title: Optimization of specimen preparation from formalin-fixed liver tissues for liver micronucleus assays: Hepatocyte staining with fluorescent dyes.
abstract: The liver micronucleus (MN) assay is an effective and important in vivo test for detecting genotoxic compounds, particularly those that require metabolic activation. For this assay, hepatocytes (HEPs) can be isolated by collagenase treatment but without requirement for in situ liver perfusion. Consequently, the liver MN assay can be integrated into a general repeated-dose (RD) toxicity study. The method is also applicable to liver MN assays involving partial hepatectomy or the use of juvenile rats. Here, we propose an improved method for staining HEPs prepared from formalin-fixed liver tissues for MN assays, without collagenase treatment. HEP suspensions are prepared by treating the tissues with concentrated KOH and a fluorescent dye, SYBR(®) Gold (SYGO), is used for staining. Visualization of the MN in SYGO-stained HEPs is clearer than with Wright-Giemsa staining. We compared the induction of MN as measured with our new method versus the conventional method using collagenase dispersion. Our method not only enables the integration of the liver MN assay into a general RD toxicity study but also allows it to be conducted retrospectively.
authors.affiliation: LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan. Electronic address: shigano.miyuki@ma.medience.co.jp.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Shigano
authors.affiliation: Tsukubamirai-shi, Ibaraki 300-2358, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Takashima
authors.affiliation: LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan.
authors.forename: Hironao
authors.initials: H
authors.lastname: Takasawa
authors.affiliation: LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hamada
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D001399:Azure Stains; D004052:Diethylnitrosamine / Q000633:toxicity; D005404:Fixatives / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D022781:Hepatocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D015162:Micronucleus Tests / Q000379:methods*; D008856:Microscopy, Fluorescence; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D016707:Tissue Fixation / Q000379:methods*; D018675:Toxicity Tests / Q000379:methods
chemical_list: D000477:Alkylating Agents; D001399:Azure Stains; D005404:Fixatives; D005456:Fluorescent Dyes; D005557:Formaldehyde; D004052:Diethylnitrosamine
keywords: Collagenase; Fluorescent dye; Formalin-fixed method; Hepatocyte; Liver micronucleus assay