Repeated dose liver micronucleus assay using adult mice with multiple genotoxicity assays concurrently performed as a combination test

Hagio, Soichiro; Furukawa, Satoshi; Abe, Masayoshi; Kuroda, Yusuke; Hayashi, Seigo; Ogawa, Izumi

doi:10.2131/jts.39.437

Cited by 7 publications

(8 citation statements)

References 27 publications

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“…Regarding assessments of assay specificity, presumed non‐genotoxicants that cause various types of liver injury need to be studied. It will also be important to consider different treatment/harvest schedules, especially those that facilitate combining multiple genotoxicity endpoints into one acute study, or else integrating MNHEP assessment into ongoing repeat‐dose toxicology studies, as these two strategies greatly reduce resource requirements and number of animals tested (Pfuhler et al, ; Hagio et al, ; Hamada et al, ). The availability of an automated scoring method provides greater efficiencies for all such investigations, especially in terms of high quality, objective data acquisition.…”

Section: Resultsmentioning

confidence: 99%

Flow cytometric method for scoring rat liver micronuclei with simultaneous assessments of hepatocyte proliferation

Avlasevich

Khanal

Singh

et al. 2018

Environ and Mol Mutagen

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The current report describes a newly devised method for automatically scoring the incidence of rat hepatocyte micronuclei (MNHEP) via flow cytometry, with concurrent assessments of hepatocyte proliferation-frequency of Ki-67-positive nuclei, and the proportion of polyploid nuclei. Proof-of-concept data are provided from experiments performed with 6-week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) or quinoline (QUIN) for 3 or 14 consecutive days. Non-perfused liver tissue was collected 4 days after cessation of treatment in the case of 3-day studies, or 1 day after last administration in the case of 14-day studies for processing and flow cytometric analysis. In addition to livers, blood samples were collected one day after final treatment for micronucleated reticulocyte (MN-RET) measurements. Dose-dependent increases in MNHEP, Ki-67-positive nuclei, and polyploidy were observed in 3- and 14-day DEN studies. Both treatment schedules resulted in elevated %MNHEP for QUIN-exposed rats, and while cell proliferation effects were subtle, appreciable increases to normalized liver weights were observed. Whereas DEN caused markedly higher %MNHEP when exposure was extended to two weeks, QUIN-induced MNHEP were slightly increased with protracted dosing. Parallel microscopy-based MNHEP frequencies were highly correlated with flow cytometry-based measurements (four study/aggregate R = 0.80). No increases in MN-RET were seen in any of the four studies. Collectively, these results suggest liver micronuclei are amenable to an automated scoring technique that provides objective analyses and higher information content relative to conventional microscopy. Additional work is needed to expand the number and types of chemicals tested, identify the most advantageous treatment schedules, and test the transferability of the method. Environ. Mol. Mutagen. 59:176-187, 2018. © 2018 Wiley Periodicals, Inc.

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Section: Resultsmentioning

confidence: 99%

Flow cytometric method for scoring rat liver micronuclei with simultaneous assessments of hepatocyte proliferation

Avlasevich

Khanal

Singh

et al. 2018

Environ and Mol Mutagen

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“…The mitotic index of HEPs is usually observed at very low levels in the juvenile rat [ 30 , 31 , 32 , 33 ] or in the RDLMN method [ 10 , 20 ]. Otherwise, Hagio et al [ 9 ] reported that HEP proliferation could be evaluated by detecting Phospho-Histone H3 antibody-positive cells, a marker of cell proliferation, in the RDLMN assay using adult mice with DEN treatments. Therefore, mitotic index would be not a good indicator for HEP proliferation, and another indicator might be useful for evaluation of cell proliferation in rodent liver.…”

Section: Discussionmentioning

confidence: 99%

“…The MN assay using the liver, a major target for carcinogenesis and a key organ for drug metabolism [ 9 ], is a useful method for detecting hepatocarcinogens and genotoxic chemicals that are mainly activated by hepatic metabolic enzymes, e.g., cytochrome P450 (CYP). Since cell proliferation for the production of micronuclei in the adult animal liver is time-consuming, liver MN assays have been developed in various aspects.…”

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confidence: 99%

“…Since cell proliferation for the production of micronuclei in the adult animal liver is time-consuming, liver MN assays have been developed in various aspects. These improvements include treatment with mitogens [ 5 ], performing partial hepatectomy (PH) [ 12 , 13 , 35 , 36 ] and using juvenile rats [ 29 , 30 , 31 , 32 , 33 ], young adult rats [ 10 , 20 , 21 , 34 ] or mice [ 9 ] with repeated treatments. Careful consideration should be given when using the co-treatment method with mitogens, since there may be interactions with the test chemicals.…”

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confidence: 99%

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Effectiveness of the liver micronucleus assay using juvenile mice

Nagasue

Murata

Sasaki

et al. 2017

The Journal of Veterinary Medical Science

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This study investigated the effectiveness of the liver micronucleus (MN) assay using juvenile mice. Therefore, we analyzed various hepatic cytochrome P450 (CYP)- mediated activities of ethoxyresorufin O-deethylation, pentoxyresorufin O-dealkylation, tolbutamide hydroxylation, bufuralol 1’-hydroxylation, aniline hydroxylation and midazolam 4-hydroxylation by CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A, respectively, in non-treated male ICR mice aged between 3 and 8 weeks. The enzyme efficiency levels in 3- and 4-week-old mice were approximately similar to or higher than those in 8-week-old mice, except for CYP1A and CYP2E in 3- and 4-week-old mice, respectively. Since these results suggest that juvenile mice have sufficient activities for most CYP enzymes, we also conducted a liver MN assay using diethylnitrosamine (DEN), a rodent hepatocarcinogen, on male ICR mice aged between 3 and 6 weeks. A peripheral blood (PB) MN assay was performed simultaneously in 4-week-old mice. Assays incorporating DEN produced positive results in 3- and 4-week-old mice and showed a dose-dependent increase in the micronucleated hepatocyte frequencies at 4 weeks. Both the liver MN assay in 5- and 6-week-old mice and the PB MN assay had negative results when using DEN. These results suggest that 3- and 4-week-old mice have micronuclei-inducing potential in the liver to detect genotoxic compounds using the liver MN assay.

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“…The in vivo comet assay was conducted according to a previously published method, with some modifications (Hagio et al, 2014). For the thyroid gland, one side of the excised thyroid gland was used for the in vivo comet assay.…”

Section: Comet Assay Methodsmentioning

confidence: 99%

Comet assay of thyroid gland in rats treated with ethyl methanesulfonate

Tsuji

Hagio

Takeuchi

et al. 2022

Fundam. Toxicol. Sci.

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In vivo comet assays are often used to evaluate genotoxicity. The advantage of an in vivo comet assay is that it can be applied to a variety of organs. In general, the liver and stomach are used in an in vivo comet assay. However, there have also been some reports on the in vivo comet assay of the thyroid gland, which is a common target organ for carcinogens in rodents. Ethyl methanesulfonate (EMS) is listed as a compound likely to be the positive control compound of choice in an in vivo comet assay, since it is known to cause DNA damage in a variety of tissues, including the liver, stomach, kidneys, and bone marrow. In contrast, there have been no reports on in vivo comet assays of the thyroid gland in rats treated with EMS. In the present study, we examined whether EMS also exhibits DNA damage in the in vivo comet assay of the thyroid gland in rats. EMS was administered orally at a dose of 200 mg/kg. The results showed a significant increase in mean % tail DNA in the thyroid gland, the extent of which was similar to that in the liver. Since histopathological examination of the thyroid gland revealed no histological findings, the increase in mean % tail DNA was most likely due to DNA damage rather than tissue damage. These results suggest that EMS can be used as a positive control compound in the in vivo comet assay of the thyroid gland in rats.

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Repeated dose liver micronucleus assay using adult mice with multiple genotoxicity assays concurrently performed as a combination test

Cited by 7 publications

References 27 publications

Flow cytometric method for scoring rat liver micronuclei with simultaneous assessments of hepatocyte proliferation

Flow cytometric method for scoring rat liver micronuclei with simultaneous assessments of hepatocyte proliferation

Effectiveness of the liver micronucleus assay using juvenile mice

Comet assay of thyroid gland in rats treated with ethyl methanesulfonate

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