Sub-Lethal Concentrations of Graphene Oxide Trigger Acute-Phase Response and Impairment of Phase-I Xenobiotic Metabolism in Upcyte® Hepatocytes

Abstract

The impact of graphene oxide on hepatic functional cells represents a crucial evaluation step for its potential application in nanomedicine. Primary human hepatocytes are the gold standard for studying drug toxicity and metabolism; however, current technical limitations may slow down the large-scale diffusion of this cellular tool for in vitro investigations. To assess the potential hepatotoxicity of graphene oxide, we propose an alternative cell model, the second-generation upcyte® hepatocytes, which show metabolic and functional profiles akin to primary human hepatocytes. Cells were acutely exposed to sub-lethal concentrations of graphene oxide (≤80 μg/ml) for 24 h and stress-related cell responses (such as apoptosis, oxidative stress, and inflammatory response) were evaluated, along with a broad investigation of graphene oxide impact on specialized hepatic functions. Results show a mild activation of early apoptosis but not oxidative stress or inflammatory response in our cell model. Notably, while graphene oxide clearly impacted phase-I drug-metabolism enzymes (e.g., CYP3A4, CYP2C9) through the inhibition of gene expression and metabolic activity, conversely, no effect was observed for phase-II enzyme GST and phase-III efflux transporter ABCG2. The GO-induced impairment of CYP3A4 occurs concomitantly with the activation of an early acute-phase response, characterized by altered levels of gene expression and protein production of relevant acute-phase proteins (i.e., CRP, Albumin, TFR, TTR). These data suggest that graphene oxide induces an acute phase response, which is in line with recent in vivo findings. In conclusion, upcyte® hepatocytes appear a reliable in vitro model for assessing nanomaterial-induced hepatotoxicity, specifically showing that sub-lethal doses of graphene oxide have a negative impact on the specialized hepatic functions of these cells. The impairment of the cytochrome P450 system, along with the activation of an acute-phase response, may suggest potential detrimental consequences for human health, as altered detoxification from xenobiotics and drugs.