Short-term in vivo exposure to graphene oxide can cause damage to the gut and testis

Journal of Hazardous Materials 328 (2017) 80–89

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Short-term in vivo exposure to graphene oxide can cause damage to the gut and testis a,∗ ˛ Marta Dziewiecka , Julia Karpeta-Kaczmarek a , Maria Augustyniak a , Magdalena Rost-Roszkowska b a b

Department of Animal Physiology and Ecotoxicology, University of Silesia in Katowice, Bankowa 9, PL 40-007 Katowice, Poland Department of Animal Histology and Embryology, University of Silesia in Katowice, Bankowa 9, PL 40-007 Katowice, Poland

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• The in vivo toxicity graphene oxide (GO) administrated with food was measured. • Stress parameters were measured in A. domesticus after exposure to graphene oxide. • Administration of GO and GO + Mn2+ with food had an effect on the organism. • Many histological changes were found in gut and testis of A. domesticus.

a r t i c l e

i n f o

Article history: Received 13 July 2016 Received in revised form 4 January 2017 Accepted 8 January 2017 Available online 9 January 2017 Keywords: Graphene oxide Toxicity Histology Gonads Acheta domesticus

a b s t r a c t Graphene oxide (GO) has unique physicochemical properties and also has a potentially widespread use in every field of daily life (industry, science, medicine). Demand for nanotechnology is growing every year, and therefore many aspects of its toxicity and biocompatibility still require further clarification. This research assesses the in vivo toxicity of pure and manganese ion-contaminated GO that were administrated to Acheta domesticus with food (at 200 mg kg−1 of food) throughout their ten-day adult life. Our results showed that short-term exposure to graphene oxide in food causes an increase in the parameters of oxidative stress of the tested insects (catalase – CAT, total antioxidant capacity – TAC), induces damage to the DNA at a level of approximately 35% and contributes to a disturbance in the stages of the cell cycle and causes an increase of apoptosis. Moreover, upon analyzing histological specimens, we found numerous degenerative changes in the cells of the gut and testis of Acheta domesticus as early as ten days after applying GO. A more complete picture of the GO risk can help to define its future applications and methods for working with the material, which may help us to avoid any adverse effects and damage to the animal. © 2017 Elsevier B.V. All rights reserved.

∗ Corresponding author. ˛ E-mail address: [email protected] (M. Dziewiecka). http://dx.doi.org/10.1016/j.jhazmat.2017.01.012 0304-3894/© 2017 Elsevier B.V. All rights reserved.

M. Dziewi˛ecka et al. / Journal of Hazardous Materials 328 (2017) 80–89

1. Introduction Even though nanotechnology is a relatively new field, nanoparticles (NP) are not. The first use of nanoparticles dates back to the 10th century BC [1]. Nanoparticles are a connecting link between bulk materials and atomic or molecular structures. The properties of many popular materials change upon the formation or addition of nanoparticles. This is possible because nanomolecules have a surface area to volume ratio that is larger than bigger particles. As a result, the chemical reactivity of the materials increases [2]. From a scientific point of view, the most attractive materials are from the graphene family [3]. One of these is graphene oxide (GO), which is a derivative of graphene sheets that have a large number of oxygen-containing hydrophilic groups in their structure [4]. The presence of hydroxyl, carbonyl or carboxylic groups makes GO a suitable material for functionalization and chemical modifications. GO is hydrophilic and thus can create a stable suspension in water. Due to its ability to bind different molecules, the practical application of GO in many different fields of science has become more popular [5]. Graphene oxide can be used in industry to produce graphene-based composites, but also has other applications such as advanced electronics, hydrogen storage, transparent film production and catalysis [6–10]. GO is also potentially the best candidate from the graphene family for biological and medical utilization. The use of GO as drug carriers, biosensors and vectors for gene or cancer therapy is now being considered [11–14]. However, before graphene oxide is used on a large scale in the medical field, a thorough understanding of its toxicology at various levels of physiological reactivity is needed [15]. Several in vivo and in vitro studies have revealed that GO exposure increases oxidative stress in the cells, enhances apoptosis and decreases cell viability [16–19]. In our previous studies, we focused on the in vivo effects of GO after intentionally injecting it into the body cavity of Acheta domesticus. The results indicated that graphene oxide can already increase the level of reactive oxygen species (ROS) within 48 h after the injection [20]. In the subsequent study, we decided to estimate the in vivo toxicity of two types of graphene oxide (pure and ones contaminated with manganese ions), which were administrated to Acheta domesticus with food throughout the ten days of their adult life. We assumed that this natural way of applying nanoparticles could show the effects of both potential medical treatment as well as any accidental contact with GO in the environment. Moreover, it appears to be highly possible that changes at the cellular level during this period may also be crucial and noticeable. The main aim of this study was to assess the effects of GO at the cellular and tissue level after a relatively short-term (ten days) exposure. The selected stress parameters – catalase (CAT), total antioxidant capacity (TAC) and the level of DNA damage – were measured every two days. The cell cycle, the level of apoptosis and the total oxidative stress were checked at the beginning (second day) and at the end of the experiment (tenth day) using flow cytometry. A histological assessment of the gut and male gonad (testis) was performed after ten days of treatment using Transmission Electron Microscopy (TEM). 2. Materials and methods

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conditions (temperature: 29.4 ± 3.5 ◦ C; photoperiod: 12 h: 12 h; humidity: 46.52 ± 9.43%) with unlimited access to water and food. The diet contained two different types of graphene oxide (pure and ones contaminated with manganese ions). The graphene oxide nanoparticles were manufactured according to modified Hummer’s method at the Wielkopolska Centre of Advanced Technology ´ Poland). The level of manganese impurity in both GO sam(Poznan, ples was estimated using electron paramagnetic resonance method (EPR). According to our calculations, the concentration of manganese ions in the GO + Mn2+ sample reached 0.23 wt%. Some trace amounts of manganese ions were detected in the pure GO sample, but only at a low temperature (4.2–40 K). The molar concentrations of manganese ions for GO with a C/O ratio equal to 1.67 were 1.94 0−2 mol% in the GO + Mn2+ sample and ∼1.26 0−4 mol% in the pure sample. The detailed characteristics of the GO samples used in this experiment as well as the AFM (Atomic Force Microscopy) images ˛ of the flakes are presented in Dziewiecka et al. [20] and Majchrzycki et al. [22]. The food with graphene oxide was prepared by grinding the standard food and mixing it with GO suspended in distilled water [23]. The final concentration of nanoparticles in the diet was set at 200 ␮g g−1 for the GO and GO + Mn2+ groups (pure graphene oxide or graphene oxide with manganese ions were used, respectively). Such a concentration of GO was determined basing on the initial pilot tests, in which survivability and growth rate of the insects were assessed. Concentration of 200 ␮g g−1 did not cause a significant increase in mortality but at the same time, the growth of insects slowed. The insects from the control group consumed uncontaminated food. Twenty-two randomly selected individuals were chosen at each of the five time points: 2, 4, 6, 8 and 10 days after the beginning of the experiment. The individuals were dissected and the hemolymph, gut and gonads of the males were isolated and prepared for further analysis. Stress parameters and the level of DNA damage were measured every two days. Health status of cells was checked at the beginning (second day) and at the end of the experiment (tenth day). A histological assessment of the gut and testis was performed after ten days of treatment (Scheme 1).

2.2. Stress parameters The insects were lightly anesthetized on ice and then the hemolymph and gastrointestinal tract were isolated. A forty ␮L of hemolymph was mixed with an anticoagulant buffer at a 1:1 ratio. The gastrointestinal tract was homogenized on ice in a phosphate buffer (pH 7.4; 4 ◦ C) and centrifuged in order to obtain a submitochondrial fraction [20,26].

2.2.1. Total antioxidant capacity assay (TAC) The antioxidant capacity parameter (TAC) is the total amount of antioxidants that is present in the plasma and body fluids [24]. The level of TAC was measured through the decolourization of 2,2 Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium ˛ salt (ABTS) at 734 nm, as was previously described by Dziewiecka et al. [20].

2.1. Experimental model Acheta domesticus, a house cricket, is frequently used as a model organism in studies because its biology and physiology are well-known [21]. Adult insects from a laboratory stock population (kept at the University of Silesia in Katowice, Poland) were divided into three groups, transferred to separate plastic insectaries (46 × 31 × 17.5 cm; 110 individuals in each) and bred in standard

2.2.2. Catalase assay (CAT) Catalase (CAT) [EC 1.11.1.6] plays the main role in the decomposition of hydrogen peroxide into water and oxygen in living organisms. CAT activity was assessed by measuring the rate of H2 O2 removal from the samples. All of the procedures were carried out as first described by Aebi [25] with the minor modifications that ˛ et al. [20]. were introduced by Dziewiecka

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Fig. 1. Activity of the stress parameters in the gut and hemolymph of the Acheta domesticus exposed to graphene oxide – pure (GO) and manganese ion contaminated (GO + Mn2+ ) – during the ten days of the experiment. (a) activity of catalase (CAT) in the gut (b) activity of catalase (CAT) in the hemolymph (c) the level of total antioxidant capacity (TAC) in the gut (d) the level of total antioxidant capacity (TAC) in the hemolymph. Different letters – significant differences between treated groups (GO, GO + Mn2+ ) and control group at each time point separately (LSD test, ANOVA, p < 0.05).

2.3. Health status of cells Six randomly chosen adult insects were anesthetized on ice and the gastrointestinal tract was isolated. A cell suspension was prepared by gently shaking the tissue with a 0.1 M phosphate buffer ® (pH 7.4) in a homogenizer (Minilys , Bertin Technologies). All of ® the measurements were performed using a Muse Cell Analyzer (Millipore, Billerica, MA, USA) flow cytometer according to the manufacturer’s protocols after standardization for A. domesticus tissues. The total level of oxidative stress in the cells was measured using ® Muse Oxidative Stress Kit. The level of apoptosis was evaluated ® ® using a Muse Annexin V & Dead Cell assay and a Muse Multi ® Caspase Assay Kit. The cell cycle was measured using a Muse Cell Cycle Assay Kit. 2.4. The level of the DNA damage The Single Cell Gel Electrophoresis assay SCGE was used to detect DNA damage [27–29]. The genotoxic effect of graphene oxide was tested in the hemocytes of six randomly chosen adult insects according to Karpeta-Kaczmarek et al. [30]. 2.5. Histological analysis The gonads of male A. domesticus and the gastrointestinal tract were inspected. The tissues were isolated from six adult insects on day ten of the experiment and prepared for analysis appropriately. 2.6. Gastrointestinal tract and male gonads The digestive system divided into sections – the foregut, midgut, hindgut and male gonads was fixed in 2.5% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4) for 1.5 h at 4 ◦ C – gut, or for 2 h – testis. After washing in a 0.1 M phosphate buffer (3 × 15 min, RT), it was postfixed in 1% OsO4 for 1.5 h (RT) – gut or 2 h – testis. After

dehydration in a graded concentration series of ethanol (50, 70, 90, 95 and 100% for 15 min each) and acetone (15 min), the material was embedded in epoxy resin (Epoxy Embedding Medium Kit, Sigma). Ultrathin sections (70 nm) – gut, or semi-thin (700 ␮m) and ultra-thin sections (80 nm) – testis, were cut on a Leica Ultracut UCT25 ultramicrotome. Semi-thin sections were stained with 1% methylene blue in 1% borax and then analyzed using an OLYMPUS BX60 light microscope. Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Hitachi H500 transmission electron microscope at 75 kV. 2.7. Statistical procedures In order to check the normality of the data for all of the parameters, the Kolmogorov-Smirnov and Shapiro-Wilk tests were used. The homogeneity of the variances was tested using the Levene and Brown-Forsythe tests. Parametric (LSD test, ANOVA; p < 0.05) or nonparametric (Kruskal-Wallis test; p < 0.05) tests were conducted to evaluate the significance of the differences among the experimental groups. Statistical analysis was performed using Statistica 12 (StatSoft, Inc. 2011). 3. Results 3.1. Stress parameters: catalase activity (CAT), and total antioxidant capacity (TAC) GO treatment did not induce any significant changes in CAT activity in the hemolymph after 2–8 days of the experiment. A significant elevation of catalase activity was observed in this tissue only after ten days of exposure (Fig. 1b). The activity of catalase in the gut revealed a different tendency than in the hemolymph of the insects on the diet with graphene oxide. A statistically significant increase in the CAT activity in the gut was observed on the 4th and 10th day of experiment in both GO-treated groups compared to the control group. The activity of the enzyme on the 8th day of exposure was significantly higher only in the gut of the insects that had con-

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Scheme 1. Design of experiment.

sumed food that had been contaminated with GO and manganese ions (Fig. 1a). Moreover, the total antioxidant capacity (TAC) level was only significantly higher after ten days of treatment. This tendency was visible in the gut (Fig. 1c) and in the hemolymph (Fig. 1d) of A. domesticus in both experimental groups (GO and GO + Mn2+ ). 3.2. The level of DNA damage The administration of graphene oxide with food during the ten days of experiment induced significant genotoxic effects in the hemocytes of A. domesticus. The amount of DNA in the comet tail (TDNA) was always significantly higher in the organisms that were exposed to both types of graphene oxide compared with the control group. The percentage of DNA in the comet tail of the hemocytes of insects from both experimental groups ranged between 20% and 35% during the whole experiment (Fig. 2a). Tail length (TL) as well as Olive tail moment (OTM) revealed similar trends (Fig. 2b, c). Graphene oxide, both pure and contaminated, caused a significant increase in both of these parameters from the beginning of the experiment (second day) until the end (tenth day). No significant differences between the two treated groups were observed in any of the analyzed parameters (Fig. 2a–c).

3.3. Health status of cells On the second day of exposure, the level of reactive oxygen species (ROS)in the gut of insects from the GO + Mn2+ group tended to be higher than in the individuals from the GO or control groups (Fig. 3a). On the last day of the experiment, the total level of ROS in the cells was significantly higher in both experimental groups comparing to the control group (Fig. 3a, b). During the experiment, the level of apoptosis increased in the gut cells of the insects treated with pure GO and GO + Mn2+ . On the tenth day of exposure, the level of live cells in both experimental groups reached about 78% of the total cells, whereas the live cells in the control group were about 95%. After 10 days, the differences between treated groups and control group were statistically sig® nificant (Fig. 3c, d). The Muse Multi Caspase Assay test showed that the ratio of dead cells to live cells was significantly higher on the tenth day compared to the second day of exposure to GO or GO + Mn2+ (Fig. 3e, f). Moreover, the cell cycle analysis indicated significant differences between the second and the tenth day of the experiment. After two days of exposure to GO or GO + Mn2+ , more cells stopped in the G0/G1 phase. Ten days after the exposure to both types of graphene oxide, the number of cells remaining in the

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Fig. 2. DNA damage (a) based on the percentage of DNA in the tail, (b) based on the tail length parameter, (c) based on the olive tail moment parameter in the hemocytes of the Acheta domesticus that were exposed to graphene oxide – pure (GO) and manganese ion contaminated (GO + Mn2+ ) – during the ten days of the experiment. Different letters – significant differences between treated groups (GO, GO + Mn2+ ) and control group at each time point separately (Kruskal-Wallis test; p < 0.05).

G2/M phase increased to about 10% of the cells measured. At the same time, the participation of the S phase in the cells obtained from the GO-treated insects decreased to about 6% of the total cells measured (Fig. 3g, h).

disappear while some lipid droplets appear. The most distinct feature of the foregut epithelium is the presence of hemocytes among the epithelial cells. Hemocytes that entered the foregut epithelium were not observed in the specimens of the control group. No changes in the structure of the cuticle were observed (Fig. 4A, B).

3.4. Histological analysis 3.4.1. Foregut The foregut monolayered epithelium lies on the non-cellular basal lamina and is composed of an epithelial cell whose surface is covered with a cuticle. The precise ultrastructure of the foregut epithelial cells was described in our previous paper [23]. 3.4.1.1. Graphene oxide (GO). The cytoplasm of the epithelial cells is electron medium and has a small number of organelles. Mitochondria that are devoid of mitochondrial cristae and cisterns of the rough endoplasmic reticulum are the main organelles of the epithelial cells. The microvilli are poorly developed. Glycogen granules

3.4.1.2. Graphene oxide contaminated with manganese ions (GO + Mn2+ ). The cytoplasm of epithelial cells is electron lucent and devoid of organelles. Only sporadic mitochondria that are devoid of mitochondrial cristae and an electron-lucent matrix were observed. The nucleus has a lobular shape and shrinks. The heterochromatin forms electron-dense patches that are located near the central region of the nucleoplasm. The microvilli disappear completely. Reserve materials such as glycogen granules were not observed. The hemocytes, which appear between the epithelial cells of the foregut in the specimens treated with graphene, were not observed in this experimental group. No changes in the structure of the cuticle were observed (Fig. 4A, C).

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Fig. 3. The results of the Muse Cell Analyzer (a–b) the level of oxidative stress (c–d) the results of the Annexin V (e–f) the results of the Multi Caspase (g–h) cell cycle phases in the gut cells in Acheta domesticus after 2 and 10 days of exposure to pure (GO) and manganese ion contaminated (GO + Mn2+ ) graphene oxide. The different letters indicate there are significant differences among the experimental groups at each time point (Kruskal-Wallis test, p < 0.05).

3.4.2. Midgut The midgut is divided into two distinct regions − the anterior midgut and the posterior midgut. The epithelium of both regions is formed by digestive and regenerative cells. The precise ultrastructure of the midgut digestive and regenerative cells was described in our previous papers [30–32]. No differences were observed between the anterior and posterior regions of the midgut, and therefore the description presented below concerns both regions of the midgut. 3.4.2.1. Graphene oxide (GO). The apical cytoplasm of the digestive cells showed some small differences in comparison to the control group, while the basal cytoplasm showed no differences. It started to become electron lucent. Single vacuoles, lipid droplets, autophagosomes and electron-dense granules, which are probably lysosomes, were observed (Fig. 5A, B). No changes were observed at the ultrastructural level of any of the midgut regenerative cells. 3.4.2.2. Graphene oxide contaminated with manganese ions (GO + Mn2+ ). The entire cytoplasm of the digestive cells started to become electron lucent. The number of autophagosomes, vacuoles and electron-dense granules (probably lysosomes) increased significantly. Numerous multivesicular bodies were observed (Fig. 5A, C). No changes were observed at the ultrastructural level in any of the midgut regenerative cells.

3.4.4.1. Graphene oxide (GO). Transmission electron microscopy showed that the cytoplasm of the male germ cells (spermatogonia, spermatocytes, spermatids and sperms) started to become electron lucent, according to their ultrastructure in the control specimens (Fig. 7A–F). Marginalization of the chromatin was observed in all of the nuclei. Therefore, the chromatin formed a distinct layer near the nuclear envelope (Fig. 7E). Numerous degenerating mitochondria were observed and the cytoplasm became poor in the organelles. However, the centrioles and axoneme structure underwent no alteration (Fig. 7C, F). Mitotic divisions were observed in the follicle of the testis (not shown). 3.4.4.2. Graphene oxide contaminated with manganese ions (GO + Mn2+ ). Transmission electron microscopy clearly showed that the entire cytoplasm of the male germ cells (spermatogonia, spermatocytes, spermatids and sperms) became electron lucent according to their ultrastructure in the control specimens and the specimens treated with pure graphene oxide (Fig. 7A–I). The nucleus was devoid of electron-dense heterochromatin (Fig. 7H). All of the mitochondria were degenerated, the number of organelles decreased and the membranes of the majority of the cells were broken (Fig. 7I). However, no alterations of the centrioles and axoneme were observed. Mitotic divisions were not detected in the follicle of the testis. 4. Discussion

3.4.3. Hindgut The monolayered epithelium of the ectodermal hindgut, which lies on the non-cellular basal lamina, was formed by an epithelial cell whose surface was covered with a cuticle. The precise ultrastructure of these epithelial cells was described in our previous paper (Karpeta- Kaczmarek et al.) [23]. 3.4.3.1. Graphene oxide (GO) or (GO + Mn2+ ). No changes were observed at the ultrastructural level in any of the hindgut epithelial cells (Fig. 6A–C). 3.4.4. Testis

Nowadays, oxidative stress is the most commonly accepted mechanism for the toxicity of nanoparticles [19,33]. Some in vitro and in vivo experiments have indeed shown that a wide range of nanomaterials can contribute to the production of ROS in cells [17,34–38]. Injection of graphene oxide into the body cavity of Acheta domesticus, which we had previously applied, caused a series of important changes that occurred during the first three days and indicated an intensification of the production of reactive oxygen species [39]. In the present study, Acheta domesticus was exposed to the same nanoparticles as in the previous experiment (pure and ones contaminated with manganese ions) at a concentration of

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Fig. 4. The foregut of adult specimens of A. domesticus. (A) The control specimen. Cross section. TEM. Bar = 1.2 ␮m. (B) Specimen treated with pure graphene oxide (GO). Cross section. TEM. Bar = 1.2 ␮m. (C) Specimen treated with graphene oxide manganese ion contaminated (GO + Mn2+ ). Cross section. TEM. Bar = 1.4 ␮m. Foregut lumen (l), cuticle (cu), mitochondria (m), nucleus (n), visceral muscles (mc), hemocytes (h), lipid droplets (lp), microvilli (arrows).

200 ␮g g−1 ; however, the method of application and the time of exposure were different. Exposure of the insects to GO with food for ten days also induced oxidative stress, which was confirmed by an increase in the CAT activity and TAC level (Fig. 1a–d). The degree and the trend of changes were the same for both forms of GO. It should be noted, however, that significant differences were mainly observed on the last day of exposure. In this model of the experiment, the gut was the first line of antioxidative defense as the insects were directly exposed to graphene oxide in food. Therefore,

Fig. 5. The midgut of adult specimens of A. domesticus. (A) The control specimen. Cross section. TEM. Bar = 0.7 ␮m. (B) Specimen treated with pure graphene oxide (GO). Cross section. TEM. Bar = 0.9 ␮m. (C) Specimen treated with graphene oxide manganese ion contaminated (GO + Mn2+ ). Cross section. TEM. Bar = 1.4 ␮m. Cisterns of RER (RER), mitochondria(m), nucleus (n), multivesicular bodies (mb), microvilli (mv), vacuoles (v), autophagosomes (au), electron-dense granules (arrows).

the relatively late intensification of CAT and TAC as well as the lack of distinct differences between both analyzed tissues may be surprising. Perhaps the nature of GO that determines its toxicity is crucial. After the contact of GO with natural organic matter during food preparation and later during the digestion of food in the intestine a variety of fast chemical reactions on the GO surface undoubtedly occur. One of the most plausible scenarios includes

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Fig. 6. The hindgut of adult specimens of A. domesticus. (A) The control specimen. Cross section. TEM. Bar = 1.4 ␮m. (B) Specimen treated with pure graphene oxide (GO). Cross section. TEM. Bar = 0.9 ␮m. (C) Specimen treated with graphene oxide manganese ion contaminated (GO + Mn2+ ). Cross section. TEM. Bar = 2 ␮m. Mitochondria (m), cisterns of RER (RER), nucleus (n).

a reduction of graphene oxide in the gut cells to insoluble reduced graphene oxide (rGO). Both GO and rGO influence the structural integrity of the plasma membrane as a result of the interaction of the nanoparticles with the phospholipid bilayer. The sharp edges of the nanosheets from the graphene family nanomaterials may cause physical damage to the cell membranes, thereby resulting in the destruction of cell structures [3,40]. The severe damage in the structure of gut cells, which we especially observed in the foregut of the A. domesticus that consumed the GO-contaminated food (Figs. 4–6), confirms the above-mentioned mechanism of GO toxicity. The samples obtained from insects consuming food contaminated with GO + Mn did not show the presence of hemocytes in foregut. At this stage of knowledge the result is difficult to explain. It may be assumed that manganese ions reduce the reactivity of GO (less oxidative damage). This hypothesis requires additional research on the process of reduction of graphene

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oxide in presence of various organic substances and manganese ions. Although distinct GO-dependent changes in the CAT activity and TAC level were observed after ten days of exposure, the level of DNA damage increased beginning on the second day of experiment and was similar in both of the tested nanoparticles (Fig. 2). Interestingly, the level of DNA damage was similar during the whole experiment. Most probably, GO and GO derivatives can penetrate through the epithelium into the body cavity and cause a destabilization in the functioning of various tissues in the body. Manganese ion impurities in nanoparticles, at the concentration as in the samples used in our research, do not have any significant influence on the process. Manganese is an indispensible element for organisms [49]. It means that the mechanisms of homeostasis for the element, within a certain range of concentrations, have to be very efficient. Our material apparently did not exceed the range of concentrations. The accumulation of reactive oxygen species in the cells can lead to DNA damage through a free radical attack [33]. The potential mechanisms may also include an indirect influence on the DNA repair processes because of energy allocation towards the fast regeneration of the epithelium. The presence of hemocytes among the destroyed epithelial cells, which we observed in the histological slides of the foregut (Fig. 4), may be a symptom of the regeneration process. Among the various functions of hemocytes, besides their role in inflammation-like responses, their participation in wound healing is critical and substantial. Additionally, the active migration of hemocytes through the epithelium in case of repairing tissues damages has been described in numerous epithelial tissues [41,42,46]. It cannot be excluded that nanoparticles may directly interact with DNA after entering the nucleus via nuclear pores. As a result of all of the above-mentioned events, some disturbance in the cell cycle and apoptosis may occur [2]. Our results, ® which were obtained using Muse Cell tests, showed some disproportion in the cell cycle phases that were dependent on the length of exposure and the type of nanoparticles. After two days of exposure to both pure and contaminated GO, more cells were stopped in the G0/G1 phase. Different types of mutations and/or adverse environmental factors may cause a block in the G1 phase. This type of checkpoint prevents the replication of defective genetic material in the S phase [43]. After ten days of exposure to graphene oxide, a slight arrest of cells in the G2/M phase was observed. The G2/M DNA damage checkpoint provides the opportunity to repair damaged DNA after replication, and before entering mitosis [44]. ® Muse Cell tests revealed an intensification of the oxidative stress level and apoptosis in the gut cells of the insects treated with GO (Fig. 3). All cytometric tests as well as the biochemical, DNA damage and histological assays confirmed that the consumption of food contaminated with graphene oxide (both pure one and contaminated one) causes numerous changes in the tissues of the subjected organism. Therefore, this study shows the in vivo toxicity of GO and is consistent and complementary with the results of in vitro researches [15,17,33]. Transmission Electron Microscopy (TEM) showed numerous changes in the ultrastructure of the gut. The first section of the gut – the foregut – was in an especially bad condition after graphene oxide treatment. Karpeta-Kaczmarek et al. [23] showed that nanodiamonds offered in food at a dose of 200 ␮g g−1 also led to damage and dysfunction in the gut. The most damaged section, however, was the midgut [23]. The shape, the size of flakes and the chemical composition and functionalization of the investigated nanomaterials most probably determine this. Yang et al. [19] demonstrated that the toxic effects of carbon nanotubes result mostly from mechanical injury and less from the generation of oxidative stress. Our studies revealed that the number of autophagosomes, vacuoles and electron-dense granules (probably lysosomes) increased in the cytoplasm of the midgut epithelial cells in specimens of both exper-

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Fig. 7. Fragments of testis of A. domesticus. (A–C) The control specimen. (A) Light microscope. Bar = 70 ␮m. (B) TEM. Bar = 1 ␮m. (C) TEM. Bar = 0.5 ␮m. (D–F) Specimen treated with pure graphene oxide GO. (D) Light microscope. Bar = 20 ␮m. (E) TEM. Bar = 1 ␮m. (F) TEM. Bar = 0.5 ␮m. (G–I) Specimen treated with graphene oxide manganese ion contaminated (GO + Mn2+ ). (G) Light microscope. Bar = 20 ␮m. (H) TEM. Bar = 1 ␮m. (I) TEM. Bar = 0.5 ␮m. Centrioles (c), nucleus (n), testis follicle (t).

imental groups. The activation of autophagy has been described as the mechanism that participates in regulating the turnover and removal of organelles and proteins. Therefore, it protects the cell against extracellular factors [47,48]. The numerous degenerative changes that we observed in the structure of the male germ cells of the crickets that consumed the food with graphene oxide was the most important finding of this study: the cytoplasm became electron-lucent and the number of organelles decreased. Literature information about the effects of graphene oxide on reproductive structures are extremely scarce. Zhao et al. [43] demonstrated that GO can reduce reproductive ability by inducing damage during the development of the gonad of Caenorhabditis elegans. Cell cycle arrest, apoptosis and increased DNA damage were also observed. Moreover, graphene oxide caused a slight delay in the hatching of zebrafish embryos [45]. Apparently, the GO that is consumed with food by crickets can reach both the

primary targeted organs such as the intestinal cells and the secondary ones such as reproductive or other cells in the body. We can therefore assume that graphene oxide can migrate via the epithelium to hemolymph and is then distributed throughout the whole organism. 5. Conclusions Short-term exposure to GO in food caused as follows: an increase in the parameters of oxidative stress, damage to the DNA, and numerous degenerative changes in the tissues of Acheta domesticus. Both pure and Mn2+ contaminated GO caused the same effects. Uncontrolled use of GO in nanotechnology might lead to unpredictable consequences for various populations, especially for the organisms at the top of the food chain. Gathering more complete information about the GO risks may help us to avoid a hazard similar to the one that was caused by pesticides and other xenobiotics.

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Acknowledgement The authors thank Michele Simmons for the language correction.

[25] [26]

References

[27]

[1] R. Brayner, The toxicological impact of nanoparticles, Nano Today 3 (2008) 48–55, http://dx.doi.org/10.1016/S1748-0132(08)70015-X. [2] A. Elsaesser, C.V. Howard, Toxicology of nanoparticles, Adv. Drug Deliv. Rev. 64 (2012) 129–137, http://dx.doi.org/10.1016/j.addr.2011.09.001. [3] X. Guo, N. Mei, Assessment of the toxic potential of graphene family nanomaterials, J. Food Drug Anal. 22 (2014) 105–115, http://dx.doi.org/10. 1016/j.jfda.2014.01.009. [4] Y. Gao, X. Zou, J.X. Zhao, Y. Li, X. Su, Graphene oxide-based magnetic fluorescent hybrids for drug delivery and cellular imaging, Colloids Surf. B Biointerfaces 112 (2013) 128–133, http://dx.doi.org/10.1016/j.colsurfb.2013. 07.020. [5] J. Liu, L. Cui, D. Losic, Graphene and graphene oxide as new nanocarriers for drug delivery applications, Acta Biomater. 9 (2013) 9243–9257, http://dx.doi. org/10.1016/j.actbio.2013.08.016. [6] A. Cao, Z. Liu, S. Chu, M. Wu, Z. Ye, Z. Cai, et al., A facile one-step method to produce graphene-CdS quantum dot nanocomposites as promising optoelectronic materials, Adv. Mater. 22 (2010) 103–106, http://dx.doi.org/ 10.1002/adma.200901920. [7] J. Wang, C. Liu, Y. Shuai, X. Cui, L. Nie, Controlled release of anticancer drug using graphene oxide as a drug-binding effector in konjac glucomannan/sodium alginate hydrogels, Colloids Surf. B Biointerfaces 113 (2014) 223–229, http://dx.doi.org/10.1016/j.colsurfb.2013.09.009. [8] G. Eda, G. Fanchini, M. Chhowalla, Large-area ultrathin films of reduced graphene oxide as a transparent and flexible electronic material, Nat. Nanotechnol. 3 (2008) 270–274, http://dx.doi.org/10.1038/nnano.2008.83. [9] D.A. Dikin, S. Stankovich, E.J. Zimney, R.D. Piner, G.H.B. Dommett, G. Evmenenko, et al., Preparation and characterization of graphene oxide paper, Nature 448 (2007) 457–460, http://dx.doi.org/10.1038/nature06016. [10] G.M. Scheuermann, L. Rumi, P. Steurer, W. Bannwarth, R. Mülhaupt, Palladium nanoparticles on graphite oxide and its functionalized graphene derivatives as highly active catalysts for the Suzuki-Miyaura coupling reaction, J. Am. Chem. Soc. 131 (2009) 8262–8270, http://dx.doi.org/10.1021/ja901105a. [11] N.T.K. Thanh, L.W. Green, Functionalisation of nanoparticles for biomedical applications, Nano Today 5 (2010) 213–230, http://dx.doi.org/10.1016/j. nantod.2010.05.003. [12] Y. Wang, Z. Li, J. Wang, J. Li, Y. Lin, Graphene and graphene oxide: biofunctionalization and applications in biotechnology, Trends Biotechnol. 29 (2011) 205–212, http://dx.doi.org/10.1016/j.tibtech.2011.01.008. [13] X. Sun, Z. Liu, K. Welsher, J.T. Robinson, A. Goodwin, S. Zaric, et al., Nano-graphene oxide for cellular imaging and drug delivery, Nano Res. 1 (2008) 203–212, http://dx.doi.org/10.1007/s12274-008-8021-8. [14] Z. Liu, J.T. Robinson, X. Sun, H. Dai, PEGylated nano-graphene oxide for delivery of water insoluble cancer drugs (b), J. Am. Chem. Soc. 130 (2008) 10876–10877, http://dx.doi.org/10.1021/ja803688x. [15] C.F. Jones, D.W. Grainger, In vitro assessments of nanomaterial toxicity, Adv. Drug Deliv. Rev. 61 (2009) 438–456, http://dx.doi.org/10.1016/j.addr.2009.03. 005. [16] Q. Wu, Y. Zhao, J. Fang, D. Wang, Immune response is required for the control of in vivo translocation and chronic toxicity of graphene oxide, Nanoscale 6 (2014) 5894–5906, http://dx.doi.org/10.1039/c4nr00699b. [17] Y. Chang, S.-T. Yang, J.-H. Liu, E. Dong, Y. Wang, A. Cao, et al., In vitro toxicity evaluation of graphene oxide on A549 cells, Toxicol. Lett. 200 (2011) 201–210, http://dx.doi.org/10.1016/j.toxlet.2010.11.016. [18] N. Lewinski, V. Colvin, R. Drezek, Cytotoxicity of nanopartides, Small 4 (2008) 26–49, http://dx.doi.org/10.1002/smll.200700595. [19] H. Yang, C. Liu, D. Yang, H. Zhang, Z. Xi, Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition, J. Appl. Toxicol. 29 (2009) 69–78, http://dx.doi.org/10.1002/jat.1385. ˛ [20] M. Dziewiecka, J. Karpeta-Kaczmarek, M. Augustyniak, Ł. Majchrzycki, M.A. Augustyniak-Jabłokow, Evaluation of in vivo graphene oxide toxicity for Acheta domesticus in relation to nanomaterial purity and time passed from the exposure, J. Hazard. Mater. 305 (2016) 30–40, http://dx.doi.org/10.1016/j. jhazmat.2015.11.021. [21] J. Szelei, J. Woodring, M.S. Goettel, G. Duke, F.X. Jousset, K.Y. Liu, et al., Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics, J. Invertebr. Pathol. 106 (2011) 394–399, http://dx.doi.org/10.1016/j.jip.2010.12.009. ´ [22] Ł. Majchrzycki, M.a. Augustyniak-Jabłokow, R. Strzelczyk, M. Mackowiak, Magnetic centres in functionalized graphene, Acta Phys. Pol. A 127 (2015) 540–542, http://dx.doi.org/10.12693/APhysPolA.127.540. [23] J. Karpeta-Kaczmarek, M. Augustyniak, M. Rost-Roszkowska, Ultrastructure of the gut epithelium in Acheta domesticus after long-term exposure to nanodiamonds supplied with food, Arthropod Struct. Dev. (2016) 1–12, http://dx.doi.org/10.1016/j.asd.2016.02.002. [24] A. Ghiselli, M. Serafini, F. Natella, C. Scaccini, Total antioxidant capacity as a tool to assess redox status: critical view and experimental data, Free Radic.

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41] [42]

[43]

[44]

[45]

[46]

[47]

[48] [49]

89

Biol. Med. 29 (2000) 1106–1114, http://dx.doi.org/10.1016/S08915849(00)00394-4. H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121–126. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254, http://dx.doi.org/10.1016/00032697(76)90527-3. M. Pavlica, G.I.V. Klobuˇcar, N. Mojaˇs, R. Erben, D. Papeˇs, Detection of DNA damage in haemocytes of zebra mussel using comet assay, Mutat. Res. Genet. Toxicol. Environ. Mutagen. 490 (2001) 209–214, http://dx.doi.org/10.1016/ S1383-5718(00)00162-5. M. Klaude, S. Eriksson, J. Nygren, G. Ahnström, The comet assay: mechanisms and technical considerations, Mutat. Res. DNA Repair 363 (1996) 89–96, http://dx.doi.org/10.1016/0921-8777(95)00063-1. ˛ M. Augustyniak, M. Gladysz, M. Dziewiecka, The Comet assay in insects – status, prospects and benefits for science, Mutat. Res. Mutat. Res. (2015) 1–10, http://dx.doi.org/10.1016/j.mrrev.2015.09.001. ˛ J. Karpeta-Kaczmarek, M. Dziewiecka, M. Augustyniak, M. Rost-Roszkowska, M. Pawlyta, Oxidative stress and genotoxic effects of diamond nanoparticles, Environ. Res. 148 (2016) 264–272, http://dx.doi.org/10.1016/j.envres.2016. 03.033. M. Rost-Roszkowska, Ultrastructural changes in the midgut epithelium of Acheta domesticus (Orthoptera: Gryllidae) during degeneration and regeneration, Morphol. Histol. Fine Struct. 101 (2008) 151–158, http://dx.doi. org/10.1603/0013-8746(2008)101[151:UCITME]2.0.CO;2. M. Rost-Roszkowska, I. Poprawa, A. Chachulska-Zymełka, Apoptosis and autophagy in the midgut epithelium of Acheta domesticus (Insecta, Orthoptera, Gryllidae), Zool. Sci. 27 (2010) 740–745, http://dx.doi.org/10. 2108/zsj.27.740. Z. Magdolenova, A. Collins, A. Kumar, A. Dhawan, V. Stone, M. Dusinska, Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles, Nanotoxicology 8 (2014) 233–278, http://dx.doi. org/10.3109/17435390.2013.773464. ˛ A.M. Jastrzebska, A.R. Olszyna, The ecotoxicity of graphene family materials: current status, knowledge gaps and future needs, J. Nanoparticle Res. 17 (2015), http://dx.doi.org/10.1007/s11051-014-2817-0. ˛ A.M. Jastrzebska, P. Kurtycz, A.R. Olszyna, Recent advances in graphene family materials toxicity investigations, J. Nanoparticle Res. 14 (2012) 1–21, http:// dx.doi.org/10.1007/s11051-012-1320-8. O. Akhavan, E. Ghaderi, A. Akhavan, Size-dependent genotoxicity of graphene nanoplatelets in human stem cells, Biomaterials 33 (2012) 8017–8025, http:// dx.doi.org/10.1016/j.biomaterials.2012.07.040. N. Asare, C. Instanes, W.J. Sandberg, M. Refsnes, P. Schwarze, M. Kruszewski, et al., Cytotoxic and genotoxic effects of silver nanoparticles in testicular cells, Toxicology 291 (2012) 65–72, http://dx.doi.org/10.1016/j.tox.2011.10.022. O. Akhavan, E. Ghaderi, H. Emamy, F. Akhavan, Genotoxicity of graphene nanoribbons in human mesenchymal stem cells, Carbon N. Y. 54 (2013) 419–431, http://dx.doi.org/10.1016/j.carbon.2012.11.058. ˛ M. Dziewiecka, J. Karpeta-Kaczmarek, M. Augustyniak, Ł. Majchrzycki, M.A. Augustyniak-Jabłokow, Evaluation of in vivo graphene oxide toxicity for Acheta domesticus in relation to nanomaterial purity and time passed from the exposure, J. Hazard. Mater. 305 (2015) 30–40, http://dx.doi.org/10.1016/j. jhazmat.2015.11.021. S. Liu, T.H. Zeng, M. Hofmann, E. Burcombe, J. Wei, R. Jiang, et al., Antibacterial activity of graphite, graphite oxide, graphene oxide, and reduced graphene oxide: membrane and oxidative stress, ACS Nano 5 (2011) 6971–6980, http:// dx.doi.org/10.1021/nn202451x. M.R. Strand, Insect hemocytes and their role in immunity, Insect Immunol. 32 (2008) 25–47, http://dx.doi.org/10.1016/B978-012373976-6.50004-5. A.F. Rowley, N.A. Ratcliffe, A histological study of wound healing and hemocyte function in the wax-moth Galleria mellonella, J. Morphol. 157 (1978) 181–199, http://dx.doi.org/10.1002/jmor.1051570206. Y. Zhao, Q. Wu, D. Wang, An epigenetic signal encoded protection mechanism is activated by graphene oxide to inhibit its induced reproductive toxicity in Caenorhabditis elegans, Biomaterials 79 (2016) 15–24, http://dx.doi.org/10. 1016/j.biomaterials.2015.11.052. A.R. Cuddihy, M.J. O’Connell, Cell-cycle responses to DNA damage in G2, Int. Rev. Cytol. 222 (2003) 99–140, http://dx.doi.org/10.1016/s00747696(02)22013-6. L. Chen, P. Hu, L. Zhang, S. Huang, L. Luo, C. Huang, Toxicity of graphene oxide and multi-walled carbon nanotubes against human cells and zebrafish, Sci. China Chem. 55 (2012) 2209–2216, http://dx.doi.org/10.1007/s11426-0124620-z. F. Parisi, M. Vidal, Epithelial delamination and migration: lessons from Drosophila, Cell Adhes. Migr. 5 (2011) 366–372, http://dx.doi.org/10.4161/ cam.5.4.17524. T. Yoshimori, Autophagy A regulated bulk degradation process inside cells, Biochem. Biophys. Res. Commun. 313 (2004) 453–458, http://dx.doi.org/10. 1016/j.bbrc.2003.07.023. N. Kourtis, N. Tavernarakis, Autophagy and cell death in model organisms, Cell Death Differ. 16 (2009) 21–30, http://dx.doi.org/10.1038/cdd.2008.120. M. Aschner, T.R. Guilarte, J.S. Schneider, W. Zheng, Manganese Recent advances in understanding its transport and neurotoxicity, Toxicol. Appl. Pharmacol. 221 (2007) 131–147, http://dx.doi.org/10.1016/j.taap.2007.03. 001.