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.

Ginsenoside Rg3 Reduces the Toxicity of Graphene Oxide Used for pH-Responsive Delivery of Doxorubicin to Liver and Breast Cancer Cells

“…CYP1A1 and CYP39A1 genes encode members of the cytochrome P450 superfamily of enzymes, which catalyze reactions involved in drug metabolism. It was previously shown that GO interferes with drug metabolism/detoxification in the body at the level of phase I cytochrome-P450 system by the inhibition of gene expression and metabolic activity [ 64 ]. This influence of GO is clinically relevant, since altered drug metabolism can significantly contribute to the variability of drug responses and be associated with an increased risk of adverse effects along with altered detoxification.…”

Environmental and Health Impacts of Graphene and Other Two-Dimensional Materials: A Graphene Flagship Perspective

“…In an alternative cell model to primary human hepatocytes, GO (with average size of 360 nm) at 80 μg/mL for 24 h was found to activate early apoptosis but not oxidative stress or inflammation. 312 In addition, this GO material impaired cytochrome P450 phase-I drug metabolism enzymes, but it had no effect on phase-II enzyme GST or phase-III efflux transporter ABCG2. Phase-I drug metabolism enzyme alteration was associated with the alteration of gene expression and protein levels for several acute-phase proteins.…”

“…GO also stimulated protein expression of αSMA, a biomarker of fibrosis via modulation of the TGFβ pathway. In an alternative cell model to primary human hepatocytes, GO (with average size of 360 nm) at 80 μg/mL for 24 h was found to activate early apoptosis but not oxidative stress or inflammation . In addition, this GO material impaired cytochrome P450 phase-I drug metabolism enzymes, but it had no effect on phase-II enzyme GST or phase-III efflux transporter ABCG2.…”

Polycarbonate nanoplastics and the in vitro assessment of their toxicological impact on liver functionality

“…Second-generation UHHs (derived from the donor 653–03; upcyte® Technologies GmbH, Hamburg, DE) were cultured as previously reported by Romaldini et al 44 After thawing, cells were expanded for 2 passages in collagen-coated tissue culture flasks (VWR, Radnor, PA, USA) and, at passage 3 (P3), were treated with the PC-NP dispersions in collagen-coated flat bottom 24- or 96-well plates (Corning Incorporated, Corning, NY, USA) as described below. At P3, the number of viable cells was higher than 92.9%, as revealed by the trypan blue (Sigma Aldrich-Merck KGaA, Darmstadt, DE) exclusion test.…”