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Detection of hepatocarcinogens by combination of liver micronucleus assay and histopathological examination in 2-week or 4-week repeated dose studies

Detection of hepatocarcinogens by combination of liver micronucleus assay and histopathological... 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: hamada.shuichi@bozo.co.jp 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) [19]. This About 60% of carcinogens are hepatocarcinogens [1], 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 [20]. The possibility of and decreased metabolic activity for partial hepatectomy predicting hepatocarcinogenicity of test chemicals based [16], risk of drug interactions for mitogen treatment on the results of 2- or 4-week repeated-dose studies was [17], and a lack of maturation for metabolic activation in assessed using a reanalysis of a previous collaborative juvenile rats [18]. 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 + [2]+ [2] + kid, lun, vsc, tes [22] N-Nitrosopyrrolidine NPYR 930–55-2 + [2]+ [2] + kid, vsc, tes [22, 23] 4,4′-Methylenedianiline MDA 101–77-9 + [2]+ [2] + thy [24] N-Nitrosodipropylamine NDPA 621–64-7 + [2] ND + eso, nas [22] 2,4-Dinitrotoluene 2,4-DNT 121–14-2 + [2]+ [2] + ski, mgl [22] 2,6-Dinitrotoluene 2,6-DNT 606–20-2 + [2]+ [2]+ – [22] Quinoline QUN 91–22-5 + [2]+ [2]+ – [25] p-Dimethylaminoazobenzene DAB 60–11-7 + [2]+ [2]+ – [22] 2-Nitropropane 2-NP 79–46-9 + [2]+ [2]+ – [26] Monocrotaline MCT 315–22-0 + [2]+ [2]+ – [22] N-Nitrosomorpholine NMOR 59–89-2 + [2] ND + vsc [22] 2-Acetylaminofluorene 2-AAF 53–96-3 + [2]+ [2] + ski, mgl [22] Sudan I (C.I.solvent yellow 14) Sudan I 842–07-9 + [21]ND + – [22] Thioacetamide TAA 62–55-5 + [21]+ [21]+ – [22] Group B Mitomycin C MMC 50–07-7 + [2]+ [2] – per [22] Cyclophosphamide H2O CP 6055-19-2 – [2]ND – ub, lym, ner [27] Potassium bromate KBrO3 7758-01-2 – [2] – [2] – kid, per, thy [22] N-Methyl-N′-nitro-N-nitrosoguanidine MNNG 70–25-7 – [2] – [2] – eso, smi, sto [22] Methyl methanesulfonate MMS 66–27-3 + [2] – [2] – hmo, lun, ner [22, 26] Kojic acid KA 501–30-4 – [2] – [2] – thy (mouse) [22] 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 [45]. 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 [20], 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 [2]. 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 [50]. 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. [2]. 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. 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Detection of hepatocarcinogens by combination of liver micronucleus assay and histopathological examination in 2-week or 4-week repeated dose studies

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Abstract

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: hamada.shuichi@bozo.co.jp 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) [19]. This About 60% of carcinogens are hepatocarcinogens [1], 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 [20]. The possibility of and decreased metabolic activity for partial hepatectomy predicting hepatocarcinogenicity of test chemicals based [16], risk of drug interactions for mitogen treatment on the results of 2- or 4-week repeated-dose studies was [17], and a lack of maturation for metabolic activation in assessed using a reanalysis of a previous collaborative juvenile rats [18]. 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 + [2]+ [2] + kid, lun, vsc, tes [22] N-Nitrosopyrrolidine NPYR 930–55-2 + [2]+ [2] + kid, vsc, tes [22, 23] 4,4′-Methylenedianiline MDA 101–77-9 + [2]+ [2] + thy [24] N-Nitrosodipropylamine NDPA 621–64-7 + [2] ND + eso, nas [22] 2,4-Dinitrotoluene 2,4-DNT 121–14-2 + [2]+ [2] + ski, mgl [22] 2,6-Dinitrotoluene 2,6-DNT 606–20-2 + [2]+ [2]+ – [22] Quinoline QUN 91–22-5 + [2]+ [2]+ – [25] p-Dimethylaminoazobenzene DAB 60–11-7 + [2]+ [2]+ – [22] 2-Nitropropane 2-NP 79–46-9 + [2]+ [2]+ – [26] Monocrotaline MCT 315–22-0 + [2]+ [2]+ – [22] N-Nitrosomorpholine NMOR 59–89-2 + [2] ND + vsc [22] 2-Acetylaminofluorene 2-AAF 53–96-3 + [2]+ [2] + ski, mgl [22] Sudan I (C.I.solvent yellow 14) Sudan I 842–07-9 + [21]ND + – [22] Thioacetamide TAA 62–55-5 + [21]+ [21]+ – [22] Group B Mitomycin C MMC 50–07-7 + [2]+ [2] – per [22] Cyclophosphamide H2O CP 6055-19-2 – [2]ND – ub, lym, ner [27] Potassium bromate KBrO3 7758-01-2 – [2] – [2] – kid, per, thy [22] N-Methyl-N′-nitro-N-nitrosoguanidine MNNG 70–25-7 – [2] – [2] – eso, smi, sto [22] Methyl methanesulfonate MMS 66–27-3 + [2] – [2] – hmo, lun, ner [22, 26] Kojic acid KA 501–30-4 – [2] – [2] – thy (mouse) [22] 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 [45]. 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 [20], 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 [2]. 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 [50]. 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. [2]. 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. 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Genes and EnvironmentSpringer Journals

Published: Jan 4, 2022

Keywords: Micronucleus assay; Liver; Hepatocarcinogen; Histopathology; Early responses

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