Access the full text.
Sign up today, get DeepDyve free for 14 days.
Background: Currently, revisions to the ICH S1 guidance on rodent carcinogenicity testing are being proposed. Application of this approach would reduce the use of animals in accordance with the 3Rs principles (reduce/refine/ replace). The method would also shift resources to focus on more scientific mechanism-based carcinogenicity assessments and promote safe and ethical development of new small molecule pharmaceuticals. In the revised draft, findings such as cellular hypertrophy, diffuse and/or focal cellular hyperplasia, persistent tissue injury and/or chronic inflammation, preneoplastic changes, and tumors are listed as histopathology findings of particular interest for identifying carcinogenic potential. In order to predict hepatocarcinogenicity of test chemicals based on the results from 2- or 4-week repeated dose studies, we retrospectively reanalyzed the results of a previous collaborative study on the liver micronucleus assay. We focused on liver micronucleus induction in combination with histopathological changes including hypertrophy, proliferation of oval cells or bile duct epithelial cells, tissue injuries, regenerative changes, and inflammatory changes as the early responses of hepatocarcinogenesis. For these early responses, A total of 20 carcinogens, including 14 genotoxic hepatocarcinogens (Group A) and 6 non-liver- targeted genotoxic carcinogens (Group B) were evaluated. Results: In the Group A chemicals, 5 chemicals (NPYR, MDA, NDPA, 2,6-DNT, and NMOR) showed all of the 6 early responses in hepatocarcinogenesis. Five chemicals (DMN, 2,4-DNT, QUN, 2-AAF, and TAA) showed 4 responses, and 4 chemicals (DAB, 2-NP, MCT, and Sudan I) showed 3 responses. All chemicals exhibited at least 3 early responses. Contrarily, in the Group B chemicals (6 chemicals), 3 of the 6 early responses were observed in 1 chemical (MNNG). No more than two responses were observed in 3 chemicals (MMC, MMS, and KA), and no responses were observed in 2 chemicals (CP and KBrO3). Conclusion: Evaluation of liver micronucleus induction in combination with histopathological examination is useful for detecting hepatocarcinogens. This assay takes much less time than routine long-term carcinogenicity studies. Keywords: Micronucleus assay, Liver, Hepatocarcinogen, Histopathology, Early responses * Correspondence: firstname.lastname@example.org BoZo Research Center Inc, 1-3-11 Hanegi, Setagaya-ku, Tokyo 156-0042, Japan Full list of author information is available at the end of the article © The Author(s). 2021 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://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hamada et al. Genes and Environment (2022) 44:2 Page 2 of 9 Introduction for evaluating liver micronuclei. The approach used 2- The liver is an important tissue not only in general toxi- or 4-week repeated-dose treatment for the accumulation cological studies, but also in carcinogenicity studies. of micronucleated hepatocytes (MNHEPs) . This About 60% of carcinogens are hepatocarcinogens , method facilitates the integration of the liver micronu- suggesting that development a new evaluation method cleus assay into repeated-dose general toxicity studies to targeting the liver is meaningful. In addition to the rou- simultaneously assess genotoxicity and histopathological tinely used erythropoietic micronucleus in rodents, the endpoints with the same animals used for the overall liver micronucleus assay has been developed to detect evaluation of chemical risk. genotoxic hepatocarcinogens that require metabolic acti- Routine long-term carcinogenicity studies are time vation [2–6]. consuming and costly and require large numbers of ani- The liver micronucleus assay targets the primary organ mals. Revision to the ICH S1 guidelines is being dis- for drug metabolism; however, it is not commonly used cussed to address these issues. In a revised draft, due to slow hepatocyte proliferation in adult rats. Partial histopathological findings such as cellular hypertrophy, hepatectomy [7–9], mitogen treatment [10, 11], and the diffuse and/or focal cellular hyperplasia, persistent tissue use of juvenile rats [12–15] have been introduced to ad- injury and/or chronic inflammation, preneoplastic dress this drawback. Unfortunately, these methods have changes, and tumors are listed as particular interest for disadvantages, including complex surgical procedures identifying carcinogenic potential . The possibility of and decreased metabolic activity for partial hepatectomy predicting hepatocarcinogenicity of test chemicals based , risk of drug interactions for mitogen treatment on the results of 2- or 4-week repeated-dose studies was , and a lack of maturation for metabolic activation in assessed using a reanalysis of a previous collaborative juvenile rats . Recently, a repeated-dose liver micro- study of the liver micronucleus assay [2, 21] in combin- nucleus assay (RDLMN) was developed as a new method ation with histopathological examination. Table 1 Liver MN assay results in the collaborative study by CSGMT/JEMS MMS and rat carcinogenicity data for the test chemicals Group Chemical Abbreviation CAS no. In vivo MN assay (Liver) Rat carcinogenicity 2 weeks Ref. 4 weeks Ref. Liver Other sites Ref. Group A Dimethylnitrosamine DMN 62–75-9 + +  + kid, lun, vsc, tes  N-Nitrosopyrrolidine NPYR 930–55-2 + +  + kid, vsc, tes [22, 23] 4,4′-Methylenedianiline MDA 101–77-9 + +  + thy  N-Nitrosodipropylamine NDPA 621–64-7 +  ND + eso, nas  2,4-Dinitrotoluene 2,4-DNT 121–14-2 + +  + ski, mgl  2,6-Dinitrotoluene 2,6-DNT 606–20-2 + + + –  Quinoline QUN 91–22-5 + + + –  p-Dimethylaminoazobenzene DAB 60–11-7 + + + –  2-Nitropropane 2-NP 79–46-9 + + + –  Monocrotaline MCT 315–22-0 + + + –  N-Nitrosomorpholine NMOR 59–89-2 +  ND + vsc  2-Acetylaminofluorene 2-AAF 53–96-3 + +  + ski, mgl  Sudan I (C.I.solvent yellow 14) Sudan I 842–07-9 + ND + –  Thioacetamide TAA 62–55-5 + + + –  Group B Mitomycin C MMC 50–07-7 + +  – per  Cyclophosphamide H2O CP 6055-19-2 – ND – ub, lym, ner  Potassium bromate KBrO3 7758-01-2 –  –  – kid, per, thy  N-Methyl-N′-nitro-N-nitrosoguanidine MNNG 70–25-7 –  –  – eso, smi, sto  Methyl methanesulfonate MMS 66–27-3 +  –  – hmo, lun, ner [22, 26] Kojic acid KA 501–30-4 –  –  – thy (mouse)  MN assay: micronucleus assay +: positive; −: negative; ND: no data; kid: kidney; lun: lung; vsc: vascular system; tes: testes; thy: thyroid gland; eso: esophagus; nas: nasal cavity; ski: skin; mgl: mammary gland; per: peritoneal cavity; ub: urinary bladder; lym: lymphocyte; ner: nervous system; smi: small intestine; sto: stomach; hmo: hematopoietic system; pan: pancreas Group A, Genotoxic hepatocarcinogens; Group B, Genotoxic carcinogens but non-liver-targeted Hamada et al. Genes and Environment (2022) 44:2 Page 3 of 9 Materials and methods Histopathological examination was performed by a path- Classification of chemicals and previous collaborative ologist using light microscopy. study by CSGMT/JEMS MMS Twenty genotoxic carcinogens examined in a previous Reanalysis of pathological findings and application to the collaborative study by CSGMT/JEMS MMS were classi- hepatocarcinogenesis process fied into two groups: Group A consisted of 14 genotoxic Common markers for a precancerous stage in hepato- hepatocarcinogens and Group B consisted of 6 non- carcinogenesis include (i) transformation of normal he- liver-targeted genotoxic carcinogens. Liver micronucleus patocytes into preneoplastic hepatocytes, (ii) selection of assay data were then integrated (Table 1). preneoplastic hepatocytes for growth, and (iii) isolation Male Crl:CD (SD) rats used in the previous report of preneoplastic hepatocytes from normal hepatic tissue. [28–44] were purchased from Charles River Japan Inc. Transformation, selection, and isolation are thus general (Atsugi, Hino or Tsukuba, Japan) and used at the age of processes for the progression of preneoplastic hepato- 6 weeks. The animals were housed in an air-conditioned cytes into malignant cells . With references to this room with a 12-h light/dark cycle and allowed free ac- report and the histopathology findings of particular cess to food and water. The animal experiments were interest for identifying carcinogenic potential pointed approved by the Institutional Animal Care and Use out in the draft S1 guidelines , changes in each car- Committee of each testing facility in advance. The rats cinogenic process were roughly divided into 10 categor- (5/group) were given each chemical repeatedly by oral ies: mutation (including liver micronucleus induction), gavage for 14 or 28 consecutive days. Twenty-four hours hypertrophy, tissue injuries, proliferation of oval cells or after the last administration, the rats were euthanized bile duct epithelial cells, regenerative changes, inflamma- under thiopental anesthesia. Livers were removed and a tory changes, focus of altered hepatocytes, non- part of each liver (left lateral lobe) was used for the liver regenerative or regenerative hyperplasia, adenoma, and micronucleus assay [28–44]. The remaining tissue was liver cancer (Fig.1). fixed with 10% phosphate-buffered formalin, embedded We used the above information to reanalyze the pres- in paraffin, thin-sectioned, and stained with hematoxylin ence of 9 liver pathological responses based on the find- and eosin according to standard protocols. ings from the previous collaborative study. Each of the 20 Fig. 1 Processes in multistage carcinogenesis theory and pathological findings in proposed changes to ICH S1 guidance Hamada et al. Genes and Environment (2022) 44:2 Page 4 of 9 chemicals was reassessed. The grades of findings and fre- bile duct epithelial cells (50% [7/14]), regenerative quency of appearance were disregarded to simplify the changes (71% [10/14]), inflammatory changes (50% [7/ evaluation. Judgment was used only for the presence or 14]), focus of altered hepatocytes (21% [3/14]), and ad- absence determination. Except for accidental findings, enomas (7% [1/14]). Non-regenerative or regenerative findings judged to result from toxic insult were compre- hyperplasia and liver cancer were not observed. hensively evaluated. Mutation was identified via induction One chemical (2,6-DNT) demonstrated 7 of the 10 of liver micronuclei. Chemicals evaluated in 14- and 28- aforementioned responses. Five chemicals (NPYR, MDA, day repeated dose studies were judged to be “with find- NDPA, NMOR, and 2-AAF) displayed 6 responses, 5 ings” if chemical-related toxicity was observed in either chemicals (DMN, 2,4-DNT, QUN, 2-NP, and TAA) ex- time frame. Chemicals without findings in either time hibited 4 responses, and 3 chemicals (DAB, MCT, and frame were judged to be “without findings”. Sudan I) showed 3 responses. No chemical showed fewer than three responses. Results Group A chemicals (genotoxic hepatocarcinogens) Group B chemicals (genotoxic carcinogens but not liver We evaluated 14 Group A chemicals for 10 markers of targeted) the carcinogenic pathways (9 liver pathological responses We evaluated 6 Group B chemicals (Fig.3). The response and liver micronucleus induction) (Fig.2). The liver mi- frequencies for these chemicals were liver micronucleus cronucleus induction was most frequently observed induction (33% [2/6]), hypertrophy (33% [2/6]), tissue in- (100% [14/14]) followed by hypertrophy (93% [13/14]), juries (17% [1/6]), regenerative changes (17% [1/6]), and tissue injuries (79% [11/14]), proliferation of oval cells or inflammatory changes (17% [1/6]). Fig. 2 Liver micronucleus induction and histopathological changes observed in 14-day and/or 28-day repeated-dose studies – genotoxic hepatocarcinogens Hamada et al. Genes and Environment (2022) 44:2 Page 5 of 9 Fig. 3 Liver micronucleus induction and histopathological changes observed in 14-day and/or 28-day repeated-dose studies – genotoxic carcinogens but not liver targeted Group B chemicals did not cause proliferation of oval cells chemical Group A or B exhibited non-regenerative or or bile duct epithelial cells, focus of altered hepatocytes, regenerative hyperplasia or liver cancer. The latter pa- non-regenerative or regenerative hyperplasia, adenoma, or rameters are recognized as the most credible indicators liver cancer. MNNG showed 3 responses, but 3 chemicals of hepatocarcinogenesis [46–48]. The present study was (MMC, MMS, and KA) showed only one or two responses. a retrospective survey of short-term study with 14- or CP and KBrO did not show any targeted responses. 28-day repeated dose design, and such findings are not expected. Thus, we selected 6 responses that expected to Discussion occur very early in the process of carcinogenesis, includ- Few Group A chemicals caused the focus of altered he- ing hypertrophy, proliferation of oval cells or bile duct patocytes (21% [3/14]) or adenoma (7% [1/14]). No epithelial cells, tissue injuries, mutation (including liver Hamada et al. Genes and Environment (2022) 44:2 Page 6 of 9 micronucleus induction), regenerative changes, and in- micronucleus induction was not observed in 4 out of 6 flammatory changes (Table 2). chemicals. These chemicals demonstrated varying re- All 14 Group A chemicals were positive for liver mi- sponses, including hypertrophy (50% [2/4]), proliferation cronucleus assay; only 2 of 6 Group B chemicals induced oval cells or bile duct epithelial cells (0% [0/4]), tissue in- micronuclei. These 2 chemicals, namely, MMC and juries (25% [1/4]), regenerative changes (0% [0/4]), and MMS, are carcinogens but are not liver-targeted. Both inflammatory changes (25% [1/4]). Further, hypertrophy, are direct-acting genotoxic chemicals that are used as proliferation oval cells or bile duct, tissue injuries, and positive controls in genotoxicity tests and induce micro- inflammatory were not observed in the two chemicals nuclei in various tissues, including the liver . There- that were positive for liver micronucleus induction. Only fore, liver micronucleus induction was considered to be regenerative changes were observed for one of these che- a useful indicator for possible hepatocarcinogenesis. micals. Thus, even if a chemical is found to be positive Speculatively, the chromothripsis could involve fragmen- for liver micronucleus induction, negative results for all tation and subsequent reassembly of a single chromatid other pathological findings indicative of early stages of from a micronucleus [49, 50]. Chromothripsis is a new carcinogenesis suggest a low probability of cancer devel- concept for mutational process; it involves genome opment in the liver. reorganization associated with micronuclei. This process Much debate has occurred over the issue of whether might elucidate the mechanisms for the production of hypertrophy is a key early response in hepatotoxicity or micronuclei and genome instability and cellular evolu- hepatocarcinogenicity in rodent toxicity studies [51–54]. tion essential in complex diseases such as cancer . We suggest that hypertrophy in the liver without mi- In addition to liver micronucleus induction, many cronucleus induction does not predict future hepatocar- Group A chemicals exhibited two or more of the other cinogenesis. Hypertrophy with micronucleus induction five responses assumed to be early predictors of carcino- is, however, closely related to hepatocarcinogenesis. Clo- genesis. Contrarily, in Group B chemicals, liver fibrate is a typical non-genotoxic hepatocarcinogen that Table 2 Histopathological changes and induction of liver micronuclei seen as very early responses of hepatocarcinogenesis Group A: Genotoxic hepatocarcinogens, Group B: Genotoxic carcinogens but not liver targeted Mut: Liver MN induction, Ht: Hypertrophy, Pob: Proliferation oval cell or bile duct, TI: Tissue injuries, RC: Regenerative change, Inf: Inflammatory Hamada et al. Genes and Environment (2022) 44:2 Page 7 of 9 induces hepatocyte hypertrophy and liver micronuclei Competing interests The authors declare that they have no competing interests. . A recently developed formalin fixation method for the Author details liver micronucleus assay [21, 55] enables retrospective BoZo Research Center Inc, 1-3-11 Hanegi, Setagaya-ku, Tokyo 156-0042, Japan. LSIM Safety Institute Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki evaluation using formalin-fixed liver samples from gen- 314-0255, Japan. Rakuno Gakuen University, 582 midorimachi, Bunkyoudai, eral toxicity and carcinogenicity studies completed in 4 Ebetsu-shi, Hokkaido 069-8501, Japan. Yakult Honsha Co., Ltd, 5-11 Izumi, the past. With this method, the prediction of hepatocar- Kunitachi-shi, Tokyo 186-8650, Japan. National Institute of Technology and Evaluation, 2-49-10 Nishihara, Shibuya-ku, Tokyo 151-0066, Japan. makoto cinogenicity of a test substance with accuracy is possible international consulting, 4-23-3-1 Kamiimaizumi, Ebina-shi, Kanagawa using data from 2- and 4-week repeated-dose toxicity 243-0431, Japan. studies, including previously published work. Received: 4 August 2021 Accepted: 19 October 2021 Conclusion Liver micronucleus induction can be employed to pre- References dict hepatocarcinogenesis. The combination of this assay 1. Fukushima S. Medium-term tests for carcinogens and carcinogenic hazard evaluation for humans. Journal of Occupational Safety and Health. 2008;1(2): with histopathological findings observed in the early 141–9. https://doi.org/10.2486/josh.1.141. stages of the carcinogenic process (hypertrophy, prolifer- 2. Hamada S, Ohyama W, Takashima R, Shimada K, Matsumoto K, Kawakami S, ation of oval cells or bile duct epithelial cells, tissue in- et al. Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of juries, regenerative changes, and inflammatory changes) the collaborative study by the Collaborative Study Group for the can increase the accuracy of the prediction even in a Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society short-term repeated dose study of 2 or 4 weeks. (JEMS) - Mammalian Mutagenicity Study Group (MMS). Mutat Res. 2015; 780–781:2–17. 3. Natarajan AT, Tates AD, Van Buul PP, Meijers M, De Vogel N. Cytogenetic Abbreviations effects of mutagens/carcinogens after activation in a microsomal system DMN: Dimethylnitrosamine; NPYR: N-Nitrosopyrrolidine; MDA: 4,4′- in vitro I. induction of chromosome aberrations and sister chromatid Methylenedianiline; NDPA: N-Nitrosodipropylamine; 2,4-DNT: 2,4- exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in Dinitrotoluene; 2,6-DNT: 2,6-Dinitrotoluene; QUN: Quinoline; DAB: p- CHO cells in the presence of rat-liver microsomes. Mutat Res. 1976;37(1):83– Dimethylaminoazobenzene; 2-NP: 2-Nitropropane; MCT: Monocrotaline; 90. https://doi.org/10.1016/0027-5107(76)90057-9. NMOR: N-Nitrosomorpholine; 2-AAF: 2-Acetylaminofluorene; Sudan 4. Ashby J, Tennant RW. Definitive relationships among chemical structure, I: C.I.solvent yellow 14; TAA: Thioacetamide; MMC: Mitomycin C; carcinogenicity and mutagenicity for 301 chemicals tested by the U. S NTP CP: Cyclophosphamide H O; KBrO : Potassium bromate; MNNG: N-Methyl-N 2 3 Mutat Res. 1991;257(3):229–306. https://doi.org/10.1016/0165-1110(91)90003- ′-nitro-N-nitrosoguanidine; MMS: Methyl methanesulfonate; KA: Kojic acid; E. ICH: The International Council for Harmonisation of Technical Requirements 5. Morita T, Asano N, Awogi T, Sasaki YF, Sato S, Shimada S, et al. Evaluation of for Pharmaceuticals for Human Use; IARC: International Agency for Research the rodent micronucleus assay in the screening of IARC carcinogens (group on Cancer; JEMS: The Japanese Environmental Mutagen Society; 1, 2A and 2B). The summary report of the 6th collaborative study by CSGM MMS: Mammalian Mutagenicity Study Group; CSGMT: The Collaborative T/JEMS∙MMS. Mutat Res. 1997;389(1):3–122. https://doi.org/10.1016/S1383- Study Group for the Micronucleus Test 5718(96)00070-8. 6. George E, Westmoreland C. Evaluation of the in vivo genotoxicity of the Acknowledgements structural analogues 2,6-diaminotoluene using the rat micronucleus test The authors thank the support by MMS/JEMS and also all participants who and rat liver UDS assay. Carcinogenesis. 1991;12(12):2233–7. https://doi.org/1 worked mainly on a voluntary basis. 0.1093/carcin/12.12.2233. 7. Tates AD, Neuteboom I, Hofker M, Den Engelse L. A micronucleus Authors’ contributions technique for detecting clastogenic effects of mutagens/carcinogens (DEN, SH, MS, and WO performed liver micronucleus assay of compounds and DMN) in hepatocytes of rat liver in vivo. Mutat Res. 1980;74(1):11–20. statistical analysis of the results obtained in the assay. YW and KK performed https://doi.org/10.1016/0165-1161(80)90187-9. histopathological examination. SH, WO, TM1, and MH performed 8. Tates AD, Den Engelse L. The role of short-lived lesions in the induction of comprehensive evaluation of all laboratory data. SH, KS, TM2 and TF created micronuclei in rat liver by ethylnitrosourea and methyl methanesulfonate: table, fig, and manuscript. All authors have read and approved the final the importance of experimental design. Mutat Res. 1989;210(2):271–9. manuscript. TM1: Takeshi Morita, TM2: Tatsuya Mitsumoto. https://doi.org/10.1016/0027-5107(89)90088-2. 9. Angelosanto FA. Tissues other than bone marrow that can be used for cytogenetic analysis. Environ Mol Mutagen. 1995;25(4):338–43. https://doi. Funding org/10.1002/em.2850250412. Not applicable. 10. Braithwaite I, Ashby J. A non-invasive micronucleus assay in the rat liver. Mutat Res. 1988;203(1):23–32. https://doi.org/10.1016/0165-1161(88)90004-0. Availability of data and materials 11. Ashby J, Lefevre PA. The rat-liver carcinogen N-nitrosomorpholine initiates All data generated or analyzed during this study are included in this unscheduled DNA synthesis and induces micronuclei in the rat liver in vivo. published article. Mutat Res. 1989;225(4):143–7. https://doi.org/10.1016/0165-7992(89)90111-5. 12. Suzuki H, Ikeda N, Kobayashi K, Terashima Y, Shimada Y, Suzuki T, et al. Declarations Evaluation of liver and peripheral blood micronucleus assays with 9 chemicals using young rats. A study by the collaborative study Group for Ethics approval the Micronucleus Test (CSGMT)/Japanese environmental mutagen society The animal experiments were approved by the Institutional Animal Care and (JEMS)-mammalian mutagenicity study group (MMS). Mutat Res. 2005;583(2): Use Committee of each testing facility prior to conducting the experiments. 133–45. https://doi.org/10.1016/j.mrgentox.2005.03.012. 13. Suzuki H, Takasawa H, Kobayashi K, Terashima Y, Shimada Y, Ogawa I, et al. Consent for publication Evaluation of a liver micronucleus assay with 12 chemicals using young rats Not applicable. (II): a study by the collaborative study Group for the Micronucleus Test/ Hamada et al. Genes and Environment (2022) 44:2 Page 8 of 9 Japanese environmental mutagen society-mammalian mutagenicity study 32. Uno F, Tanaka J, Ueda M, Nagai M, Fukumuro M, Natsume M, et al. group. Mutagenesis. 2009;24(1):9–16. https://doi.org/10.1093/mutage/gen04 Repeated-dose liver and gastrointestinal tract micronucleus assays for 7. quinoline in rats. Mutat Res. 2015;780–781:51–5. https://doi.org/10.1016/j. 14. Takasawa H, Suzuki H, Ogawa I, Shimada Y, Kobayashi K, Terashima Y, et al. mrgentox.2015.01.003. Evaluation of a liver micronucleus assay in young rats (III): a study using 33. Shimada Y, Sui H, Wako Y, Kawasako K. The evaluation of the repeated-dose nine hepatotoxicants by the collaborative study Group for the Micronucleus liver micronucleus assay with p-dimethylaminoazobenzene. Mutat Res. 2015; Test (CSGMT)/Japanese environmental mutagen society (JEMS)-mammalian 780–781:56–9. https://doi.org/10.1016/j.mrgentox.2014.10.004. mutagenicity study group (MMS). Mutat Res. 2010;698(1-2):30–7. https://doi. 34. Kawakami S, Araki T, Nakajima M, Kusuoka O, Uchida K, Sato N, et al. org/10.1016/j.mrgentox.2010.02.009. Repeated-dose liver micronucleus assay: an investigation with 2- 15. Takasawa H, Suzuki H, Ogawa I, Shimada Y, Kobayashi K, Terashima Y, et al. nitropropane, a hepatocarcinogen. Mutat Res. 2015;780–781:60–3. https:// Evaluation of a liver micronucleus assay in young rats (IV): a study using a doi.org/10.1016/j.mrgentox.2014.06.005. double-dosing/single-sampling method by the collaborative study Group 35. Takashima R, Takasawa H, Wako Y, Kawasako K, Yasunaga K, Hattori A, et al. for the Micronucleus Test (CSGMT)/Japanese environmental mutagen Micronucleus induction in rat liver and bone marrow by acute vs. repeat society (JEMS)-mammalian mutagenicity study group (MMS). Mutat Res. doses of the genotoxic hepatocarcinogen monocrotaline. Mutat Res. 2015; 2010;698(1-2):24–9. https://doi.org/10.1016/j.mrgentox.2010.02.010. 780–781:64–70. https://doi.org/10.1016/j.mrgentox.2014.12.008. 16. Rossi AM, Romano M, Zaccaro L, Pulci R, Salmona M. DNA synthesis, mitotic 36. Hayashi A, Kosaka M, Kimura A, Wako Y, Kawasako K, Hamada S. Evaluation indices, drug-metabolising systems and cytogenetic analyses in of the repeated-dose liver micronucleus assay using N-nitrosomorpholine in regenerating rat liver. Mutat Res. 1987;182(2):75–82. https://doi.org/10.1016/ young adult rats: report on collaborative study by the collaborative study 0165-1161(87)90056-2. Group for the Micronucleus Test (CSGMT)/Japanese environmental mutagen 17. Parton JW, Garriott ML. An evaluation of micronucleus induction in bone society (JEMS) – mammalian mutagenicity study (MMS) group. Mutat Res. marrow and in hepatocytes isolated from collagenase perfused liver or from 2015;780–781:71–5. https://doi.org/10.1016/j.mrgentox.2014.04.002. formalin-fixed liver using four-week-old rats treated with known clastogens. 37. Matsumura S, Ikeda N, Hamada S, Ohyama W, Wako Y, Kawasako K, et al. Environ Mol Mutagen. 1997;29(4):379–85. https://doi.org/10.1002/(SICI)1 Repeated-dose liver and gastrointestinal tract micronucleus assays with CI 098-2280(1997)29:4<379::AID-EM6>3.0.CO;2-5. solvent yellow 14 (Sudan I) using young adult rats. Mutat Res. 2015;780– 18. Kato R, Yamazoe Y. Sex-specific cytochrome P450 as a cause of sex and 781:76–80. https://doi.org/10.1016/j.mrgentox.2014.09.002. species-related differences in drug toxicity. Toxicol Lett. 1992;64/65:661–7. 38. Sui H, Matsumoto H, Wako Y, Kawasako K. Evaluation of in vivo genotoxicity 19. Narumi K, Ashizawa K, Takashima R, Takasawa H, Katayama S, Tsuzuki Y, by thioacetamide in a 28-day repeated-dose liver micronucleus assay using et al. Development of repeated-dose liver micronucleus assay using adult male young adult rats. Mutat Res. 2015;780–781:81–4. https://doi.org/10.101 rats: an investigation of diethylnitrosamine and 2, 4-diaminotoluene. Mutat 6/j.mrgentox.2014.10.001. Res. 2012;747(2):234–9. https://doi.org/10.1016/j.mrgentox.2012.05.012. 39. Matsumoto K, Zaizen K, Miyamoto A, Wako Y, Kawasako K, Ishida H. 20. ICH HARMONISED GUIDELINE. S1B(R1), ADDENDUM TO THE GUIDELINE ON Evaluation of the repeated dose liver micronucleus assay using young adult TESTING FOR CARCINOGENICITY OF PHARMACEUTICALS, draft version, rats with cyclophosphamide monohydrate: a report of a collaborative study endorsed on 10 may 2021. Currently under public consultation. by CSGMT/JEMS MMS. Mutat Res. 2015;780–781:90–3. https://doi.org/10.101 21. Hamada S, Shigano M, Kawakami S, Ueda M, Sui H, Yamada K, et al. 6/j.mrgentox.2014.05.004. Evaluation of the novel liver micronucleus assay using formalin-fixed tissues. 40. Okada E, Fujiishi Y, Narumi K, Kado S, Wako Y, Kawasako K, et al. Evaluation Genes Environ. 2019;41(1):13. https://doi.org/10.1186/s41021-019-0128-5. of repeated dose micronucleus assays of the liver and gastrointestinal tract using potassium bromate: a report of the collaborative study by CSGMT/ 22. L S. Gold, The carcinogenic potency database (CPDB). 2011. https://files. JEMS MMS. Mutat Res. 2015;780–781:94–9. https://doi.org/10.1016/j. toxplanet.com/cpdb/indices.html. mrgentox.2014.03.002. 23. Greenblatt M, Lijinsky W. Nitrosamine studies: neoplasms of liver and genital mesothelium N-nitrosopyrrolidine-treated MRC rats. J Natl Cancer Inst. 1972; 41. Takayanagi T, Wako Y, Kawasako K, Hori H, Fujii W, Ohyama W. Repeated 48(6):1687–96. dose liver and gastrointestinal tract micronucleus assays using N-methyl-N’- 24. IARC, IARC Monographs on the evaluation of the carcinogenic risk of nitro-Nnitrosoguanidine in young adult rats. Mutat Res. 2015;780–781:100–6. chemicals to humans. Some Chemicals Used in Plastic and Elastomers 1986; 42. Muto S, Yamada K, Kato T, Wako Y, Kawasako K, Iwase Y, Uno Y. Assessment 39:347–365. of methyl methanesulfonate using the repeated-dose liver micronucleus assay in young adult rats. Mutat Res. 2015;780–781:107–10. 25. Hirao K, Shinohara Y, Tsuda H, Fukushima S, Takahashi M, Ito N. 43. Takayanagi T, Takashima R, Wako Y, Kawasako K, Tanaka Y, Hori H, Fujii W. Carcinogenic activity of quinoline on rat liver. Cancer Res. 1976;36:329–35. Repeated dose liver micronucleus assay using clofibrate in young adult rats. 26. IARC, IARC Monographs on the evaluation of the carcinogenic risk of Mutat Res. 2015;780–781:117–22. chemicals to humans, Re-evaluation of Some Organic Chemicals Hydrazine and Hydrogen Peroxide 1999;71:1059–1078. 44. Inoue K, Ochi A, Koda A, Wako Y, Kawasako K, Doi T. The 14-day repeated 27. IARC, IARC Monographs on the evaluation of the carcinogenic risk of dose liver micronucleus test with methapyrilene hydrochloride using young chemicals to humans, A Review of Human Carcinogens: Pharmaceuticals adult rats. Mutat Res. 2015;780–781:123–7. https://doi.org/10.1016/j. 2012;100A: 63–90. mrgentox.2014.04.004. 28. Takashima R, Takasawa H, Kawasako K, Ohyama W, Okada E, Narumi K, et al. 45. Ogawa K. Molecular pathology of early stage chemically induced Evaluation of a repeated dose liver micronucleus assay in rats treated with hepatocarcinogenesis. Pathol Int. 2009;59(9):605–22. https://doi.org/10.1111/ two genotoxic hepatocarcinogens, dimethylnitrosamine and 2- j.1440-1827.2009.02416.x. acetylaminofluorene: the possibility of integrating micronucleus tests with 46. Sugitani S, Sakamoto M, Ichida T, Genda T, Asakura H, Hirohashi S. multiple tissues into a repeated dose general toxicity study. Mutat Res. Hyperplastic foci reflect the risk of multicentric development of human 2015;780–781:18–24. https://doi.org/10.1016/j.mrgentox.2014.10.007. hepatocellular carcinoma. J Hepatol. 1998;28(6):1045–53. https://doi.org/10.1 016/S0168-8278(98)80355-3. 29. Ogawa I, Hagio S, Furukawa S, Abe M, Kuroda Y, Hayashi S, et al. Evaluation of 47. Farber E. Similarities in the sequence of early histological changes induced repeated dose micronucleus assays of the liver using N-nitrosopyrrolidine: a in the liver of the rat by ethionine, 2-acetylamino-fluorene, and 3′-methyl-4- report of the collaborative study by CSGMT/JEMS MMS. Mutat Res. 2015;780– dimethylaminoazobenzene. Cancer Res. 1956;16(2):142–8. 781:25–30. https://doi.org/10.1016/j.mrgentox.2014.05.007. 30. Terashima Y, Yokoi R, Takakura I, Saitou E, Wako Y, Kawasako K, et al. 48. Laconi S, Pani P, Pillai S, Sarma DSR, Laconi E. A growth-constrained Detection of micronuclei in hepatocytes isolated from young adult rats environment drives tumor progression in vivo. Proc Natl Acad Sci U S A. repeatedly treated with N-nitrosodi-n-propylamine. Mutat Res. 2015;780– 2001;98(14):7806–11. https://doi.org/10.1073/pnas.131210498. 781:36–40. https://doi.org/10.1016/j.mrgentox.2014.04.001. 49. Zhang CZ, Spektor A, Cornils H, Francis JM, Jackson EK, Liu S, et al. 31. Maeda A, Tsuchiyama H, Asaoka Y, Hirakata M, Miyoshi T, Oshida K, et al. Chromothripsis from DNA damage in micronuclei. Nature. 2015;522(7555): Evaluation of the repeated-dose liver micronucleus assay using 2,4- 179–84. https://doi.org/10.1038/nature14493. dinitrotoluene: a report of a collaborative study by CSGMT/JEMS MMS. 50. Ye CJ, Sharpe Z, Alemara S, Mackenzie S, Liu G, Abdallah B, et al. Mutat Res. 2015;780–781:41–5. https://doi.org/10.1016/j.mrgentox.2014.05. Micronuclei and genome chaos: changing the system inheritance. Genes. 002. 2019;10(5):366. https://doi.org/10.3390/genes10050366. Hamada et al. Genes and Environment (2022) 44:2 Page 9 of 9 51. Hall AP, Elcombe CR, Foster JR, Harada T, Kaufmann TW, Knippel A, et al. Liver hypertrophy: a review of adaptive (adverse and non-adverse) changes —conclusions from the 3rd international ESTP expert workshop. Toxicol Pathol. 2012;40(7):971–94. https://doi.org/10.1177/0192623312448935. 52. Robert RM, Yoshizawa K, Nyska A, Harada T, Flake G, Mueller G, et al. Hepatic enzyme induction: histopathology. Toxicol Pathol. 2010;38(5):776– 95. https://doi.org/10.1177/0192623310373778. 53. Elcombe CR, Peffer RC, Wolf DC, Bailey J, Bars R, Bell D, et al. Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: a case study with phenobarbital as a model constitutive androstane receptor (CAR) activator. Crit Rev Toxicol. 2014;44(1):64–82. https://doi.org/10.3109/1 0408444.2013.835786. 54. Rusyn I, Peters JM, Cunningham ML. Modes of action and species-specific effects of di-(2-ethylhexyl)phthalate in the liver. Effects of DEHP in the Liver: Modes of Action and Species-Specific Differences Crit Rev Toxicol. 2006; 36(5):459–79. https://doi.org/10.1080/10408440600779065. 55. Shigano M, Takashima R, Takasawa H, Hamada S. Optimization of specimen preparation from formalin-fixed liver tissues for liver micronucleus assays: hepatocyte staining with fluorescent dyes. Mutat Res. 2016;800:35–9. https:// doi.org/10.1016/j.mrgentox.2016.03.004. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Genes and Environment – Springer Journals
Published: Jan 4, 2022
Keywords: Micronucleus assay; Liver; Hepatocarcinogen; Histopathology; Early responses
Access the full text.
Sign up today, get DeepDyve free for 14 days.