Development of a co-culture model of mouse primary hepatocytes and splenocytes to evaluate xenobiotic genotoxicity using the medium-throughput Comet assay

To date, only a limited number of toxicological studies have focused on the establishment and validation of in vitro genotoxicity screening systems using primary hepatocytes, and the results of these studies have been inconsistent. Therefore, the aim of this study was to develop an effective co-culture model of mouse-derived primary hepatocytes and splenocytes for screening chemicals for genotoxicity using the medium-throughput Comet assay. This cocultured model was constructed and verified using known genotoxic and non-genotoxic compounds as positive and negative controls, respectively. Cytotoxicity was measured using Cell Counting Kit-8 and lactate dehydrogenase methods. DNA damage was detected using both alkaline and formamidopyrimidine DNA glycosylase (FPG) Comet assays. Compared with the controls, DNA strand breaks and FPG-sensitive sites showed significant concentration-dependent increases in genotoxic-agent-treated groups. In contrast, DNA damage remained unchanged in non-genotoxic-agent-treated groups. In addition, different types of genotoxic agents resulted in different time-dependent DNA lesions. Our results indicated that the % tail DNA indicating both DNA strand breaks and FPG-sensitive sites might be effective markers for predicting chemical-induced DNA damage and oxidative DNA damage using the cocultured model of hepatocytes and splenocytes. Collectively, these findings provide reliable experimental data for the establishment of in vitro genotoxicity screening methods.