Chimeric mice with human hepatocytes: A new system for genotoxicity studies

Highlights

• Chimeric mice with human hepatocytes can be a model material for genotoxicity assays.

• Chimeric mice with humanized livers can be a model material for genotoxicity assays.

• Chimeric mice with humanized livers can be an animal model for genotoxicity assays.

Abstract

Genotoxicity assays are characterized by a method, an in vitro or in vivo target, and an endpoint. Many cell types have been used as targets, including bacterial cells, cultured mammalian cells, and rodent cells in vivo. Human cells are the most important target for evaluating the risk to humans associated with exposure to chemicals. Almost exclusively, the human cells used in genotoxicity tests have been cultured cells. Here, we have tested human hepatocytes in PXB-mice^®, chimeric mice in which the liver has been repopulated with human hepatocytes, as a source of target cells for in vivo genotoxicity assays. We applied the single-cell gel electrophoresis (comet) assay to detect DNA damage and the micronucleus assay to evaluate chromosomal aberrations. These chimeric mice can serve as a valuable model system for genotoxicity assays.

Introduction

Established human cell lines have been widely employed to evaluate the genotoxicity of chemicals in vitro. Although the use of primary human cells for chemical safety evaluation is restricted, primary cultures of human peripheral blood lymphocytes are frequently used in chromosomal aberration assays to evaluate the clastogenicity of chemicals [1]. Human cells also serve as the starting material for microsomal fractions and as an exogenous metabolic activation system (S9 fraction) for bacterial gene mutation assays and in vitro genotoxicity assays [2]. In the latter case, the cells are intended to reproduce human metabolism of pro-mutagens.

The humanized chimeric mouse was developed and established as a model for evaluation of metabolism and other features of human hepatocytes in the environment of the mammalian body [[3], [4], [5], [6], [7]]. Chimeric mice with human hepatocytes have been tested in many fields, including pharmacology and toxicology, especially to evaluate the human metabolism of chemicals when human liver models are needed. These mice provide a way to use human hepatocytes as an in vivo target that maintains their original characteristics. Technical protocols to establish a chimeric mouse with human hepatocytes have been published, and stable mass production has been reported [8,9]. In fact, we previously succeeded in producing chimeric mice with the humanized liver from albumin enhancer/promoter-urokinase-type plasminogen activator-transgenic/SCID (uPA/SCID) mice (PXB-mice^®) [3]. Examination of the mRNA expression levels of ˜82% of the genes in the human hepatocytes from the chimeric mice yielded results similar to, or within a 2-fold difference from, levels in the human liver [8]. In addition, the protein expression levels of cytochromes P450 (CYPs) and transporters in the livers of the chimeric mice were similar to, or within a 4-fold difference of, levels in the human liver [10]. Nonetheless, chimeric uPA/SCID mice present some disadvantages, because the human hepatocyte replacement index (RI) in the mouse liver is decreased as a result of deleted transgenes and the animals’ small body size. To overcome these disadvantages, we recently developed a new type of host mouse: the albumin enhancer/promoter-urokinase-type plasminogen activator-cDNA transgenic/SCID (cDNA-uPA/SCID) mouse [7]. To construct uPA/SCID mice, five genomic murine uPA genes were introduced in tandem; whereas to generate cDNA-uPA/SCID mice, only a single copy of the murine uPA-cDNA was introduced. Chimeric mice generated from the cDNA-uPA/SCID mice have better characteristics relative to the chimeric mice generated from uPA/SCID mice, including increased body weight and higher stable-repopulation rates of human hepatocytes [7]. In the present study, we generated chimeric mice from both the original host uPA/SCID mice and this new type of host, cDNA-uPA/SCID mice.

Genotoxicity assays are regularly performed during safety evaluations of many types of chemicals, such as pharmaceutical drugs, agricultural chemicals, food additives and related compounds, industrial chemicals, etc. The target materials used in genotoxicity assays include bacterial cells [11], cultured mammalian cells [1], and rodents [12,13]. Some chemicals require metabolic activation before they interact with DNA; to test this property, different exogenous metabolic activation systems are employed. The standard system is the rodent liver S9 fraction. Although S9 fraction prepared from human materials recently became available [2], it is not yet widely used. Application of human cells as a target for risk assessment would be a major breakthrough, and the proposed chimeric-mouse system may enable a more accurate assessment of human risk. In this study, we evaluated these chimeric mice as a tool for the genotoxicity assessment of representative genotoxic chemicals.

We aimed to evaluate the validity of a chimeric mouse with human hepatocytes in its liver, for mimicking the actual human liver. Nevertheless, some experiments did not fulfill the conditions required by the test guidelines; for example, a small number of animals only were used here. Further experiments are needed, involving chemicals that are activated more effectively by human than by rodent liver cells, to test the possible advantages of this chimeric-mouse model.