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Flow cytometric method for scoring rat liver micronuclei with simultaneous assessments of hepatocyte proliferation
Author(s) -
Avlasevich Svetlana L.,
Khanal Sumee,
Singh Priyanka,
Torous Dorothea K.,
Bemis Jeffrey C.,
Dertinger Stephen D.
Publication year - 2018
Publication title -
environmental and molecular mutagenesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 87
eISSN - 1098-2280
pISSN - 0893-6692
DOI - 10.1002/em.22168
Subject(s) - micronucleus test , flow cytometry , hepatocyte , reticulocyte , micronucleus , biology , cell growth , andrology , genotoxicity , toxicity , pathology , medicine , microbiology and biotechnology , biochemistry , in vitro , messenger rna , gene
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 2 = 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.