Exploring the use of spheroid cultures of human liver cells for the mechanistic testing of carcinogenic compounds

Background and objectives
Before any new chemical can be placed on the market, its carcinogenic potential, along with many other toxicological endpoints, must be thoroughly investigated. However, the current carcinogenicity assessment has limitations in predicting the potential risk associated with non-genotoxic carcinogenic compounds. As a result, new predictive models for more efficient and reliable hazard identification are urgently needed. Spheroid cultures of liver cells have been considered promising models in this regard.

Material and methods
Spheroid cultures consisting of human hepatocyte C3A cells and human liver stellate LX-2 cells in ratio of 10:1 were set up. The spheroid cultures were exposed to 4 different concentrations of genotoxic liver carcinogenic compounds (benzo[a]pyrene, hydroquinone and aflatoxin B1), non-genotoxic liver carcinogenic compounds (dioctyl phthalate, acetamide and methapyrilene) and non-carcinogenic compounds (D-mannitol, tolbutamide and clonidine) for 72 hours. The spheroid cultures were monitored for oxidative stress using 2′,7′- dichlorodihydrofluorescein diacetate (Incucyte and flow cytometry), mitochondrial dysfunction using Rhodamine 123 (Incucyte and flow cytometry), endoplasmic reticulum stress using Thioflavin T (plate reader and flow cytometry), apoptosis (caspase 3/7 activity) and inflammatory cytokines (enzyme-linked immunosorbent assay).

Results
Hydroquinone and aflatoxin B1 consistently affected the spheroid cultures through increased production of reactive oxygen species, induced depolarization of mitochondrial membrane potential, increased endoplasmic reticulum stress and increased caspase-3/7 activity. No effects induced by other compounds in any of the parameters were observed.

Discussion and conclusion
There are no in vitro tests validated by the regulatory authorities for identifying the non-genotoxic carcinogenic compounds. The C3A–LX-2 spheroid model could be further explored for this purpose. Therefore, the 3D spheroid cultures of liver cells could be valuable tools for mechanistic testing of carcinogenic effects.