Influence of Selected Carbon Nanostructures on the CYP2C9 Enzyme of the P450 Cytochrome

Abstract

Carbon nanostructures have recently gained significant interest from scientists due to their unique physicochemical properties and low toxicity. They can accumulate in the liver, which is the main expression site of cytochrome P450 (CYP450) enzymes. These enzymes play an important role in the metabolism of exogenous compounds, such as drugs and xenobiotics. Altered activity or expression of CYP450 enzymes may lead to adverse drug effects and toxicity. The objective of this study was to evaluate the influence of three carbon nanostructures on the activity and expression at the mRNA and protein levels of CYP2C9 isoenzyme from the CYP2C subfamily: Diamond nanoparticles, graphite nanoparticles, and graphene oxide platelets. The experiments were conducted using two in vitro models. A microsome model was used to assess the influence of the three-carbon nanostructures on the activity of the CYP2C9 isoenzyme. The CYP2C9 gene expression at the mRNA and protein levels was determined using a hepatoma-derived cell line HepG2. The experiments have shown that all examined nanostructures inhibit the enzymatic activity of the studied isoenzymes. Moreover, a decrease in the expression at the mRNA and protein levels was also observed. This indicates that despite low toxicity, the nanostructures can alter the enzymatic function of CYP450 enzymes, and the molecular pathways involved in their expression.The liver is the main organ involved in biotransformation and the main expression site of enzymes from the cytochrome P450 family (CYP450). These enzymes constitute a large and multifunctional protein family, involved in many reactions related to the metabolism of exogenous compounds like xenobiotics and carcinogens. They also play a crucial role in the inactivation and activation of drugs [9]. CYP450 are monooxygenases, and they catalyze the incorporation of the oxygen atom to the substrate molecule with the simultaneous formation of a water molecule [10][11][12]. There are two phases of the biotransformation of drugs and xenobiotics in the human body. CYP450 enzymes play an important role in the course of the first-phase reactions. In these reactions, the introduction or unveiling of the -OH, -COOH, -SH, or -NH 2 functional groups occurs, which in turn, leads to an increase in the hydrophilicity of the drug/xenobiotic and allows second-phase reactions and consequently, expulsion from the body [13].One of the most important CYP450 subfamilies is the CYP2C subfamily, which metabolizes about 20% of all clinically used drugs and several endogenous compounds. Four isoenzymes are distinguished within this subfamily: 2C8, 2C9, 2C18, and 2C19 [14][15][16]. Of all the isoenzymes from the CYP2C subfamily, the CYP2C9 isoform is the most strongly expressed. It represents 50% of the entire subfamily and metabolizes about 15% of clinically used drugs, for example, non-steroidal anti-inflammatory drugs and diuretics [17,18]. As CYP enzymes are involved in the metabolism of exogenous compounds, their dysfunction may lead to slower elimination of dr...

Effect of Graphene Family Materials on Multiple Myeloma and Non-Hodgkin’s Lymphoma Cell Lines

“…On the other hand, the results obtained from the ATP assay suggested a high toxic effect in all groups, starting from the lowest used concentration ( Figure 7 ). Interestingly, the highest cytotoxic effect was observed for GO, while oxidized forms of graphene are generally more biocompatible, and GO had a low toxic effect against other cell lines, such as liver-derived cells, in our previous studies [ 23 , 39 ]. Furthermore, natural graphene or reduced forms of graphene oxides exhibited not only a cytotoxic effect against other cancer cells [ 17 , 40 ], but also more hemolytic character when compared to GO [ 41 ].…”

“…Furthermore, natural graphene or reduced forms of graphene oxides exhibited not only a cytotoxic effect against other cancer cells [ 17 , 40 ], but also more hemolytic character when compared to GO [ 41 ]. However, it was also found that GO and other carbon nanostructures, such as carbon nanotubes, might non-specifically inhibit enzymatic reactions by binding to the structure of a protein [ 24 , 39 , 42 ]. Considering the nature of the ATP assay, where the reaction should occur very rapidly [ 43 ], it may be possible that the obtained results are due to the mentioned interactions between large graphene oxide platelets and luciferase, which inhibited the oxidation of luciferin, or between the luciferin and GO.…”

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

“…In that work, although using two different in vitro approaches (such as the differentiated HepaRG™ cells for monitoring CYP gene expression and a microsomal-based model for assessing CYP metabolic activity), it has been reported a strong reduction of transcription of many CYP isoforms (CYP3A4, CYP2B6, CYP1A2, and CYP2E1) and the inhibition of the catalytic activity of CYP3A4, CYP1A2, and CYP2D6 by GO ( Strojny et al, 2018 ). Similarly, Sekretarska et al showed, using a microsomal-based model, that GO (50 and 100 μg/ml) caused inhibition of CYP2C9 catalytic activity higher than 40% compared to the control, but, in HepG2 cells, GO did not affect the relative gene expression ( Sekretarska et al, 2019 ). It is worthwhile mentioning that the presented data on CYP gene expression and metabolic activity were obtained using the same cell model and, hence, provided for the first time a complete description of the CYP-inhibition effect of GO in functional, differentiated hepatocytes.…”

Comparison of In Vitro Models for Drug-Induced Liver Injury Assessment

“…There is also evidence that HepG2 cells have low expression of CYP2C9, an important enzyme involved in drug metabolism [44][45][46]. This low expression affects their suitability as a model for studying hepatotoxicity and drug metabolism [47,48]. It is important to consider this limitation when using HepG2 cells as a model system for drug metabolism research.…”