Evaluation of functional candidate biomarkers of non-genotoxic hepatocarcinogenicity in human liver spheroid co-cultures

Validated in vitro assays for testing non-genotoxic carcinogenic potential of chemicals are currently not available. Consequently, the two-year rodent bioassay remains the gold standard method for the identification of these chemicals. Transcriptomic and proteomic analyses have provided a comprehensive understanding of the non-genotoxic carcinogenic processes, however, functional changes induced by effects at transcriptional and translational levels have not been addressed. The present study was set up to test a number of proposed in vitro biomarkers of non-genotoxic hepatocarcinogenicity at the functional level using a translational 3-dimensional model. Spheroid cultures of human hepatocytes and stellate cells were exposed to 5 genotoxic carcinogenic, 5 non-genotoxic carcinogenic, and 5 non-carcinogenic chemical compounds and assessed for oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, apoptosis, and inflammation. The spheroid model could capture many of these events triggered by the genotoxic carcinogenic chemicals, particularly aflatoxin B1 and hydroquinone. Nonetheless, no clear distinction could be made between genotoxic and non-genotoxic hepatocarcinogenicity. Therefore, spheroid cultures of human liver cells may be appropriate in vitro tools for mechanistic investigation of chemical-induced hepatocarcinogenicity, however, these mechanisms and their read-outs do not seem to be eligible biomarkers for detecting non-genotoxic carcinogenic chemicals.

Automated summary

Currently, there are no validated in vitro assays for testing the non-genotoxic carcinogenic potential of chemicals. Therefore, the two-year rodent bioassay remains the standard method for identifying these chemicals. Transcriptomic and proteomic analyses have provided a comprehensive understanding of non-genotoxic carcinogenesis, but functional changes at the transcriptional and translational levels have not been addressed. This study tested proposed in vitro biomarkers of non-genotoxic hepatocarcinogenicity using a 3-dimensional model, and found that spheroid cultures of human liver cells may be suitable tools for investigating chemical-induced hepatocarcinogenicity, but the mechanisms and read-outs studied are not eligible biomarkers for detecting non-genotoxic carcinogens.