Elsevier

Carbon

Volume 95, December 2015, Pages 309-317
Carbon

Dose-dependent effects of nanoscale graphene oxide on reproduction capability of mammals

https://doi.org/10.1016/j.carbon.2015.08.017Get rights and content

Abstract

In vivo dose-dependent effects of nanoscale graphene oxide (NGO) sheets on reproduction capability of Balb/C mice were investigated. Biodistribution study of the NGO sheets (intravenously injected into male mice at dose of ∼2000 μg/mL or 4 mg/kg of body weight) showed a high graphene uptake in testis. Hence, in vivo effects of the NGO sheets on important characteristics of spermatozoa (including their viability, morphology, kinetics, DNA damage and chromosomal aberration) were evaluated. Significant in vivo effects was found at the injected concentrations ≥200 μg/mL after (e.g., ∼45% reduction in sperm viability and motility at 2000 μg/mL). Observation of remarkable DNA fragmentations and chromosomal aberrations of the spermatozoa after ∼8 weeks from the first weekly injection were assigned to the involvement of the NGO in spermatogenesis of the mice. The uptake of the NGO in the testis could also increase the generation of reactive oxygen species in semen of the mice. Moreover, semen of the NGO-treated mice (containing the damaged spermatozoa) might disturb the hormone secretion and pregnant functionality of female mice (∼44, 35 and 59% reduction in fertility, gestation ability and multi-production capability) and also viability of the next generation (∼15% reduction in postnatal viability of delivered pups).

Introduction

Graphene (as the thinnest two-dimensional sheet of sp2-hybridized carbon atoms) has surprisingly provided fascinating developments in science [1], [2], [3], [4] and technology [5], [6], [7], [8], [9]. This resulted in mass production of graphene [10], [11] as well as fast increasing requests for its application especially in biotechnology and medicine [12]. Hence, the potential toxic effects of graphene on cells [13], microorganisms [14], animals [15], [16], [17], [18] and environment [19], [20] have attracted many attentions.

Concerning the cytotoxic effects of graphene on cells, dose-dependent cytotoxicity of graphene in human erythrocytes and skin fibroblasts was studied by Liao et al. [21]. In addition, dose- and time-dependent cytotoxicity of graphene and graphene oxide (GO) on neural pheochromocytoma-derived PC12 [22], A549 [23], and human mesenchymal stem cells (hMSCs) [24] were investigated and the main mechanism of cytotoxicity was assigned to the generation of reactive oxygen species (ROS). The cell membrane damage induced by physically interaction of the cells with the extremely sharp edges of GO [25], [26], change in the chemical state of GO sheets into reduced graphene oxide (rGO) ones under metabolic activity of microorganisms [27] and entrapping microorganisms by aggregating the rGO sheets in a suspension [28] have also been proposed as other mechanisms of cytotoxicity. It was reported that nanoscale graphene (e.g., graphene nanoplatelets and nanoribbons) can induce DNA fragmentations and chromosomal aberrations in hMSCs [24], [29]. Recently, in vitro cyto- and geno-toxic effects of GO and rGO suspensions on spermatozoa were also reported [30]. Nevertheless, there have been limited investigations about the effects of graphene on animals. For example, Chen et al. [31] found slight hatching delay of zebrafish embryos at high concentrations of GO (∼50 μg/mL), although no significant increase of apoptosis was observed in embryos. Blood circulation and biodistribution of nanoscale graphene oxide (NGO) in tumor and normal organs of mice were also studied [32], [33].

In recent years, in spite of all current discussions on the potential toxicity and/or biocompatibility of graphene, it has extensively been applied in imaging, targeting and therapy of cancers [32], [33], [34], [35], [36], stem cell-based tissue engineering [37], [38], neural network regeneration (as scaffolds to support cellular attachment, proliferation and differentiation) [39], [40], [41], [42], [43], drug delivery (as effective functionalizable transporters) [44], [46], [45], and DNA/RNA extraction [47], [48], [49]. In this regard, nanoscale graphene has attracted more attentions in, e.g., in vivo ultra-low laser power cancer therapy (as a high NIR absorbent) [32], [33] and delivery of water-insoluble cancer drugs (as an effective and apparently biocompatible transporter) [45]. However, despite of such in vivo promising applications of graphene, no investigations about the probable long-term effects of graphene on bioactivity of animals and humans (including fertility and viability of the next generations) have been studied yet. In fact, the nanoscale graphene dispersed in a body can pass through biological membranes including cell membranes, blood–brain barrier, and blood-testis barrier, and subsequently, affect the physiology of cells in the body. In this regard, it was found that intravenous injection of NGO [32], [33] results in its accumulation in various organs such as heart, lung, muscle, bone, skin, stomach, intestine, spleen, kidney, thyroid, liver and brain. This means that graphene can also accumulate in testis (as one of the active organs) and subsequently affect the spermatogenesis in males as well as epigenesis in females. Very recently, Fu et al. [50] reported the negative effects of GO on female mice orally exposed to GO, including delayed developments of offspring mice in lactation period and decreases in the length of the intestinal villus of the filial mice.

In this work, at first, in vivo biodistribution of intravenously injected NGO sheets in various organs of mice (especially testis, as the center of spermatogenesis) was studied. Then, in vivo dose-dependent effects of NGO sheets on the important parameters of the spermatozoa (such as viability, morphology, kinetics and DNA as well as chromosomal properties of the spermatozoa) were studied. Moreover, the influence of the NGO sheets accumulated in testis on ROS level of semen of the mice was examined. After successful insemination of the female mice by the NGO-treated males, the indirect effects of the NGO sheets on the pregnant functionality of the females (including fertility, gestation ability, hormone secretion, and multi-production capability) and postnatal viability of the delivered pups were investigated. This procedure is schematically presented in Fig. 1.

Section snippets

Synthesis of NGO sheets

Graphene oxide powder was synthesized through a modified Hummers' method using natural graphite powder with particle diameter of ≤40 μm (Sigma Aldrich) according to our previously reported protocol [51]. In a typical procedure, 100 mL H2SO4 was added into a beaker containing 2 g graphite at room temperature. The beaker was cooled down to 0 °C by using an ice bath. Then, 6 g KMnO4 was slowly added to the mixture while warmed up to room temperature. The suspension was stirred continuously for 2 h

Results and discussion

Fig. 2a shows one of the typical AFM images of the NGO sheets deposited on a Si(100) substrate. The sheet-like features of the NGO is clearly seen in Fig. 2a. The lateral size histogram and height profile distribution of the nanosheets are presented in Fig. 2b and c, respectively. The lateral dimension of the nanosheets was found substantially <100 nm with the maximum frequency at ∼25 nm. The first peak in the height profile distribution (associated with the roughness fluctuations of the Si

Conclusions

Biodistribution of the intravenously injected NGO sheets showed high uptakes of the NGO in testis of mice, in addition to the residual organs (such as liver and spleen), kidneys and thyroids. Then, the NGO sheets affected the important parameters of the spermatozoa including their viability, morphology, kinetics and chromosomes. In addition, the NGO sheets accumulated in the testis could increase the ROS level of semen of the mice. Then, the semen of the NGO-treated mice (containing the damaged

Acknowledgment

The authors would like to thank the Research Council of Sharif University of Technology (Grant No. G930207) for supporting the work.

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