Optimization of specimen preparation from formalin-fixed liver tissues for liver micronucleus assays: Hepatocyte staining with fluorescent dyes

Shigano, Miyuki; Takashima, Rie; Takasawa, Hironao; Hamada, Shuichi

doi:10.1016/j.mrgentox.2016.03.004

Cited by 10 publications

(11 citation statements)

References 16 publications

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“…However, there currently is no direct evidence of the mechanism of chemical carcinogenesis originating from MN formation, since few carcinogens have genotoxic potential that manifests only as CAs. In addition, animals have lower tolerance for long periods of aneugen administration (Shigano et al 2016) and detection sensitivity of MNs is lower in chemicaltreated animal models. Since in vivo and in vitro cancer cells obtained from human cancers are already transformed (Zhang et al 2018), animal models are essential for rigorous investigation of the involvement of CAs in carcinogenesis including tumor initiation.…”

Section: Discussionmentioning

confidence: 99%

“…For each sample, around 2000 parenchymal HEPs were analyzed with a magnification of ≥ 200 ×. The number of micronucleated HEPs (MNHEPs) was recorded as previously reported (Shigano et al 2016;Hamada et al 2019). In the current study, large micronucleated HEPs (LMNHEPs) were defined as hepatocytes with MN that showed diameters 1/2 to 1/4 that of primary nuclei.…”

Section: Liver Micronucleus Assaymentioning

confidence: 99%

“…Indeed, an in vivo MN test, a common tool for detecting the clastogenic 1 3 or aneugenic potential of chemicals, can successfully detect MN formation in target organs of carcinogenic agents, such as skin, lung, gastrointestinal tracts and liver (Uno et al 2015a;Hamada et al 2019). Notably, recent studies reported that the MN assay in liver has high sensitivity and specificity for detecting hepatocarcinogenicity of both genotoxic and nongenotoxic carcinogens (Shigano et al 2016;Hamada et al 2019), suggesting that the CAs underlie the mechanisms of various chemical-induced hepatocarcinogenesis. However, the involvement of CAs via MN formation in chemical carcinogenesis remains to be fully elucidated, largely because examination of the characteristics of MN in animal models has been more limited.…”

Section: Introductionmentioning

confidence: 99%

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Chromosome aberrations induced by the non-mutagenic carcinogen acetamide involve in rat hepatocarcinogenesis through micronucleus formation in hepatocytes

et al. 2021

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Chromosome aberrations (CAs), i.e. changes in chromosome number or structure, are known to cause chromosome rearrangements and subsequently tumorigenesis. However, the involvement of CAs in chemical-induced carcinogenesis is unclear. In the current study, we aimed to clarify the possible involvement of CAs in chemical carcinogenesis using a rat model with the non-mutagenic hepatocarcinogen acetamide. In an in vivo micronucleus (MN) test, acetamide was revealed to induce CAs specifically in rat liver at carcinogenic doses. Acetamide also induced centromere-positive large MN (LMN) in hepatocytes. Immunohistochemical and electron microscopic analyses of the LMN, which can be histopathologically detected as basophilic cytoplasmic inclusion, revealed abnormal expression of nuclear envelope proteins, increased heterochromatinization, and massive DNA damage. These molecular pathological features in LMN progressed with acetamide exposure in a timedependent manner, implying that LMN formation can lead to chromosome rearrangements. Overall, these data suggested that CAs induced by acetamide play a pivotal role in acetamide-induced hepatocarcinogenesis in rats and that CAs can cause chemical carcinogenesis in animals via MN formation.

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Section: Discussionmentioning

confidence: 99%

Section: Liver Micronucleus Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Chromosome aberrations induced by the non-mutagenic carcinogen acetamide involve in rat hepatocarcinogenesis through micronucleus formation in hepatocytes

et al. 2021

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“…HEP-specimens were prepared from the formalin-fixed liver tissues with a slightly modified version of the previously reported method [16, 19]. In brief, a small portion of the fixed-liver tissue was cut into approximately 3 mm-cubes with a razor and thoroughly washed with water.…”

Section: Methodsmentioning

confidence: 99%

“…The advantages of liver micronucleus assay are made more obvious when it is incorporated into general toxicity studies. In a recently improved formalin-fixed method, procedures to prepare samples for liver micronucleus assays have been provided [19]. Because this method enables retrospective evaluation of micronucleus induction in the formalin fixed liver tissues obtained in general toxicity studies completed in the past, clastogenic potential of chemicals from the materials obtained in the general toxicity studies are able to be easily evaluated.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of the novel liver micronucleus assay using formalin-fixed tissues

Hamada¹,

Shigano²,

Kawakami

et al. 2019

Genes and Environ

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Background The repeated-dose liver micronucleus (RDLMN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly for those that require metabolic activation to show genotoxicity. In a collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) – Mammalian Mutagenicity Study Group (MMS), micronucleus induction of 22 chemicals with the RDLMN assay employing the collagenase digestion method was examined and reported on. Recently, we have developed a method which enables retrospective evaluation of micronucleus induction in formalin-fixed liver tissues (the formalin-fixed method) obtained in general toxicity studies completed in the past. Using this method, we were able to easily evaluate clastogenic potential of chemicals from the formalin-fixed tissues obtained in the general toxicity studies. In this study, to evaluate the usefulness of the formalin-fixed method, we have conducted a liver micronucleus assay using the formalin-fixed liver samples obtained from the above collaborative study (18 of 22 test chemicals) and carried out a comparison with the results obtained by the collagenase digestion method. Results Comparison of the collagenase digestion and formalin-fixed methods was conducted using the results of the micronucleus assays with a total of 18 test chemicals which included 12 genotoxic hepatocarcinogens (Group A), 4 genotoxic carcinogens but not liver targeted (Group B), and 2 nongenotoxic hepatocarcinogens (Group C). The formalin-fixed method obtained the similar results as the collagenase digestion method in 10 out of the 12 chemicals of Group A, and all chemicals of Group B and Group C. Although the results were statistically contradictive due to different levels of concurrent negative control, the 2 other chemicals of Group A showed comparable responses between the two methods. Conclusion The present study shows that the formalin-fixed method is capable of detecting liver carcinogens with sensitivity equal to or higher than that of the collagenase digestion method. We recommend use of the formalin-fixed method because of its capability of enabling retrospective evaluation of micronucleus induction in the formalin-fixed liver tissues obtained in general toxicity studies completed in the past.

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In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT)

Kirkland¹,

Uno²,

Luijten

et al. 2019

Mutation Research/Genetic Toxicology and Environmental Mutagene

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Optimization of specimen preparation from formalin-fixed liver tissues for liver micronucleus assays: Hepatocyte staining with fluorescent dyes

Cited by 10 publications

References 16 publications

Chromosome aberrations induced by the non-mutagenic carcinogen acetamide involve in rat hepatocarcinogenesis through micronucleus formation in hepatocytes

Chromosome aberrations induced by the non-mutagenic carcinogen acetamide involve in rat hepatocarcinogenesis through micronucleus formation in hepatocytes

Evaluation of the novel liver micronucleus assay using formalin-fixed tissues

In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT)

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