The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part I: Isolation, structural, genetic, and biochemical characterization
- DOI
- Language of the publication
- English
- Date
- 2018-12-27
- Type
- Article
- Author(s)
- Cox, Julie A.
- Zwart, Edwin P.
- Luijten, Mirjam
- White, Paul A.
- Publisher
- Wiley
Abstract
To develop an improved in vitro mammalian cell gene mutation assay, it is imperative to address the known deficiencies associated with existing assays. Primary hepatocytes isolated from the MutaMouse are ideal for an in vitro gene mutation assay due to their metabolic competence, their "normal" karyotype (i.e., neither transformed nor immortalized), and the presence of the MutaMouse transgene for rapid and reliable mutation scoring. The cells were extensively characterized to confirm their utility. Freshly isolated cells were found to have a hepatocyte-like morphology, predominantly consisting of binucleated cells. These cells maintain hepatocyte-specific markers for up to 3 days in culture. Analyses revealed a normal murine hepatocyte karyotype with a modal ploidy number of 4n. Fluorescence in situ hybridization analysis confirmed the presence of the lambda shuttle vector on chromosome 3. The doubling time was determined to be 22.5 ± 3.3 h. Gene expression and enzymatic activity of key Phase I and Phase II metabolic enzymes were maintained for at least 8 and 24 h in culture, respectively. Exposure to β-naphthoflavone led to approximately 900- and 9-fold increases in Cyp1a1 and Cyp1a2 gene expression, respectively, and approximately twofold induction in cytochrome P450 (CYP) 1A1/1A2 activity. Exposure to phenobarbital resulted in an approximately twofold increase in CYP 2B6 enzyme activity. Following this characterization, it is evident that MutaMouse primary hepatocytes have considerable promise for in vitro mutagenicity assessment. The performance of these cells in an in vitro gene mutation assay is assessed in Part II. Environ. Mol. Mutagen. 60:331-347, 2019.
Plain language summary
Health Canada is responsible for evaluating the safety of chemicals in commerce; all evaluations include an assessment of a substance’s ability to damage genetic material (i.e., genetic toxicity). Genetic toxicity tests using cultured cells (i.e., in vitro tests) provide a cost- and time-effective alternative to more resource-intensive animal tests. Moreover, in response to the worldwide trend to reduce animal testing, there is an acute need to develop novel, robust in vitro genetic toxicity tests. Currently available in vitro genetic toxicity assays are not always reliable. This is, in part, due to their limited ability to simulate mammalian metabolism, which is often critical for accurate assessment of chemical safety. This project involved the development of a novel, metabolically-competent in vitro genetic toxicity assay based on a well-validated mutation scoring system. More specifically, a gene mutation test was developed that employs liver cells (i.e., hepatocytes) isolated from the transgenic MutaMouse, a mouse strain wherein chemically-induced mutations can be easily quantified. The hepatocytes were grown in the laboratory and characterized to fully assess their utility for routine detection of chemically-induced mutations. Advanced microscopy was used to examine growth and morphological changes of the cultured hepatocytes. Further analysis showed that the cells are genetically stable. The isolated hepatocytes grow at a rate that is relevant to mutation scoring, and enzymes involved in mammalian metabolism were present and active. The results show that MutaMouse hepatocytes are well suited for in vitro assessment of genetic toxicity. The isolated hepatocytes behave like normal liver cells, are capable of metabolizing chemicals, and offer the reliable MutaMouse mutation scoring system. A companion manuscript employed a set of test chemicals to evaluate the performance of the assay.
Subject
- Health,
- Health and safety