed using seven clastogens and two genotoxic- non-carcinogens, and their sensitivity and specific - ity were confirmed (Table  2). Furthermore, the present study revealed that aneugens can be detected as positive, References and non-genotoxic non-carcinogens can be evaluated 1. ICH. ICH harmonized tripartite guideline S2(R1): guidance on genotoxic- as negative. 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Tweats DJ, Blakey D, Heflich RH, Jacobs A, Jacobsen SD, Morita T, et al. rapid publication on acceptance Report of the IWGT working group on strategies and interpretation support for research data, including large and complex data types of regulatory in vivo tests I. increases in micronucleated bone mar- • gold Open Access which fosters wider collaboration and increased citations row cells in rodents that do not indicate genotoxic hazards. Mutat Res. 2007;627:78–91. https:// doi. org/ 10. 1016/j. mrgen tox. 2006. 10. 005. maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- Learn more biomedcentral.com/submissions lished maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genes and Environment Springer Journals

Evaluation of a 4-day repeated-dose micronucleus test in rat glandular stomach and colon using aneugens and non-genotoxic non-carcinogens

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Abstract

Background: We previously developed a rodent gastrointestinal (GI) tract micronucleus (MN) test using the glan- dular stomach and/or colon, and evaluated this test method using several genotoxic carcinogens (clastogens) and genotoxic non-carcinogens; we demonstrated that this test method could detect genotoxic stomach and/or colon carcinogens with target organ specificity. In the present study, we further evaluated the sensitivity and specificity of the MN test for the rat glandular stomach and colon using three aneugens (colchicine, vinblastine sulfate, and doc- etaxel hydrate) and two non-genotoxic non-carcinogens (sodium chloride and sucrose). Results: Male Crl:CD (SD) rats were administered test compounds through clinical administration route (orally or intravenously) for four consecutive days and then examined for the micronucleated cell frequencies in the glandular stomach and colon. We observed that all three aneugens significantly and dose-dependently increased the micro - nucleated cell frequencies in the stomach and colon. In contrast, neither of the two non-genotoxic non-carcinogens increased the micronucleated cell frequency in these tissues. Notably, an increase in cell proliferation was observed in the glandular stomach of rats administered a stomach toxicant, sodium chloride, but this increase did not affect the induction of micronuclei in the gastric cells. Conclusions: In the present study, it was demonstrated that the glandular stomach and colon MN tests could detect aneugens as positive and could adequately evaluate non-genotoxic non-carcinogens as negative, including a chemi- cal that enhances cell proliferation. These results provide important evidence supporting good performance of the rat glandular stomach and colon MN tests with a 4-day treatment regimen. Keywords: Micronucleus test, Gastrointestinal tract, Glandular stomach, Colon, 4-day treatment regimen, Aneugen showing a positive result in an in  vitro genotoxicity test Introduction gives a negative result in the erythrocyte MN test, it is The erythrocyte micronucleus (MN) test using rodent necessary to conduct follow-up evaluation with a second bone marrow/peripheral blood is one of the stand- in  vivo test using appropriate target tissues and/or end- ard genotoxicity tests in regulatory use and is valuable points based on factors such as exposure, metabolism, for evaluating the clastogenicity and aneugenicity of and distribution in the drug development [1]. Regarding chemicals in  vivo [1–3]. However, when a test chemical evaluation of orally administered substances, the gas- trointestinal (GI) tract is useful being a direct contact *Correspondence: emiko-okada@yakult.co.jp site for the substances. In particular, the stomach is con- Yakult Central Institute, Yakult Honsha Co., Ltd., 5-11 Izumi, Kunitachi-shi, Tokyo 186-8650, Japan sidered to be important because of its high exposure to © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Okada et al. Genes and Environment (2022) 44:12 Page 2 of 11 orally administered substances. The colon is also useful inter-laboratory reproducibility was confirmed using for evaluating the genotoxicity of substances that reach negative- and positive-control substances with a 4-day the intestine via intestinal contents and/or bloodstream treatment regimen [19]. Furthermore, the glandular as well as substances that are metabolized by intesti- stomach and colon MN tests showed good performance nal microbiota. For genotoxicity tests targeting the GI even with the long-term RD regimen of 14 and/or 28 days tract, the comet assay to detect DNA damage and the [19, 20]. This evidence suggests that the GI tract MN test transgenic rodent gene mutation assay to detect gene could be incorporated into a 28-day RD toxicity test, sim- mutations have already been established and listed in ilar to the erythrocyte MN test. Based on these findings, the OECD Test Guidelines [4, 5]; however, the GI tract the GI tract MN test was concluded to be a promising MN test to detect chromosome damage has not yet been method to examine the clastogenicity of test chemicals in standardized for regulatory use. the stomach and/or colon of rats in the 6th and 7th Inter- Micronuclei are formed when cells with aberrations national Workshop on Genotoxicity Testing (IWGT) [21, of chromosomes and/or mitotic apparatus divide into 22]. However, it was also mentioned that further data two daughter cells. The GI tract epithelium that has would be needed to identify the sensitivity and specific - high cell proliferative activity is a suitable tissue for ity of this test, in particular, validation of sensitivity using analysis of micronucleated (MNed) cells. GI tract MN aneugens and specificity using both genotoxic and non- tests using mice or rats have been developed by several genotoxic non-carcinogens. In response to these require- researchers [6–13]. They have shown that the GI tract ments, we further evaluated the specificity of the test MN test could detect genotoxic stomach and/or colon using two genotoxic non-carcinogens, amaranth (AM) carcinogens with target organ specificity and that the and quercetin dihydrate (QN) in a 4- and/or 28-day treat- MNed cells in the stomach and colon increased rela- ment regimen, and two non-genotoxic non-carcinogens, tively early after the administration of genotoxic agents NaCl and sucrose (SUC) in a 28-day treatment regimen (mainly clastogens). In our previous study using rats, [15, 20]. However, aneugens and non-genotoxic non-car- the maximum frequency of MNed cells was observed cinogens have not been evaluated with a 4-day treatment at 48–72 h (glandular stomach) and 96 h (colon) after regimen that allows setting higher doses. a single oral administration of genotoxic-carcinogens To obtain additional data to validate the performance [13]. These time-response patterns of MN induction of the GI tract MN test in the present study, we con- were considered to be ascribed to the relatively short ducted rat glandular stomach, colon, and erythrocyte turnover time (approximately 2–4 days) of the GI tract MN tests using three aneugens—colchicine (COL), vin- epithelium [14]. blastine sulfate (VBS), and docetaxel hydrate (DOC)— Based on such time-response patterns of MN induc- and two non-genotoxic non-carcinogens—NaCl (also tion, we developed a rat GI tract MN test combined stomach toxicant) and SUC. These tests were per - with the bone marrow MN test with a 4-day treatment formed with a 4-day treatment regimen using higher regimen [15]. The advantage of this regimen is that MN doses than those in a long-term treatment study. In the induction in cells of the glandular stomach, colon, and present study with aneugens, MN induction in hepato- bone marrow can be simultaneously evaluated in the cytes was preliminarily evaluated. same rat. The test showed good performance using seven genotoxic carcinogens (clastogens) and two geno- toxic non-carcinogens, and the results of the GI tract Materials and methods MN test correlated well with their carcinogenicity to the Animals stomach and/or colon. Notably, N-methyl-N′-nitro-N- Male Crl:CD (SD) rats were purchased from Charles nitrosoguanidine (MNNG) and N-methyl-N-nitrosoure- River Laboratories Japan, Inc. (Kanagawa, Japan), accli- thane (NMUT), genotoxic-stomach-carcinogens, which mated for a week, and used for experiments at an age of are known to be rapidly degraded by contact with high 8 weeks. Either two or three rats were housed in a cage concentrations of thiols in the gastric mucosa after oral with wood-chip bedding, under constant temperature administration [16–18], were positive only in the glandu- (20 ± 3 °C) and humidity (50 ± 20%) with alternating lar stomach, but not in the colon and bone marrow. As 12 h intervals of light and dark throughout the acclima- part of a collaborative study of the repeated-dose (RD) tion and experimental periods. The rats were provided liver MN test in the Mammalian Mutagenicity Study with food and water ad  libitum. All experiments were (MMS) group, a subgroup of the Japanese Environmen- performed according to the guidelines for the care and tal Mutagen and Genome Society (JEMS), a collaborative use of laboratory animals of the Institutional Animal trial of the GI tract MN test among six laboratories was Care and Use Committee of Yakult Central Institute, conducted. After the technical transfer of the method, and the protocols were approved by the committee. Ok ada et al. Genes and Environment (2022) 44:12 Page 3 of 11 Chemicals of 10 mL/kg body weight/day. Each animal in the test We used five test chemicals (COL, VBS, DOC, NaCl and chemical groups was administered once a day for 4 days SUC) for evaluation of the performance of the GI tract (days 1–4), at three doses, via oral gavage (po) or intra- MN test. The rationale for selecting of these chemicals is venous (iv) consistent with their administration routes as follows; COL, VBS, and DOC were selected as aneu- for humans (Table  1). The maximum dose of aneugens gens with different mechanisms of action and routes of was determined based on the toxicity data (for example, administration. COL and VBS exert their action by inhi- decrease in body weight) in a preliminary experiment. bition of tubulin polymerization [23, 24], while DOC acts For non-genotoxic non-carcinogens, the maximum mainly through inhibition of tubulin depolymerization dose was set at 2 g/kg body weight/day (the limit dose in [25]. Furthermore, route of administration for COL is ICH/OECD guidelines [1, 2]) for NaCl and 10 g/kg body oral, while that of VBS and DOC is intravenous injection weight/day (5-fold of the limit dose) for SUC; these doses [24, 26–28]. NaCl and SUC were selected as non-geno- correspond to 2/3 and 1/3 of the L D values [29], respec- toxic non-carcinogens. Additionally, NaCl is a stomach tively. The negative control animals were treated with the toxicant. Vehicles were used as negative controls and vehicle, water for injection (DW, po) or saline (iv), once a three clastogens, 1,2-dimethylhydrazine dihydrochloride day for 4 days. The positive control animals were treated (DMH), N-nitroso-N-methylurea (MNU), and mitomy- via oral gavage with DMH on day 1 and MNU on days 3 cin C (MMC), were used as positive controls. Details and 4 (P1 regimen). This regimen was demonstrated in regarding the test chemicals and positive control chemi- our preliminary test to increase the frequency of MNed cals (source, lot number, purity, and vehicle) are given in cells in the liver as well as in the glandular stomach, Table  1. Each chemical was dissolved in its vehicle and colon, and bone marrow [15], but not in the peripheral administered to rats immediately after preparation. blood. Thus, the positive control animals for the periph - eral blood MN test were intravenously treated with Dose levels and treatment MMC for 4 days (P2 regimen). Each treatment group consisted of five randomly selected During the treatment period including the nec- rats. All administrations were performed in a volume ropsy day, the animals were weighed, and their general Table 1 Chemical information Chemicals Abbreviation CAS No. Source Lot No. Purity Vehicle Dosage Route (mg/10 mL/ kg body weight/day) Aneugens Colchicine COL 64-86-8 FUJIFILM Wako Pure CTP2407 > 95% DW 4, 8, 16 po Chemical Vinblastine sulfate VBS 143-67-9 Nippon Kayaku Y30400 (PF) Saline 0.125, 0.25, 0.5 iv (Exal for Inj. 10 mg) b) Docetaxel hydrate DOC 148408-66-6 Sanofi 4D023J (PF) Saline 1, 2, 4 iv (TAXOTERE 20 mg for I.V. Infusion) Non-genotoxic non-carcinogens Sodium chloride NaCl 7647-14-5 FUJIFILM Wako Pure KPH4443 ≥99.5% DW 500, 1000, 2000 po Chemical Sucrose SUC 57-50-1 Tokyo Chemical Industry E3PLM > 99% DW 1000, 5000, 10000 po Positive control (clastogens) 1,2-Dimethylhydrazine DMH 306-37-6 Tokyo Chemical Industry VU5VG-RD 100% DW 90 po dihydrochloride a) c) N-Nitroso-N-methylurea MNU 684-93-5 Sigma-Aldrich 100 M1436 45% DW 20 po Mitomycin C MMC 50-07-7 Kyowa Hakko Kirin 569ACH (PF) Saline 1 iv (MITOMYCIN Injection 2 mg) DW Water for injection, PF Pharmaceutical formulation The remaining 55% contains stabilizer (water and acetic acid combined) As amount of docetaxel As amount of MNU Okada et al. Genes and Environment (2022) 44:12 Page 4 of 11 condition was observed once a day. On day 3, 24 h after blood samples were washed and suspended in 20 μL the second administration, approximately 0.1 mL of buffer solution. The suspensions (10 μL) were mixed with blood was collected from the tail vein for peripheral 40 μL of labeling solution containing RNase solution, rat blood MN test. Twenty-four hours after the final dosing, anti-CD71, and platelet antibody, and kept in the dark the rats were anesthetized with isoflurane and eutha - for 30 min on ice and then for 30 min at room tempera- nized by exsanguination via abdominal aorta, and their ture. The samples were stained with DNA stain solution stomachs, colons, livers, and right femurs were obtained. containing propidium iodide (PI) and immediately used for flow cytometric analysis; flow cytometric analysis Glandular stomach, colon, liver, and bone marrow MN tests acquired approximately 10,000 to 20,000 CD71-positive MN tests using the glandular stomach, colon, liver, and erythrocytes per animal to determine the frequency of bone marrow were performed according to our previous PI/CD71 double-positive erythrocytes (MNed IMEs) and reports of the MMS/JEMS collaborative study [19, 30]. percentage of CD71-positive erythrocytes among total Briefly, the glandular stomach and colon were everted erythrocytes (%IMEs). Before the analysis, a biological separately on a glass rod and incubated in a solution con- standard sample, malaria-infected erythrocytes, was used taining 1 mmol/L ethylenediaminetetraacetic acid diso- to set up and calibrate the instrument. dium salt (EDTA) and 2 mmol/L dithiothreitol for the stomach and 1 mmol/L EDTA for the colon to isolate the Ki‑67 immunohistochemistry epithelium. The liver (left lateral lobe, approximately 1 g) In the experiment using non-genotoxic non-carcinogens, was sliced and incubated in a flask containing 100 units/ cell proliferation in the glandular stomach and colon was mL of collagenase (Collagenase Yakult-S; Yakult Phar- assessed using Ki-67-positive cells as the marker. We maceutical Industry, Tokyo, Japan) to isolate hepato- considered that this assay was unsuitable for aneugens cytes. Bone marrow cells were collected by washing the known to induce G1, G2, and/or M arrest and, therefore, femur cavity with 1 mL of 10% neutral-buffered forma - did not perform the assay. lin. These cell suspensions were washed, fixed with 10% Ki-67 analysis was performed according to our previ- neutral-buffered formalin, and stored at 4 °C until further ous report [20]. Briefly, a part of the glandular stomach analysis. (containing the fundus) and colon (middle region, 1 cm) Immediately before observation, the cell suspensions were fixed in 10% neutral-buffered formalin, embedded were mixed with staining solution (acridine orange in paraffin, and cut into 4 μm sections. The sections were (AO)/4′,6-diamidino-2-phenylindole dihydrochloride deparaffinized and placed in antigen retrieval solution mixture for the stomach, colon, and liver; AO solution (Target Retrieval Solution; Agilent Technologies Inc., for the bone marrow) on glass slides. Scoring was car- Santa Clara, CA, USA) at 100 °C. Endogenous peroxidase ried out under blinded conditions using a fluorescence activity was inhibited by incubation with 3% H O . The 2 2 microscope (magnification: 600× for the stomach, colon, sections were incubated with monoclonal mouse anti-rat and bone marrow; 400× for the liver) with UV excitation Ki-67 antigen (clone MIB-5; Agilent Technologies Inc.) (365 nm) for the stomach, colon, and liver, and with blue followed by biotinylated rabbit anti-mouse immuno- excitation (490 nm) for the bone marrow. Two thousand globulin (Agilent Technologies Inc.), and subsequently cells or immature erythrocytes (IMEs) were scored per with streptavidin/horseradish peroxidase (Agilent Tech- tissue per animal to determine the frequency of MNed nologies Inc.). Staining was developed with diaminoben- cells. Additionally, 1000 erythrocytes from each rat were zidine (Agilent Technologies Inc.) and the sections were analyzed to determine the percentage of IMEs among counterstained with hematoxylin. Scoring was performed total erythrocytes (%IME). using a light microscope (600×). Thirty glands of the gastric fundus and 30 crypts of Peripheral blood MN test the colon were observed to determine the number of Ki- Peripheral blood MN test was performed using the Rat 67-positive cells per gland and crypt. A cell was scored MicroFlow Plus Micronucleus Analysis Kit (Litron Labo- positive for Ki-67 when the nucleus of the cell was dis- ratories, Rochester, MN, USA) and a flow cytometer (BD tinctively brown. ™ ™ FACSVerse flow cytometer with BD FACSuite soft- ware, Becton, Dickinson and Company, Franklin Lakes, Statistical analyses NJ, USA) following manufacturer’s instructions with Differences in the MNed cell frequency between the slight modification. Briefly, peripheral blood (80 μL) from test chemical groups or positive control group and the each animal was mixed with 250 μL anticoagulant/dilu- negative control group were analyzed statistically using ent, fixed in ultra-cold methanol, and stored at − 80 °C Kastenbaum and Bowman’s tables with an upper-tailed until further analysis. On the day of analysis, the fixed significance level of 0.05. When the frequency of MNed Ok ada et al. Genes and Environment (2022) 44:12 Page 5 of 11 cells increased, the Cochran-Armitage test for a dose- one of them died on the day of necropsy (day 5). In the related trend was also performed, with a one-sided sig- NaCl and SUC treatment groups, no changes in the mean nificance level of 0.05. body weight and general condition were observed com- The other data were analyzed for statistical signifi - pared to the negative control group. cance using two- or multiple-comparison test. Briefly, the statistical significance between two groups was Glandular stomach, colon, liver, and erythrocyte MN tests determined using Student’s t-test for homogenous data Aneugens (COL, VBS, and DOC) or Aspin-Welch test for non-homogenous data, whereas Two animals administered 16 mg/kg COL exhibited the statistical significance between multiple groups was severe toxicity (death and/or diarrhea); therefore, we determined using Dunnett’s test for homogenous data judged this dosage as inappropriate for genotoxicity eval- or Steel test for non-homogenous data, with a two-sided uation and used the samples from animals administered 4 significance level of 0.05. The variance homogeneity of and 8 mg/kg COL for MN analysis. In the glandular stom- two groups or multiple groups was examined using the ach and colon of the animals administered COL orally, F-test or Bartlett’s test, respectively, with a two-sided sig- statistically significant and dose-dependent increases in nificance level of 0.05. the MNed cell frequencies were observed at doses of 4 All analyses were performed using the SAS, version 9.4 and 8 mg/kg compared to those of the respective nega- (SAS Institute Inc., Cary, NC, USA). tive controls (Fig.  2A(a) and B(a)). In the bone marrow, no significant increase in the MNed IME frequency was Results observed at any dose; however, %IME decreased sig- Body weight and general condition nificantly at 8 mg/kg, providing evidence for the bone A significant decrease in the mean body weight was marrow exposure (Fig. 2C). In the liver, although the fre- observed in the treatment groups at middle and high quency of MNed cells increased in both the 4 and 8 mg/ doses of COL, high dose of VBS, and all doses of DOC kg groups, a statistically significant increase was observed compared to that of the respective negative control only in the 4 mg/kg group; this increase in frequency for groups (Fig.  1). No change in the general condition was both groups was not dose-dependent (Fig. 2E(a)). observed in these groups except COL 16 mg/kg body In the animals administered VBS and DOC intra- weight/day (hereafter mg/kg) treatment group; in this venously for 4 days, statistically significant and dose- group, diarrhea was observed in two rats after day 3 and dependent increases in the MNed cell frequencies were Fig. 1 Changes in body weight of different treatment groups: COL (A), VBS (B), and DOC (C). The data are expressed as the mean ± SD. ○: Negative (vehicle) control group, ■: low-dose group (4 mg/kg/day (A), 0.125 mg/kg/day (B), 1 mg/kg/day (C)), ▲: middle-dose group (8 mg/kg/day (A), 0.25 mg/kg/day (B), 2 mg/kg/day (C)), ♦: high-dose group (16 mg/kg/day (A), 0.5 mg/kg/day (B), 4 mg/kg/day (C)). Statistical significance: *p < 0.05, **p < 0.01 (Dunnett’s test) compared to the negative control (See figure on next page.) Fig. 2 Micronucleus test in the glandular stomach, colon, bone marrow, peripheral blood, and liver with aneugens. The test was performed in rats administered COL (po), VBS (iv), and DOC (iv) for 4 days. The sampling points were as follows: 24 h after 4 daily administration (stomach, colon, bone marrow, and liver), and 24 h after the second administration (peripheral blood). Each bar represents the frequency of MNed cells or MNed immature erythrocytes (IMEs) (mean ± SD). Each closed circle represents %IME (mean ± SD). The horizontal axis represents chemical and/or dosage (mg/kg/ day). N, Negative (vehicle) control; P, positive control (P1: MNU and DMH treatment, P2: MMC treatment); TOX, data were excluded due to severe # § ‡‡ toxicity; ND, not done. Statistical significance: **p < 0.01 (Kastenbaum & Bowman test), p < 0.05 (Dunnett’s test), p < 0.05 (Steel test), and p < 0.01 (Aspin-Welch test) compared to the negative control Okada et al. Genes and Environment (2022) 44:12 Page 6 of 11 Fig. 2 (See legend on previous page.) Ok ada et al. Genes and Environment (2022) 44:12 Page 7 of 11 observed in the glandular stomach and colon (Fig. 2A(b) peripheral blood but also in the glandular stomach and and B(b)). In the bone marrow, cell proliferation was colon (Fig. 2). markedly inhibited (% IME values were less than 10% of the negative control values) at all dosages after 4-day Discussion treatment; hence, the MNed cell frequencies could not We assessed the performance of the GI tract MN test be analyzed. Instead, MN induction was analyzed using for aneugens (COL, VBS, and DOC) and non-genotoxic peripheral blood collected on the day after the sec- non-carcinogens (NaCl and SUC) with a 4-day treatment ond treatment and a statistically significant and dose- regimen. The results of MN tests, in  vitro genotoxicity dependent increase in the frequency of MNed IMEs was tests, and carcinogenicity studies are shown in Table 2 on observed (Fig.  2D). The %IMEs decreased significantly several chemicals that were used in this study and past at all doses for each chemical. In the liver, statistically studies [15, 18, 30–51]. significant and dose-dependent increases in MNed cell COL, an alkaloid product of Colchicum autumnale, frequencies were observed at all doses of both VBS and inhibits tubulin polymerization, and it is used as an oral DOC after 4-day treatment. medicine to treat gout and other inflammations [26, 27]. VBS, a vinca alkaloid, and DOC, a taxoid, are known to inhibit tubulin polymerization and depolymerization, Non‑genotoxic non‑carcinogens (NaCl and SUC) respectively, and are used as anti-cancer drugs admin- In the NaCl and SUC treatment groups, no significant istered intravenously [24, 25, 28]. After administering increase in MNed cell frequency was observed in any of these aneugens to rats according to their clinical admin- the three tissues (Fig.  3). In the NaCl treatment group, istration route, the frequencies of MNed cells in the the number of Ki-67-positive cells significantly increased glandular stomach and colon significantly increased in a only in the glandular stomach at doses of 1 and 2 mg/kg, dose-dependent manner for all aneugens. It is considered while in the SUC treatment group, no change in cell pro- that intravenously administered VBS and DOC reach the liferation was observed in any of the three tissues. stomach and colon via the bloodstream and then induce MN in the cells of these organs. Being administered intra- Positive control gastrically, COL was in contact with the stomach. After In all of the P1 positive control groups (DMH + MNU absorption from the small intestine, a proportion of the treatment groups) set for the glandular stomach, colon, unchanged drug possibly reaches the stomach and colon bone marrow, and liver MN tests, statistically significant via the blood stream, and the major proportion reaches increases in MNed cell frequencies were observed in all the colon from the luminal side due to the involvement target tissues (Figs.  2, 3). In addition, in the P2 positive of P-glycoprotein in the small intestine and liver [27]. In control group (MMC treatment group) set for the periph- the present study, it was revealed that the GI tract MN eral blood MN test, statistically significant increase in test could detect aneugens in the glandular stomach the MNed cell frequencies were observed not only in the and colon of rats when administered via clinical route Fig. 3 Micronucleus test in the glandular stomach, colon, and bone marrow with non-genotoxic non-carcinogens. The test was performed in rats administered NaCl or SUC for 4 days. Each bar represents the frequency of MNed cells or MNed immature erythrocytes (IMEs) (mean ± SD). Each closed circle represents the number of Ki-67 positive cells (per gland or crypt) or %IME (mean ± SD). The horizontal axis represents chemical and/or dosage (mg/kg/day). N, Negative (vehicle) control; P1, positive control (DMH and MNU treatment). Statistical significance: **p < 0.01 (Kastenbaum & ## †† Bowman test), p < 0.01 (Dunnett’s test), and p < 0.01 (Student’s t-test) compared to the negative control Okada et al. Genes and Environment (2022) 44:12 Page 8 of 11 Table 2 Summary of the results of 4-day repeated-dose micronucleus test Classes Chemicals Route Micronucleus test with a 4‑ day treatment In vitro genotoxicity test Carcinogenicity in regimen rodent (main target) [Ref.] Glandular Colon Bone marrow Ref. Ames [Ref.] Chromosomal stomach aberration [Ref.] Aneugens COL po + + – Present study – [31] + (poly) [31] No data a) VBS iv + + Tox (PB; +) Present study – [31] + (poly) [31, 32] No data a) DOC iv + + Tox (PB; +) Present study – [33] + (poly) [33] No data Clastogens MNU po + + + [15] + [34] + [34] + (Stomach) [35, 36] 4NQO po + – + [15] + [31] + [31] + (Stomach) [37] MNNG po + – – [15] + [34] + [34] + (Stomach) [35, 38] NMUT po + – – [15] + [39] + [39] + (Stomach) [18] DMH po – + + [15] + [34] + [34] + (Colon) [35, 40] PhIP po – + + [15] + [31] + [31] + (Colon) [35, 41] KBrO po + Eq + [15, 30] + [42] + [42] + (Kidney) [35, 42] MMC iv + + + Present study + [31, 34] + [31, 34] + (Peritoneum) [35] Genotoxic AM po – – – [15] – [43] + [39] – [44, 45] non-carcinogens QN po – – – [15] + [43] + [43] – [46–48] Non-genotoxic NaCl po – – – Present study – [34] – [34], + [49] – [34, 35] non-carcinogens SUC po – – – Present study – [50] – [51], + [49] – [35] +, Positive; −, negative; Eq Equivocal, Tox Toxic, PB Peripheral blood, Ref. References MN induction in erythrocytes was analyzed using peripheral blood on the day after the second administration COL Colchicine, VBS Vinblastine sulfate, DOC Docetaxel hydrate, MNU N-nitroso-N-methylurea, 4NQO 4-nitroquinoline-1-oxide, MNNG N-methyl-N′-nitro-N- nitrosoguanidine, NMUT N-methyl-N-nitrosourethane, DMH 1,2-dimethylhydrazine dihydrochloride, PhIP 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride, KBrO Potassium bromate, MMC Mitomycin C, AM Amaranth, QN Quercetin dihydrate, NaCl Sodium chloride, SUC Sucrose (intravenous and oral administration). To date, there are present study, and dose-dependent MN induction (posi- few reports of MN induction by aneugens in GI tract tive response) was confirmed for VBS and DOC. For cells, particularly in the stomach epithelium. Therefore, COL, although the frequency of MNed cells increased the present results provide important evidence showing in both the 4 and 8 mg/kg groups, a statistically signifi - good sensitivity of the GI tract MN test to aneugens. cant increase was observed only in the 4 mg/kg group; In erythrocytes, on the other hand, MN induction this increase in frequency for both groups was not dose- could not be detected even when COL was administered dependent. The MN test using the liver, the primary tis - up to the maximum tolerated dose. Cammerer et  al. sue involved in metabolism, as well as the GI tract MN [52] reported a delayed increase in MNed erythrocytes test are considered important for in  vivo genotoxicity (peak at 96 h after the final dosing) in rats orally admin - evaluation. Therefore, various methods such as the par - istered 6 and 8 mg/kg COL for five consecutive days. In tial hepatectomy (PH) method, the juvenile/young rat addition, in the present study, since severe cytotoxicity method, and the RD method have been developed [22, was observed in the bone marrow of rats administered 53, 54]. Sensitivity verification using clastogens has been VBS or DOC for 4 days at all dosages, MNed erythro- carried out using these methods but that using aneugens cyte induction could not be assessed in the bone marrow has been mainly performed using the PH method. Cur- (data not shown). Instead, MN induction was detected in rently, the MMS group is evaluating the sensitivity of the the peripheral blood on the day after the second adminis- liver MN test with the 28-day RD method using aneu- tration. Therefore, the 4-day treatment regimen allows to gens (Shigano M et  al., manuscript in preparation). The collect peripheral blood during the dosing period or col- RD method has the potential to detect MN induction lect bone marrow cells after the recovery period to evalu- in cells in several tissues including the liver, bone mar- ate MNed erythrocyte induction by compounds such as row, and GI tract. However, the cell division cycles dif- aneugens that cause severe bone marrow toxicity and/or fer substantially among these tissues, and in the case of delay of MN induction. aneugens that induce mitotic inhibition and severe cyto- The MNed hepatocyte frequencies in rats treated toxicity, it is important to determine the optimal dos- with aneugens were preliminarily monitored in the age, dosing period, and sampling time for MN analyses Ok ada et al. Genes and Environment (2022) 44:12 Page 9 of 11 in the respective tissues. In the present study, a signifi - of genotoxicity (clastogenicity and aneugenicity) of test cant increase in MNed cell frequency was observed in chemicals. the liver in addition to the GI tract and peripheral blood in rats administered aneugens with a 4-day treatment Conclusions regimen. These results suggest that it may be possible to To further clarify the sensitivity and specificity of the rat detect the aneugenic potential of chemicals in the liver GI tract MN test, we conducted additional studies using using this 4-day treatment regimen. However, further three aneugens and two non-genotoxic non-carcinogens research is required to elucidate the performance of this with a 4-day treatment regimen. The study demonstrated short-term administration method. that the glandular stomach and colon MN tests could To investigate the specificity of the GI tract MN test detect aneugens as positive and could adequately evalu- with a 4-day treatment regimen, NaCl and SUC were ate non-genotoxic non-carcinogens as negative, includ- selected as non-genotoxic non-carcinogens. They did not ing a chemical that enhances cell proliferation. These induce MNed cells in the GI tract and bone marrow of results provide important evidence supporting the good rats when administered up to 2 g/kg of NaCl (2/3 of the performance of the rat glandular stomach and colon MN LD value) and 10 g/kg of SUC (1/3 of the LD value) 50 50 tests with a 4-day treatment regimen. [29]. No treatment-related changes were observed in the clinical signs or body weight gain. NaCl is a stomach Abbreviations toxicant that promotes the proliferation of the glandular MN: Micronucleus; GI: Gastrointestinal; RD: Repeated-dose; COL: Colchicine; VBS: stomach epithelium [55] and exerts tumor-promoting Vinblastine sulfate; DOC: Docetaxel hydrate; SUC: Sucrose; DMH: 1,2-dimethylhy- drazine dihydrochloride; MNU: N-nitroso-N-methylurea; MMC: Mitomycin C. activity [56] when orally administered to rats. SUC is known to enhance the proliferation of the colonic epithe- Acknowledgments lium of rats administered orally [57]. As an index of cell We would like to thank Dr. S. Hamada (Bozo Research Center Inc., Tokyo, Japan), the principal investigator of the MMS/JEMS collaborative study (the proliferation in the GI tract, we analyzed proliferating repeated-dose liver micronucleus study), and the MMS/JEMS members for cells in tissue sections using immunohistochemistry of advice on the study experiments. We are grateful to Drs. T. Kobayashi and S. Ki-67, a nuclear protein expressed in the cell cycle phase Kado for their encouragement throughout the study. We would like to thank Editage (www. edita ge. com) for English language editing. other than the G0 phase [58]. In the present study, the Ki- 67-positive cell counts significantly increased only in the Authors’ contributions gastric epithelium of the rats treated with NaCl, indicat- EO, YF, KN, and WO conducted micronucleus assays and performed a comprehensive evaluation. EO created the tables and figures and wrote the ing that cell proliferation was enhanced, but not in the manuscript. All authors read and approved the final manuscript. cells of all three tissues evaluated including the colon in the rats treated with SUC. It has been reported that an Funding Not applicable. increase in erythropoiesis that is not due to chemical- induced genotoxic damage causes an increase in the Availability of data and materials number of spontaneous MNed erythrocytes, resulting in All data generated or analyzed during this study are included in this published article. false-positive results in the routine erythrocyte MN test [59]. Under the conditions of the present experiment, Declarations however, it was demonstrated that both NaCl and SUC did not increase MNed cell frequencies in the glandular Ethics approval and consent to participate stomach and colon, regardless of whether cell prolifera- Animal experiments were approved by the Institutional Animal Care and Use Committee of Yakult Central Institute before the experiments were conducted. tion was enhanced. These results show that non-geno - toxic non-carcinogens including chemicals that enhance Consent for publication cell proliferation could be correctly evaluated as negative Not applicable. in the glandular stomach and colon MN tests. Competing interests In our previous study, the rat glandular stomach and The authors declare that they have no competing interests. colon MN tests with a 4-day treatment regimen were Received: 23 December 2021 Accepted: 27 March 2022 performed using seven clastogens and two genotoxic- non-carcinogens, and their sensitivity and specific - ity were confirmed (Table  2). Furthermore, the present study revealed that aneugens can be detected as positive, References and non-genotoxic non-carcinogens can be evaluated 1. ICH. ICH harmonized tripartite guideline S2(R1): guidance on genotoxic- as negative. 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Tweats DJ, Blakey D, Heflich RH, Jacobs A, Jacobsen SD, Morita T, et al. rapid publication on acceptance Report of the IWGT working group on strategies and interpretation support for research data, including large and complex data types of regulatory in vivo tests I. increases in micronucleated bone mar- • gold Open Access which fosters wider collaboration and increased citations row cells in rodents that do not indicate genotoxic hazards. Mutat Res. 2007;627:78–91. https:// doi. org/ 10. 1016/j. mrgen tox. 2006. 10. 005. maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- Learn more biomedcentral.com/submissions lished maps and institutional affiliations.

Journal

Genes and EnvironmentSpringer Journals

Published: Apr 11, 2022

Keywords: Micronucleus test; Gastrointestinal tract; Glandular stomach; Colon; 4-day treatment regimen; Aneugen

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