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

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.