Biochemical and Histopathological Evaluation of Graphene Oxide in Sprague-Dawley Rats

Research Article

Austin J Environ Toxicol. 2017; 3(1): 1021.

Biochemical and Histopathological Evaluation of Graphene Oxide in Sprague-Dawley Rats

Patlolla AK1,2*, Rondalph J3 and Tchounwou PB1,2

1NIH-RCMI Center for Environmental Health, College of Science Engineering and Technology, Jackson State University, USA

2Department of Biology CSET, Jackson State University, USA

3Jackson Public School, USA

*Corresponding author: Anita K. Patlolla, NIH-RCMI Center for Environmental Health, College of Science Engineering and Technology, Jackson State University, Jackson, MS, USA

Received: November 22, 2017; Accepted: November 30, 2017; Published: December 07, 2017


Graphene and its derivatives are promising material for important biomedical applications due to their versatility. A detailed comprehensive study of the toxicity of these materials is required in context with the prospective use in biological setting. We investigated toxicity of Graphene Oxide (GO) in rats following exposure with respect to hepatotoxicity and oxidative stress biomarkers. Four groups of five male rats were orally administered GOs, once a day for five days, with doses of 10, 20 and 40mg/Kg GO. A control group consisted of five rats. Blood and liver were collected 24h after the last treatment following standard protocols. GO’s exposure increased induction of Reactive Oxygen Species (ROS), activities of liver enzymes (Alanine ALT, Aspartate AST, Alkaline Phosphates ALP), concentration of Lipid Hydro Peroxide (LHP) and morphological alterations of liver tissue in exposed groups compared to control. The highest two doses, 20 and 40mg/kg, showed statistically significant (p<0.05) increases in the induction of ROS, activities of ALT, ALP, LHP concentration, and morphological alterations of liver tissue compared to control. However, AST activity showed no effect. The results of this study demonstrate that GO may be hepatotoxic, and its toxicity might be mediated through oxidative stress.

Keywords: Graphene Oxide; Reactive Oxygen Species; Alanine Aminotransferases; Aspartate Aminotransferases; Lipid Hydroperoxide; Sprague-Dawley Rats 


Recently, graphene and graphene-related materials are considered the future of advanced nanomaterials owing to their exemplary properties and to their applications in biotechnology and medicine. However, the information about their potential toxicity is limited, causing concern regarding potential health hazards, similar to e.g. Carbon Nano Tubes (CNT), despite the quite different 2-dimensional structure and large lateral size [1].

Graphene oxide is a single-atomic-layered nanomaterial, which is obtained by the oxidation of graphite crystals, which are inexpensive and abundant. After oxidation, the hydroxyl and carboxyl groups are formed in GO and, when conjugated, such particles can be effectively dispersed in aqueous solutions [2]. It is dispersible in water, and as a result is easy to process. Most importantly, it can be converted back into graphene.

GO can be used for the immobilization of various biomolecules, due to its large surface area and also it has been considered as a candidate for drug-delivery [1-3]. The biological applications of GO have not been well studied, however, its biocompatibility was studied successfully in fibroblast cells (L-929) [4] and it has been used as a carrier for controlled drug-delivery and the release of anticancer drugs [5,6]. In previous reports GO was shown to induce oxidative stress in neural pheochromocytoma-derived PC12 cells [7]. Liu et al. [1,3] study, reported PEGylated nano-GO could be used to deliver water insoluble anticancer drugs without any toxicity. Various studies have reported the antibacterial activity of graphene-based nanomaterials [8-12]. Chronic toxicity and lung granuloma death was reported in mice after GO administration [13]. In other reports [14,15], Dose-dependent pulmonary toxicity, granulomatous lesions, pulmonary edema fibrosis and inflammatory cell infiltrations were also found after GO administration. Schinwald et al. [16] reported a pulmonary inflammatory response in rats after BSA-capped graphene administration. The number of in vivo studies based on tissue distribution and excretion of graphene is gradually increasing.

The proposed mechanism involved in toxicity of nanomaterials is its ability to interact with biological tissues and generate reactive oxygen species [17]. They are well known to play both deleterious and a beneficial role in biological interactions. Mostly, the harmful effects of ROS on the cell are often damage to DNA, oxidation of polydesatured fatty acids in lipid (lipid peroxidation) oxidations of amino acids in proteins and oxidatively inactivate specific enzymes by oxidation of co-factors. Many different forms of fine, ultrafine and nanoscale particles, to be associated with minimal metal contamination have been shown to increase the generation of ROS [18,19].

The oxidative catabolism of polyunsaturated fatty acids, Lipid Peroxidation (LPO), is widely accepted as a general mechanism for cellular injury and death [20,21]. Free radicals and LPO generation are complex and deleterious processes that are closely related to toxicity [3,22]. LOP has been implicated in diverse pathological conditions. The extension of the oxidative catabolism of lipid membranes can be evaluated by several endpoints, but the most widely used method is the quantification of Lipid Hydroperoxide (LHP), one of the stable aldehydic products of lipoperoxidation, present in biological samples [23]. Liver is an important organ in vertebrates including humans, its plays a significant chemical metabolism. The methods normally employed for the detection of hepatotoxicity vary with the circumstances of their use. In vivo studies are essential to demonstrate a toxic agent that has in fact a demonstrable adverse effect on the liver in a setting of physiological significance. Biochemically, serum enzyme analyses have become the standard measure of hepatotoxicity during the past 25 years [24].

This study assesses the effects, after oral administration of GO on ROS induction and various hepatotoxicity markers in the rat model. The question of the health effects of GOs is quite acute and this study brings new data in a field where the largest proportion of publications have been conducted with pulmonary models. Few studies that involves GO focus on the possible pulmonary distress causing excessive inflammation [14]. Pulmonary edema and lung granulomas formation [13,25]. There is a limited knowledge relating to their environmental toxicity and biological safety profile. Extensive testing is now deemed essential for graphene-based materials to assess their biological safety profile. Therefore the results presented here are of importance for health risk assessment.

Material and Methods

Chemicals and reagents

Graphene oxide (40nm diameter) was purchased from Graphene Supermarket (Reading, MA, USA) and was dissolved in water. Xylene, ethyl alcohol, paraffin wax, hematoxylin-eosin stain, Diagnostic enzyme assay kits were obtained from Sigma, (St. Louis MO, USA). Diagnostic kit for Lipid peroxidation assays were purchased from Calbiochem (La, Jolla, CA, USA).

Animal maintenance

Healthy adult male Sprague-Dawley rats (8-10 weeks of age, with average Body Weight (BW) of 125±2g) were used in this study. They were obtained from Harlan-Sprague-Dawley Breeding Laboratories in Indianapolis, Indiana, USA. The rats were randomly selected and housed in polycarbonate cages (18.88 in x7.25 in x3.76 in) (three rats per cage) with steel wire tops and corn-cob bedding. They were maintained in a controlled atmosphere with a cyclic 12h dark/12h light cycle, a temperature of 22 ±2°C and 50-70% humidity and also with free access to pelleted feed (oval normal diet with complete balanced nutritional value for biomedical research) and fresh tap water. The rats were allowed to acclimate for 10 days before treatment.       

Doses of graphene oxide

Groups of five rats each were treated with three doses of Graphene Oxide (GO). GO was diluted with deionized water, and orally administered using feeding needles to the rats at the doses of 10, 20, 40mg/Kg BW. Each rat received a total of five doses at 24h intervals. Deionized water was used as negative control and was administered in the same manner as in the treatment groups.