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Abstract

The determination of genotoxicity is an important element in the safety assessment of various substances, with the purpose of preventing a number of chemicals from affecting human health. Genotoxicity testing is required for all classes of chemicals, drugs and biological agents, which can lead to a wide range of diseases, cancer included. In the last decades, there has been an ongoing shift towards developing new effective testing methods, since a single test is not sufficient for the detection of all relevant genotoxic aspects; consequently, a variety of complementary testing techniques and methodologies have to be used. In addition, increasing emphasis is given to alternative in vitro models, which focus on the genotoxic effects of chemicals and environmental contaminants, thereby contributing to the reduction of animals used in preclinical testing.
In toxicology, hepatocellular two-dimensional (2D) cell models are being conventionally used for determining the damaging effects of chemicals in vitro. Nevertheless, there is a demand for new approaches as currently existing models often yield misleading results because they lack expressions of important metabolic enzymes. In this respect, newly developed in vitro three-dimensional (3D) cell-based models are gaining importance as they more realistically imitate the in vivo cell behavior. In the in vitro conditions 3D models give more accurate results compared to 2D cultures; as such, they offer an attractive alternative to animal testing. Even though 3D cell models are better than 2D cell models, they lack standardization, in particular in terms of cultivation protocols and adequate characterization, which prevent their general use in the field of genotoxcicity.
This thesis aims to validate and optimize an approach for testing the genotoxic activity of chemicals on a hepatocellular in vitro 3D cell model (spheroid) developed from a human hepatocellular carcinoma (HepG2 and HepG2/C3A) cell line by the forced floating method and cultured in a dynamic bioreactor (CelVivo BAM/bioreactor) system. We showed that the newly developed 3D cell models better illustrate in vivo conditions than traditional monolayer cell cultures, since they have improved cell-matrix and cell-cell interactions, as well as preserved in vivo cell phenotypes. Moreover, we showed decreased proliferation over the cultivation period and a higher expression of liver-specific functions and genes encoding phase I and II metabolic enzymes in 3D models compared to 2D models.
In the present study, we applied novel hepatocellular in vitro 3D models cultured under static and dynamic conditions for the assessment of cytotoxicity and genotoxicity of xenobiotic compounds. Compared to 2D cell models the applied in vitro 3D cell models showed increased stability and viability, thus enabling long-term exposures, which is particularly important in studying genotoxic compounds at lower concentrations, to which humans are exposed in everyday life. Moreover, transcriptomic analyses revealed that 3D cell models express genes related to metabolism and characteristic of hepatic cells to a higher extent than 2D models, showing a higher sensitivity to the detection of indirect-acting genotoxic compounds.
We believe that the newly developed hepatocellular 3D cell models, due to their more complex structure and improved metabolic capacity, provide a suitable experimental model for genotoxicity studies as well as the regulatory testing of new chemicals and products.
Despite that, 3D cell models must be further characterized and validated in terms of cell division and response to genotoxic stress in order to better know their behavior and properties. The 3D models have the potential to bridge the gap between in vitro and animal studies.