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Micronucleus test in rodent tissues other than liver or erythrocytes: Report of the IWGT working group Yoshifumi Uno a,∗ , Takeshi Morita b , Mirjam Luijten c , Carol Beevers d , Shuichi Hamada e , Satoru Itoh f , Wakako Ohyama g , Hironao Takasawa e a
Mitsubishi Tanabe Pharma Co., Chiba, Japan National Institute of Health Sciences, Tokyo, Japan c National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands d Covance Laboratories Ltd., Harrogate, UK e LSI Medience (Formerly Mitsubishi Chemical Medience Co.), Ibaraki, Japan f Daiichi Sankyo Co., Ltd., Tokyo, Japan g Yakult Honsha Co., Ltd., Tokyo, Japan b
a r t i c l e
i n f o
Article history: Received 2 March 2015 Accepted 3 March 2015 Available online xxx Keywords: Micronucleus test Stomach Colon Gastrointestinal (GI) tract IWGT
a b s t r a c t At the 6th International Workshop on Genotoxicity Testing, the liver micronucleus test (MNT) working group briefly discussed the MNT using tissues other than liver/erythrocytes. Many tissues other than liver/erythrocytes have been studied, primarily for research purposes. They have included the colon and intestinal epithelium, skin, spleen, lung, stomach, bladder, buccal mucosa, vagina, and fetal/neonatal tissues. These tissues were chosen because they were target sites of carcinogens, and/or relevant to a specific route of exposure. Recently, there has been particular focus on the gastrointestinal (GI) tract as it is a contact site associated with high exposure following oral gavage. Furthermore GI tumors are observed with high frequency in human populations. A collaborative study of the rat glandular stomach and colon MNT was conducted in conjunction with a collaborative study of the repeated-dose liver MNT. Based on limited data currently available, the rodent MNT using the glandular stomach and/or colon seems to detect genotoxic carcinogens with GI tract target-organ specificity. The working group concluded that the GI tract MNT would be a promising method to examine clastogenicity or aneugenicity of test chemicals in the stomach and/or colon. Further data will be needed to fully establish the methods, and to identify the sensitivity and specificity of the GI tract MNT. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The micronucleus test (MNT) is most often performed using rodent hematopoietic cells (i.e., erythrocytes) in bone marrow or peripheral blood for the assessment of clastogenicity/aneugenicity of chemicals. The advantages of the use of hematopoietic cells are the ease with which newly-formed erythrocytes can be identified and micronuclei (MN) can be scored. However, in principle, the MNT is applicable to any dividing cells in any tissues or organs. Therefore, the MNT has been applied to many tissues other than erythrocytes. The application to other tissues was discussed by the in vivo MN assay working group at the 2nd International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC
∗ Corresponding author. Tel.: +81 438 52 3535; fax: +81 438 52 3556. E-mail address: [email protected] (Y. Uno).
in March 25–26, 1999 [1], and at the 4th International Workshop on Genotoxicity Testing (IWGT) held in San Francisco in September 8–9, 2005 [2]. At the 2nd IWGTP, MNTs using liver, colon, skin, spleen, lung, spermatids, and fetal/neonatal animals were reviewed and discussed. The working group recognized that these tests were useful research tools for mechanistic investigations in certain regulatory situations, but that they had not yet been standardized and validated for routine regulatory use [1]. At the 4th IWGT, the working group discussed the relevance of the liver MNT using young rats, and the group concluded that the young rat liver MNT is a good alternative method to the use of partial hepatectomy or mitogenic stimulation of the liver for detecting MN induction, but more information on the metabolic capabilities of young rats would be needed [2]. Additional studies with colon and skin MNT models have also been reported, enhancing confidence in the utility of these models [2]. After the 4th IWGT, many new studies on the liver MNT have been published, including a new methodology, using adult rats with
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repeated-dosing of chemicals [3]. Therefore, further discussion of the liver MNT was accepted as one of the topics at the 6th IWGT held in Foz do Iguac¸u, Brazil, on October 31, 2013. The working group concluded that the liver MNT is a useful and promising assay for regulatory use; however, further actions for development will be needed [3]. In addition to this topic, the rodent liver MNT working group also briefly discussed the MNT using tissues other than liver and erythrocytes in order to identify promising test systems. The gastrointestinal (GI) tract was a particular focus as it is a contact site associated with high exposure following oral gavage. Furthermore GI tumors are observed with high frequency in human populations. Many reports have recently been published for the rodent GI tract MNT, and collaborative studies of the repeated-dose GI tract MNT were carried out in conjunction with the repeated-dose liver MNT [4]. Thus, the discussions regarding the MNT in rodent GI tract are summarized in this paper. 2. Micronucleus tests in rodent tissues other than erythrocytes or liver An update of the MNT in rodent tissues other than erythrocytes, was presented to the working group at the 6th IWGT; this has been recently reviewed by Morita et al. [5]. The review revealed that since the discussions at the 4th IWGT, many tissues other than erythrocytes have been studied for MN induction. These tissues included the liver, colon and intestinal epithelium, skin, spleen, lung, stomach, bladder, buccal mucosa, vagina, and fetal/neonatal tissues. Many studies were primarily for research purposes because these tissues were target sites of carcinogens, or because of their relevance to specific routes of exposure. The main conclusions of the review paper [5] were as follows. • Colon and intestinal epithelium are important tissues for metabolism by the intestinal microflora. • Skin is a major site of direct exposure to many chemicals including pharmaceuticals or cosmetics. • In the spleen, an in vivo/in vitro method is mainly used. • The lung is an important target site for detection of local and systemic genotoxic effects by inhalation; however, intratracheal instillation is often used in the experiments. • The stomach is the first site of direct contact to many chemicals in animal studies using oral gavage administration. • The bladder is an exposure site of water-soluble chemicals excreted via the urine, and the bladder MNT using exfoliated
cells would be a good biomonitoring tool for humans exposed to carcinogens/genotoxicants. • The buccal mucosa is mainly used in human studies for biomonitoring of DNA damage potentially leading to oral cancer. • The vagina MNT might be a useful tool for screening new chemopreventive agents. • Foetal/neonatal tissues will be useful for evaluation of agents associated with reproductive and developmental toxicity.
The review concluded that the MNT using tissues other than erythrocytes are critical for risk assessment, for in situ evaluation and for studies of systemic genotoxic effects and modes of action. Protocols for the majority of these MNTs have not yet been standardized and validated for regulatory application [5]. The working group recognized the current status of the MNT using the above tissues. Although the oral cavity is important as a first contact site for chemicals in the diet or drinking water, the GI tract is the first contact site of high exposure following oral gavage dosing. The colon is also an important target site because some ultimate carcinogens are formed or metabolized to reactive forms by intestinal microflora [5]. Therefore, more recent updates on MNT using the GI tract (i.e., stomach, colon and intestinal epithelium) were investigated. A literature search was conducted using PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez); search terms included ‘micronucleus’, ‘rat’, ‘mouse’ and specific tissue name (i.e., ‘stomach’, ‘colon’, or ‘intestinal epithelium’), and English language. The search was limited to the period of time from January 2011 to September 2013. Three papers were identified [6–8], and the published results are summarized in Table 1. Although most of the earlier published data were obtained from mice [5], our search identified data for the effects of approximately ten chemicals in the GI tract MNT with rats treated by oral gavage. This increase in rat studies may reflect the ICH S2(R1) guideline [9] requirements to examine genotoxicity in a second relevant in vivo test when there are positive in vitro findings. Almost all the chemicals in Table 1 were potent carcinogens or aneugens, and were, therefore, suitable as model chemicals to establish methodology for the GI tract MNT. The data on these chemicals are important as an initial approach to establish the GI tract MNT. However, further investigations on suitable treatment regimens and more data with different chemical classes or modes of action will be needed to standardize the method.
Table 1 MN assays in GI tract in rodents (published between 2011 and September 2013). [Specific tissue/organ]
Species
Administration
Sampling time after the final treatment
Chemical
Tested
Route
Times/duration
[Colon and intestinal epithelium] Acetyl salicylic acid Carbendazim Cyclophosphamide Cylindrospermopsin 1,2-Dimethylhydrazine 1,2-Dimethylhydrazine Mitomycin C Mitomycin C Vinblastine sulfate Vinblastine sulfate
Rat Rat Rat Mouse Rat Rat Rat Rat Rat Rat
Po Po Po Po Po Po Po Ip Po Ip
Twice (daily) Twice (daily) Twice (daily) Once Twice (daily) Once Twice (daily) Twice (daily) Twice (daily) Twice (daily)
[Stomach] N-Methyl-N -nitro-N-nitrosoguanidine N-Nitroso-N-methylurea
Rat Rat
Po Po
Once Once
MN induction in the specific organ
Result
Ref.
24 h 24 h 24 h 24 h 24 h 72–120 h 24 h 24 h 24 h 24 h
−/−a −/+a +/−a −/nta +/+a +/nta −/−a +/+a −/−a +/−a
[6] [6] [6] [7] [6] [8] [6] [6] [6] [6]
24–120 h 24–120 h
+ +
[8] [8]
Abbreviations: po: per os (by gavage), ip: intraperitoneal injection, nt: not tested. a Colon/duodenum.
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Table 2 Cell preparation methods for rodent GI tract micronucleus test. Organ
Cellpreparation
Cell-sampling time after final dosing
Animal species
Ref.
Colon
Histopathology
24 h 1–3 days 1–2 days 24, 36 and 48 h 24 h 24 h 3 and 4 days 2, 3 and 4 days 1–5 days 24 h 30 h 24 h 24 h
Mouse Mouse Mouse Mouse Mouse Mouse, rat Mouse Mouse Rat Mouse Rat Rat Mouse, rat
[7] [10] [14] [17] [19] [11] [12] [15] [8] [13,20] [16] [6] [11]
3 and 4 days 1–5 days
Mouse Rat
[18] [8]
Mince EDTA
Scrape after fixed Trypsin and EDTA Trypsin Glandular Mince stomach EDTA
3. Current status of in vivo GI tract micronucleus test As the colon and stomach are high frequency sites for human carcinogenesis, it is important to assess chemical genotoxicity within these tissues. Many reports have been published for the rodent GI tract MNT, especially for the colon [6–8,10–21]. Fewer MNT tests have been performed with the glandular stomach of rats or mice [8,11,18,21]. Based on the published reports, the rodent GI tract MNT seems to demonstrate appropriate target-organ specificity for genotoxic carcinogens to the stomach and/or colon. The test methods for the GI tract MNT, in particular the cell preparation methods and the cell sampling times (Table 2), have not yet been standardized. Recently, Ohyama et al. [8] reported on a simple cell preparation method, which has now been used in a collaborative study of the rat GI tract MNT, organized by the Japanese Environmental Mutagen Society/Mammalian Mutagenicity Study Group (JEMS/MMS). In brief, the glandular stomach and colon are each everted on a glass rod and incubated in Mg2+ /Ca2+ -free Hanks’ balanced salt solution including 1 mM EDTA. The isolated epithelium is dispersed into single cells by pipetting, and cells are collected by centrifugation, fixed with 10% neutralbuffered formalin and then stained with acridine orange (AO) and 4 6-diamidino-2-phenylindole dihydrochloride (DAPI) for microscopic observation [8]. The working group felt that one advantage of the new, simple cell preparation method, when compared to the classical section method, was that intact cells are analyzed; thus, the method could be applied to the stomach and colon without interfering with histopathological examination for toxicology end-points. This suggests that the GI tract MNT could be integrated into general toxicity studies. It is reported that the peak response of MN induction in the GI tract would be 48–96 h
3
after single dosing of N-nitroso-N-methylurea (MNU), N-methylN -nitro-N-nitrosoguanidine (MNNG) and 1,2-dimethylhydrazine dihydrochloride (DMH) [8]. The authors suggested that this cell sampling time could be applied to any test chemicals after a single treatment. The working group members considered this suggestion rational because the turnover of epithelial cells is 3–4 days in the glandular stomach (mainly upper side of the gland) and colon. More recently (i.e., after September 2013 of the end of the search period in Table 1), Okada et al. [21] proposed a 4-day dosing regimen to perform the GI tract MNT combined with the BM MNT, based on the time-responses of MN induction and the cellturnover time of the GI tract epithelium and BM erythrocytes. They reported that four daily oral gavage administrations of nine test chemicals to rats showed positive or negative results in the GI tract MNT, which correlated well with their carcinogenicity to the stomach and/or colon. For example, MNNG and N-methyl-Nnitrosourethane (NMUT) were carcinogenic in the rat stomach, and the substances showed MN induction in the stomach but not in the colon and bone marrow. The update of a collaborative study of the GI tract MNT organized by JEMS/MMS was reported at the 6th IWGT meeting. The collaborative trial was conducted for the technical transfer and familiarization in 6 laboratories, and inter-laboratory reproducibility was confirmed with two positive controls, MNU and DMH, and a solvent control under the 4-day oral dosing regimen [4]. Briefly, MN cell frequencies were statistically significantly increased in both the glandular stomach and colon of rats treated by oral gavage with DMH (90 mg/kg) on day 1 and MNU (20 mg/kg/day) on days 3–4, in comparison to those of rats given distilled water on days 1–4. The ranges of the mean frequency of micronucleated cells in negative controls for the glandular stomach or colon were 0.05–0.17% or 0.07–0.17%, respectively, among the 6 laboratories. For the positive controls, the ranges of MN cell frequencies 0.58–1.20% or 0.80–2.02% for stomach and colon, respectively (Table 3) [4]. The data obtained for negative and positive control responses were encouragingly consistent across the six participant laboratories. The subsequent JEMS/MMS collaborative study used a 14- and/or 28-day repeated oral dosing regimen, and MNT assessments in the glandular stomach, colon, liver and bone marrow were conducted for nine chemicals: MNNG, NMUT (stomach carcinogens), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP; colon carcinogen), MNU (GI tract carcinogen), KBrO3 (renal carcinogen), 2-acethylaminofluorene (2AAF), dimethylnitrosamine, quinoline, and Sudan I (liver carcinogens) [4]. MNNG, NMUT and MNU gave positive MN responses in the glandular stomach, and PhIP was positive in the colon. MNNG and NMUT gave negative or equivocal results in the other tissues examined. Additionally, KBrO3 and 2AAF, which are clastogenic in in vitro chromosomal aberration tests without metabolic activation [22,23], also gave positive results in the glandular stomach. Current
Table 3 Results of a collaborative trial of the rat GI tract MNT using negative and positive control substances.a Organ
Micronucleated cell frequencies (%) (mean ± SD)
Treatment groupb
Laboratories Lab. 1
Lab. 2
Lab. 3
Lab. 4
Lab. 5
Lab. 6
Glandular stomach
Negative control Positive control
0.11 ± 0.04 1.09 ± 0.13*
0.17 ± 0.12 0.58 ± 0.12*
0.05 ± 0.04 1.09 ± 0.35*
0.07 ± 0.08 0.99 ± 0.14*
0.09 ± 0.10 1.20 ± 0.23*
0.05 ± 0.04 1.13 ± 0.28*
Colon
Negative control Positive control
0.16 ± 0.09 1.42 ± 0.49*
0.14 ± 0.11 0.82 ± 0.21*
0.17 ± 0.06 0.98 ± 0.48*
0.07 ± 0.08 2.02 ± 0.62*
0.07 ± 0.04 0.80 ± 0.18*
0.07 ± 0.08 1.30 ± 0.25*
a
Data from Fig. 2 in Hamada et al. [4] with modifications. Eight-week-old male CD (SD) rats were used for the negative (vehicle) and positive control group (5 rats/group). Negative control animals were administered distilled water by oral gavage once daily for 4 days. Positive control animals were given DMH (90 mg/kg) once on the first day (day 1) and MNU (20 mg/kg/day) twice on day 3 and day 4 via oral gavage. They were euthanized at 24 h after the last dosing, and the glandular stomach and colon were sampled for the MN analysis. * P < 0.01; significant difference from the negative control (Kastenbaum and Bowman test). b
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data indicate that the rat stomach and colon MNT could detect GI tract-targeted carcinogens, and the stomach MNT could also detect substances that give positive results in in vitro chromosomal aberration tests without metabolic activation. In practice, both of the GI tract tissues could be prepared for MN analysis at the same time as tissues are prepared for histopathological examination, suggesting that the GI tract MNT could be integrated into general toxicity studies. Finally some further issues relating to the GI tract MNT were raised. At the present moment, there are insufficient data on expected negative chemicals and, therefore, the specificity of the method is not known. In addition, a method to determine cell proliferation in GI tract is not established. Ki-67 immunohistochemistry might be useful to examine cell proliferation [21], but more data will be needed. The working group had insufficient time to fully discuss other issues, but further investigation is clearly required to establish and standardize the GI tract MNT. 4. Conclusion The working group concluded that the GI tract MNT would be a promising method to examine the clastogenicity/aneugenicity of test chemicals in the stomach and/or colon. It will be useful for hazard identification by tissue-specific clastogenic effects by chemicals and their risk evaluation. Further data will be needed to more accurately assess the sensitivity and specificity of the GI tract MNT. Conflict of interest C. Beevers is employed by a contract research laboratory that offers this assay as a service offering. Funding
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[8]
[9]
[10]
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[13]
[14]
[15]
[16]
None. [17]
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Micronucleus test in rodent tissues other than liver or erythrocytes: Report of the IWGT working group Yoshifumi Uno a,∗ , Takeshi Morita b , Mirjam Luijten c , Carol Beevers d , Shuichi Hamada e , Satoru Itoh f , Wakako Ohyama g , Hironao Takasawa e a
Mitsubishi Tanabe Pharma Co., Chiba, Japan National Institute of Health Sciences, Tokyo, Japan c National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands d Covance Laboratories Ltd., Harrogate, UK e LSI Medience (Formerly Mitsubishi Chemical Medience Co.), Ibaraki, Japan f Daiichi Sankyo Co., Ltd., Tokyo, Japan g Yakult Honsha Co., Ltd., Tokyo, Japan b
a r t i c l e
i n f o
Article history: Received 2 March 2015 Accepted 3 March 2015 Available online xxx Keywords: Micronucleus test Stomach Colon Gastrointestinal (GI) tract IWGT
a b s t r a c t At the 6th International Workshop on Genotoxicity Testing, the liver micronucleus test (MNT) working group briefly discussed the MNT using tissues other than liver/erythrocytes. Many tissues other than liver/erythrocytes have been studied, primarily for research purposes. They have included the colon and intestinal epithelium, skin, spleen, lung, stomach, bladder, buccal mucosa, vagina, and fetal/neonatal tissues. These tissues were chosen because they were target sites of carcinogens, and/or relevant to a specific route of exposure. Recently, there has been particular focus on the gastrointestinal (GI) tract as it is a contact site associated with high exposure following oral gavage. Furthermore GI tumors are observed with high frequency in human populations. A collaborative study of the rat glandular stomach and colon MNT was conducted in conjunction with a collaborative study of the repeated-dose liver MNT. Based on limited data currently available, the rodent MNT using the glandular stomach and/or colon seems to detect genotoxic carcinogens with GI tract target-organ specificity. The working group concluded that the GI tract MNT would be a promising method to examine clastogenicity or aneugenicity of test chemicals in the stomach and/or colon. Further data will be needed to fully establish the methods, and to identify the sensitivity and specificity of the GI tract MNT. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The micronucleus test (MNT) is most often performed using rodent hematopoietic cells (i.e., erythrocytes) in bone marrow or peripheral blood for the assessment of clastogenicity/aneugenicity of chemicals. The advantages of the use of hematopoietic cells are the ease with which newly-formed erythrocytes can be identified and micronuclei (MN) can be scored. However, in principle, the MNT is applicable to any dividing cells in any tissues or organs. Therefore, the MNT has been applied to many tissues other than erythrocytes. The application to other tissues was discussed by the in vivo MN assay working group at the 2nd International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC
∗ Corresponding author. Tel.: +81 438 52 3535; fax: +81 438 52 3556. E-mail address: [email protected] (Y. Uno).
in March 25–26, 1999 [1], and at the 4th International Workshop on Genotoxicity Testing (IWGT) held in San Francisco in September 8–9, 2005 [2]. At the 2nd IWGTP, MNTs using liver, colon, skin, spleen, lung, spermatids, and fetal/neonatal animals were reviewed and discussed. The working group recognized that these tests were useful research tools for mechanistic investigations in certain regulatory situations, but that they had not yet been standardized and validated for routine regulatory use [1]. At the 4th IWGT, the working group discussed the relevance of the liver MNT using young rats, and the group concluded that the young rat liver MNT is a good alternative method to the use of partial hepatectomy or mitogenic stimulation of the liver for detecting MN induction, but more information on the metabolic capabilities of young rats would be needed [2]. Additional studies with colon and skin MNT models have also been reported, enhancing confidence in the utility of these models [2]. After the 4th IWGT, many new studies on the liver MNT have been published, including a new methodology, using adult rats with
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repeated-dosing of chemicals [3]. Therefore, further discussion of the liver MNT was accepted as one of the topics at the 6th IWGT held in Foz do Iguac¸u, Brazil, on October 31, 2013. The working group concluded that the liver MNT is a useful and promising assay for regulatory use; however, further actions for development will be needed [3]. In addition to this topic, the rodent liver MNT working group also briefly discussed the MNT using tissues other than liver and erythrocytes in order to identify promising test systems. The gastrointestinal (GI) tract was a particular focus as it is a contact site associated with high exposure following oral gavage. Furthermore GI tumors are observed with high frequency in human populations. Many reports have recently been published for the rodent GI tract MNT, and collaborative studies of the repeated-dose GI tract MNT were carried out in conjunction with the repeated-dose liver MNT [4]. Thus, the discussions regarding the MNT in rodent GI tract are summarized in this paper. 2. Micronucleus tests in rodent tissues other than erythrocytes or liver An update of the MNT in rodent tissues other than erythrocytes, was presented to the working group at the 6th IWGT; this has been recently reviewed by Morita et al. [5]. The review revealed that since the discussions at the 4th IWGT, many tissues other than erythrocytes have been studied for MN induction. These tissues included the liver, colon and intestinal epithelium, skin, spleen, lung, stomach, bladder, buccal mucosa, vagina, and fetal/neonatal tissues. Many studies were primarily for research purposes because these tissues were target sites of carcinogens, or because of their relevance to specific routes of exposure. The main conclusions of the review paper [5] were as follows. • Colon and intestinal epithelium are important tissues for metabolism by the intestinal microflora. • Skin is a major site of direct exposure to many chemicals including pharmaceuticals or cosmetics. • In the spleen, an in vivo/in vitro method is mainly used. • The lung is an important target site for detection of local and systemic genotoxic effects by inhalation; however, intratracheal instillation is often used in the experiments. • The stomach is the first site of direct contact to many chemicals in animal studies using oral gavage administration. • The bladder is an exposure site of water-soluble chemicals excreted via the urine, and the bladder MNT using exfoliated
cells would be a good biomonitoring tool for humans exposed to carcinogens/genotoxicants. • The buccal mucosa is mainly used in human studies for biomonitoring of DNA damage potentially leading to oral cancer. • The vagina MNT might be a useful tool for screening new chemopreventive agents. • Foetal/neonatal tissues will be useful for evaluation of agents associated with reproductive and developmental toxicity.
The review concluded that the MNT using tissues other than erythrocytes are critical for risk assessment, for in situ evaluation and for studies of systemic genotoxic effects and modes of action. Protocols for the majority of these MNTs have not yet been standardized and validated for regulatory application [5]. The working group recognized the current status of the MNT using the above tissues. Although the oral cavity is important as a first contact site for chemicals in the diet or drinking water, the GI tract is the first contact site of high exposure following oral gavage dosing. The colon is also an important target site because some ultimate carcinogens are formed or metabolized to reactive forms by intestinal microflora [5]. Therefore, more recent updates on MNT using the GI tract (i.e., stomach, colon and intestinal epithelium) were investigated. A literature search was conducted using PubMed (http://www.ncbi.nlm.nih.gov/sites/entrez); search terms included ‘micronucleus’, ‘rat’, ‘mouse’ and specific tissue name (i.e., ‘stomach’, ‘colon’, or ‘intestinal epithelium’), and English language. The search was limited to the period of time from January 2011 to September 2013. Three papers were identified [6–8], and the published results are summarized in Table 1. Although most of the earlier published data were obtained from mice [5], our search identified data for the effects of approximately ten chemicals in the GI tract MNT with rats treated by oral gavage. This increase in rat studies may reflect the ICH S2(R1) guideline [9] requirements to examine genotoxicity in a second relevant in vivo test when there are positive in vitro findings. Almost all the chemicals in Table 1 were potent carcinogens or aneugens, and were, therefore, suitable as model chemicals to establish methodology for the GI tract MNT. The data on these chemicals are important as an initial approach to establish the GI tract MNT. However, further investigations on suitable treatment regimens and more data with different chemical classes or modes of action will be needed to standardize the method.
Table 1 MN assays in GI tract in rodents (published between 2011 and September 2013). [Specific tissue/organ]
Species
Administration
Sampling time after the final treatment
Chemical
Tested
Route
Times/duration
[Colon and intestinal epithelium] Acetyl salicylic acid Carbendazim Cyclophosphamide Cylindrospermopsin 1,2-Dimethylhydrazine 1,2-Dimethylhydrazine Mitomycin C Mitomycin C Vinblastine sulfate Vinblastine sulfate
Rat Rat Rat Mouse Rat Rat Rat Rat Rat Rat
Po Po Po Po Po Po Po Ip Po Ip
Twice (daily) Twice (daily) Twice (daily) Once Twice (daily) Once Twice (daily) Twice (daily) Twice (daily) Twice (daily)
[Stomach] N-Methyl-N -nitro-N-nitrosoguanidine N-Nitroso-N-methylurea
Rat Rat
Po Po
Once Once
MN induction in the specific organ
Result
Ref.
24 h 24 h 24 h 24 h 24 h 72–120 h 24 h 24 h 24 h 24 h
−/−a −/+a +/−a −/nta +/+a +/nta −/−a +/+a −/−a +/−a
[6] [6] [6] [7] [6] [8] [6] [6] [6] [6]
24–120 h 24–120 h
+ +
[8] [8]
Abbreviations: po: per os (by gavage), ip: intraperitoneal injection, nt: not tested. a Colon/duodenum.
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Table 2 Cell preparation methods for rodent GI tract micronucleus test. Organ
Cellpreparation
Cell-sampling time after final dosing
Animal species
Ref.
Colon
Histopathology
24 h 1–3 days 1–2 days 24, 36 and 48 h 24 h 24 h 3 and 4 days 2, 3 and 4 days 1–5 days 24 h 30 h 24 h 24 h
Mouse Mouse Mouse Mouse Mouse Mouse, rat Mouse Mouse Rat Mouse Rat Rat Mouse, rat
[7] [10] [14] [17] [19] [11] [12] [15] [8] [13,20] [16] [6] [11]
3 and 4 days 1–5 days
Mouse Rat
[18] [8]
Mince EDTA
Scrape after fixed Trypsin and EDTA Trypsin Glandular Mince stomach EDTA
3. Current status of in vivo GI tract micronucleus test As the colon and stomach are high frequency sites for human carcinogenesis, it is important to assess chemical genotoxicity within these tissues. Many reports have been published for the rodent GI tract MNT, especially for the colon [6–8,10–21]. Fewer MNT tests have been performed with the glandular stomach of rats or mice [8,11,18,21]. Based on the published reports, the rodent GI tract MNT seems to demonstrate appropriate target-organ specificity for genotoxic carcinogens to the stomach and/or colon. The test methods for the GI tract MNT, in particular the cell preparation methods and the cell sampling times (Table 2), have not yet been standardized. Recently, Ohyama et al. [8] reported on a simple cell preparation method, which has now been used in a collaborative study of the rat GI tract MNT, organized by the Japanese Environmental Mutagen Society/Mammalian Mutagenicity Study Group (JEMS/MMS). In brief, the glandular stomach and colon are each everted on a glass rod and incubated in Mg2+ /Ca2+ -free Hanks’ balanced salt solution including 1 mM EDTA. The isolated epithelium is dispersed into single cells by pipetting, and cells are collected by centrifugation, fixed with 10% neutralbuffered formalin and then stained with acridine orange (AO) and 4 6-diamidino-2-phenylindole dihydrochloride (DAPI) for microscopic observation [8]. The working group felt that one advantage of the new, simple cell preparation method, when compared to the classical section method, was that intact cells are analyzed; thus, the method could be applied to the stomach and colon without interfering with histopathological examination for toxicology end-points. This suggests that the GI tract MNT could be integrated into general toxicity studies. It is reported that the peak response of MN induction in the GI tract would be 48–96 h
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after single dosing of N-nitroso-N-methylurea (MNU), N-methylN -nitro-N-nitrosoguanidine (MNNG) and 1,2-dimethylhydrazine dihydrochloride (DMH) [8]. The authors suggested that this cell sampling time could be applied to any test chemicals after a single treatment. The working group members considered this suggestion rational because the turnover of epithelial cells is 3–4 days in the glandular stomach (mainly upper side of the gland) and colon. More recently (i.e., after September 2013 of the end of the search period in Table 1), Okada et al. [21] proposed a 4-day dosing regimen to perform the GI tract MNT combined with the BM MNT, based on the time-responses of MN induction and the cellturnover time of the GI tract epithelium and BM erythrocytes. They reported that four daily oral gavage administrations of nine test chemicals to rats showed positive or negative results in the GI tract MNT, which correlated well with their carcinogenicity to the stomach and/or colon. For example, MNNG and N-methyl-Nnitrosourethane (NMUT) were carcinogenic in the rat stomach, and the substances showed MN induction in the stomach but not in the colon and bone marrow. The update of a collaborative study of the GI tract MNT organized by JEMS/MMS was reported at the 6th IWGT meeting. The collaborative trial was conducted for the technical transfer and familiarization in 6 laboratories, and inter-laboratory reproducibility was confirmed with two positive controls, MNU and DMH, and a solvent control under the 4-day oral dosing regimen [4]. Briefly, MN cell frequencies were statistically significantly increased in both the glandular stomach and colon of rats treated by oral gavage with DMH (90 mg/kg) on day 1 and MNU (20 mg/kg/day) on days 3–4, in comparison to those of rats given distilled water on days 1–4. The ranges of the mean frequency of micronucleated cells in negative controls for the glandular stomach or colon were 0.05–0.17% or 0.07–0.17%, respectively, among the 6 laboratories. For the positive controls, the ranges of MN cell frequencies 0.58–1.20% or 0.80–2.02% for stomach and colon, respectively (Table 3) [4]. The data obtained for negative and positive control responses were encouragingly consistent across the six participant laboratories. The subsequent JEMS/MMS collaborative study used a 14- and/or 28-day repeated oral dosing regimen, and MNT assessments in the glandular stomach, colon, liver and bone marrow were conducted for nine chemicals: MNNG, NMUT (stomach carcinogens), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine hydrochloride (PhIP; colon carcinogen), MNU (GI tract carcinogen), KBrO3 (renal carcinogen), 2-acethylaminofluorene (2AAF), dimethylnitrosamine, quinoline, and Sudan I (liver carcinogens) [4]. MNNG, NMUT and MNU gave positive MN responses in the glandular stomach, and PhIP was positive in the colon. MNNG and NMUT gave negative or equivocal results in the other tissues examined. Additionally, KBrO3 and 2AAF, which are clastogenic in in vitro chromosomal aberration tests without metabolic activation [22,23], also gave positive results in the glandular stomach. Current
Table 3 Results of a collaborative trial of the rat GI tract MNT using negative and positive control substances.a Organ
Micronucleated cell frequencies (%) (mean ± SD)
Treatment groupb
Laboratories Lab. 1
Lab. 2
Lab. 3
Lab. 4
Lab. 5
Lab. 6
Glandular stomach
Negative control Positive control
0.11 ± 0.04 1.09 ± 0.13*
0.17 ± 0.12 0.58 ± 0.12*
0.05 ± 0.04 1.09 ± 0.35*
0.07 ± 0.08 0.99 ± 0.14*
0.09 ± 0.10 1.20 ± 0.23*
0.05 ± 0.04 1.13 ± 0.28*
Colon
Negative control Positive control
0.16 ± 0.09 1.42 ± 0.49*
0.14 ± 0.11 0.82 ± 0.21*
0.17 ± 0.06 0.98 ± 0.48*
0.07 ± 0.08 2.02 ± 0.62*
0.07 ± 0.04 0.80 ± 0.18*
0.07 ± 0.08 1.30 ± 0.25*
a
Data from Fig. 2 in Hamada et al. [4] with modifications. Eight-week-old male CD (SD) rats were used for the negative (vehicle) and positive control group (5 rats/group). Negative control animals were administered distilled water by oral gavage once daily for 4 days. Positive control animals were given DMH (90 mg/kg) once on the first day (day 1) and MNU (20 mg/kg/day) twice on day 3 and day 4 via oral gavage. They were euthanized at 24 h after the last dosing, and the glandular stomach and colon were sampled for the MN analysis. * P < 0.01; significant difference from the negative control (Kastenbaum and Bowman test). b
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data indicate that the rat stomach and colon MNT could detect GI tract-targeted carcinogens, and the stomach MNT could also detect substances that give positive results in in vitro chromosomal aberration tests without metabolic activation. In practice, both of the GI tract tissues could be prepared for MN analysis at the same time as tissues are prepared for histopathological examination, suggesting that the GI tract MNT could be integrated into general toxicity studies. Finally some further issues relating to the GI tract MNT were raised. At the present moment, there are insufficient data on expected negative chemicals and, therefore, the specificity of the method is not known. In addition, a method to determine cell proliferation in GI tract is not established. Ki-67 immunohistochemistry might be useful to examine cell proliferation [21], but more data will be needed. The working group had insufficient time to fully discuss other issues, but further investigation is clearly required to establish and standardize the GI tract MNT. 4. Conclusion The working group concluded that the GI tract MNT would be a promising method to examine the clastogenicity/aneugenicity of test chemicals in the stomach and/or colon. It will be useful for hazard identification by tissue-specific clastogenic effects by chemicals and their risk evaluation. Further data will be needed to more accurately assess the sensitivity and specificity of the GI tract MNT. Conflict of interest C. Beevers is employed by a contract research laboratory that offers this assay as a service offering. Funding
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[8]
[9]
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[13]
[14]
[15]
[16]
None. [17]
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