There is an increasing demand for toxicity testing of chemicals with animal alternatives using new approach methods (NAMs). At present, there are many established in vitro tests using both primary and continuous cell cultures. However, few are suitable for evaluating chemical long-term toxicity. A recent advancement using a more complex three-dimensional (3D) spheroid model, derived from rainbow trout primary hepatocytes (RT-HEP), has shown potential as it retains physiological properties over an extended period of time.
To determine its suitability within toxicity testing, an extensive characterization of the RT-3D hepatic cell culture system is required. For this, high-resolution multiphoton fluorescence microscopy (MFM) was used to examine various parameters like morphology, DNA integrity, and inner-core hypoxia. Additionally, comprehensive characterization utilizing omics techniques and various bioassays was conducted to assess RT hepatic 3D spheroids' response to toxins.
The assessment involved exposure to model chemicals representing different modes of toxicity (MoT), such as copper, 17 β-ethinyl estradiol (EE2), pyrene, 3,4-dichloroaniline (DCA), and carbonyl cyanide m-chlorophenyl hydrazone (CCCP). One of the aims of this study was to assess the RT-3D model’s sensitivity, responsiveness, and reproducibility to the above-mentioned toxins by measuring endpoints such as viability, metabolic activity, ATP synthesis, vitellogenin (Vg) induction, and oxidative stress and comparing it to in vitro literature. Furthermore, transcriptomics (RNA-seq) and untargeted metabolomics (mass spectrometry-based) approaches were employed to unravel the molecular landscape of the 3D spheroids when exposed to chemicals with diverse MoT.
Preliminary results indicate the reliability ofRT hepatic 3D spheroids in evaluating cytotoxicity, cell membrane integrity, and oxidative stress. Additionally, the absence of hypoxia at the spheroid core was confirmed. These ongoing characterizations display the potential use of RT hepatic 3D spheroids as an ecotoxicity screening tool for screening a variety of chemicals with different toxic MoT.
