In vitro cardiotoxicity evaluation of graphene oxide

https://doi.org/10.1016/j.mrgentox.2019.03.004Get rights and content

Highlights

  • Graphene oxide nanoparticles induced cytotoxicity in H9c2 cells.

  • Graphene oxide nanoparticles induced mitochondrial hyperpolarization in H9c2 cells.

  • Graphene oxide nanoparticles elicited an increase in free radicals in H9c2 cells.

  • DNA damage was related to graphene oxide nanoparticles cytotoxicity.

Abstract

Graphene is a two-dimensional (2D) monolayer of carbon atoms, tightly packed, forming a honey comb crystal lattice, with physical, chemical, and mechanical properties greatly used for energy storage, electrochemical devices, and in nanomedicine. Many studies showed that nanomaterials have side-effects on health. At present, there is a lack of information regarding graphene and its derivatives including their cardiotoxic properties. The aim of the present study was to evaluate the toxicity of nano-graphene oxide (nano-GO) in the rat cardiomyoblast cell line H9c2 and the involvement of oxidative processes. The cell viability was evaluated with the fluorescein diacetate (FDA)/propidium iodide (PI) and in the trypan blue exclusion assay, furthermore mitochondrial membrane potential and production of free radicals were measured. Genotoxicity was evaluated in comet assay and low molecular weight DNA experiment. Reduction of cell viability with 20, 40, 60, 80, and 100 μg/mL nano-GO was observed after 24 h incubation. Besides, nano-GO induced a mitochondrial hyperpolarization and a significant increase of free radicals production in the same concentrations. DNA breaks were observed at 40, 60, 80, and 100 μg/mL. This DNA damage was accompanied by a significant increase in LMW DNA only at 40 μg/mL. In conclusion, the nano-GO caused cardiotoxicity in our in vitro model, with mitochondrial disturbances, generation of reactive species and interactions with DNA, indicating the importance of the further evaluation of the safety of nanomaterials.

Introduction

Carbon-based nanomaterials received growing interest in techonology due to their unique physicochemical properties. Their use all over the world increased in recent years. Graphene is a two-dimensional (2D) monolayer of carbon atoms, tightly packed, forming a honey comb crystal lattice, with physical, chemical, and mechanical properties [1,2]. Since 2004, after the isolation of graphene by Novoselov, Geim and co-workers using a mechanical exfoliation [1], further protocols were developed for the production of this material [[3], [4], [5], [6], [7], [8]].

Different materials, such as graphite oxide, graphene oxide (GO) and nano-graphene oxide (nano-GO), are produced using graphene as raw material. At present, they are widely used due to their applications, that embrace energy storage [9,10], electrochemical devices [11,12], cell imaging and drug delivery [13], adsortion of enzyme [14], as well as in nanomedicine [15,16]. GO is absorbed by normal cells and tumors, which might serve to deliver insoluble drugs [17,18] and other molecules [19] into cells for bioimaging and therapeutic purposes [20].

Many studies showed that nanomaterials may cause side-effects in humans [[20], [21], [22], [23]]. The understanding of their biological behaviour is important to prevent adverse health effects [[20], [21], [22], [23],24,25]. Up to date there is a lack of information regarding the safety of GO and its derivates, which needs to be explored before humans are exposed to this material.

The biocompatibility of GO has been tested in different cell lines, including human fibroblasts [26,27], human lung carcinoma epithelial (A549) cells [28], human hepatoblastoma (HepG2) cells [29], human multiple myeloma (RMPI 8226) cells [30], and human erythrocytes [31]. The results collectively indicate that GO is biocompatible. However, single-layer GO sheets were internalized and sequestered in cytoplasmic membrane bound vacuoles by human lung epithelial cells and fibroblasts, and induced cytotoxicity at doses ≥ 20 μg/mL after 24 h exposure [32]. When injected intravenously into mice, 0.4 mg GO induced granulomas in the lungs, liver, spleen and kidney, and was lethal [33,34]. It was also observed that GO induces oxidative stress at low concentration in vitro and in vivo [[35], [36], [37], [38]].

The potential adverse effects of new nanomaterials can be not excluded and toxicological studies are needed. Recently, GO has gained interest as scaffold material for electrical conduction in the therapeutic of heart failure [39] and myocardial infarction [[40], [41], [42]], but little is known about the safety of its use. Therefore, the aim of this study was to evaluate the cyto and genotoxicity of nano-GO in the rat cardiomyoblast cell line H9c2, an in vitro model which was used earlier to assess cardiotoxicity. This cell line has been extensively used as an alternative to study cardiac mechanisms like arrhythmias [43], ROS production [44], and relevant clinical situations like ischemia-reperfusion injury [45].

Section snippets

Chemicals

Dulbecco Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin-EDTA, phosphate-buffered saline (PBS), sodium chloride (NaCl), triton x-100, tetramethylrhodamine ethyl ester perchlorate (TMRE), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), dimethyl sulfoxide (DMSO), fluorescein diacetate (FDA), propidium iodide (PI), agorose, and trypan blue were purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin hydrochloride was purchased from Glenmark (Osasco, SP, Brazil).

GO sample

The graphene

Induction of acute cytotoxicity in H9c2 cardiomyoblasts

The viability of the cells are depicted in Fig. 1. We observed a clear reduction (p < 0.001) of the viability after 24 h incubation with 20, 40, 60, 80, and 100 μg/mL nano-GO. Doxorubicin, used as positive control, produced an approximated 40% decreased in the cell viability.

Findings of the trypan blue exclusion assay supported the results of the FDA/PI experiment (Fig. 2). A significant reduction (p < 0.01) of cell viability was observed at 20, 40, 60, 80, and 100 μg/mL nano-GO. The positive

Discussion

The toxicity of nanomaterials can be useful to guide their potential clinical applications. To use nanosystems for drug delivery, their cytotoxicity must be low, however if the nanomaterials present a high toxicity, they could be applied to kill unwanted cells (e.g., cancer cells) [57]. Here, we studied the effects of non functionalized nano-GO and the results were similar to those found in earlier experiments with human fibroblast cells. Nano-GO at concentrations ≤ 20 μg/mL do not induce

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was realised in the framework of “Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul” (Fapergs/Proex) and National Institute of Science and Technology of Pharmaceutical Nanotechnology (INCT-Nanofarma), which is supported by “Fundação de Amparo à Pesquisa do Estado de São Paulo” (Fapesp, Brazil, grant #14/50928-2) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil, grant #465687/2014-8). M. D. Arbo is recipient of post-PhD fellowship (BJT, grant #

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