Cytochrome P450 genes expression in HepG2 cells after treatment with nanostructures of different allotropic forms of carbon

Introduction Carbon nanomaterials, such as diamond nanoparticles (DN), graphene oxide (GO) or graphite (GR) nanoplatelets have been recently studied for several biomedical applications, what results from their unique... [ view full abstract ]

Introduction

Carbon nanomaterials, such as diamond nanoparticles (DN), graphene oxide (GO) or graphite (GR) nanoplatelets have been recently studied for several biomedical applications, what results from their unique properties at nanoscale and high biocompatibility. The safety of their application depends not only on the lack of systemic toxic effects, but also on the possibility of metabolism alteration of co-administrated xenobiotics. The mechanism of carbon nanoparticles metabolism within liver, where nanoparticles are relocated after in vivo administration, is also still unknown. Thereby, effects of DN, GO and GR on expression of genes responsible mainly for drug metabolism in liver were studied on HepG2 in vitro model of liver cells.

Methods

Morphology of DN, GO and GR was assessed using transmission electron microscopy. Cytotoxic effect of DN, GO and GR against HepG2 cells was determined by MTT viability assay in doses ranging from 3,125 to 100 μg/mL. Gene expression on mRNA level was quantified using real-time PCR with ddC_T relative method, using β-actin as a calibrator. Following genes were studied: AhR, CAR, PXR receptors and CYP1A2, CYP2B6, CYP2D6, CYP2E1, CYP3A4 cytochrome P450 genes.

Results

DN did not affect the HepG2 cells viability, whereas GO and GR induced cytotoxic effect in dose-dependent manner. All used nanomaterials decreased expression of genes for receptors CAR, AhR and PXR, as well as for CYP2D6 and CYP3A4, inducing at the same time over-expression of CYP1A2 and CYP2B6. CYP2E1 expression increased in DN and GR groups, and decreased in GO group.

Discussion

Obtained results confirmed that out of the three examined nanostructures, DN are characterized by the highest biocompatibility. It is in accordance with the previous results, where toxicity of DN, GO and GR were studied on rat model during the 12-week long experiment. Alterations of indices such as glutathione and MDA levels in rat livers suggested relocation of nanoparticles into the liver. Analysis of gene expression for cytochrome P450 enzymes and regulatory receptors on in vitro liver model showed that all three carbon materials induced similar response, what may be first step toward understanding the mechanism of carbon nanoparticles metabolism.