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Cite this: DOI: 10.1039/c4nr02688h

Molecular signals regulating translocation and toxicity of graphene oxide in the nematode Caenorhabditis elegans† Qiuli Wu, Yunli Zhao, Yiping Li and Dayong Wang* Both in vitro and in vivo studies have demonstrated the toxic effects of graphene oxide (GO). However, the molecular basis for the translocation and toxicity of GO is still largely unclear. In the present study, we employed an in vivo Caenorhabditis elegans assay system to identify molecular signals involved in the control of the translocation and toxicity of GO. We identified 7 genes whose mutations altered both the translocation and toxicity of GO. Mutations of the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 genes caused greater GO translocation into the body and toxic effects on both primary and secondary targeted organs compared with wild type; however, mutations of the isp-1 and clk-1 genes resulted in significantly decreased GO translocation into the body and toxicity on both primary and secondary targeted organs compared with wild-type. Moreover, mutations of the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 genes caused increased intestinal permeability and prolonged mean defecation cycle length in GO-exposed nematodes, whereas mutations of the isp-1 and clk-1 genes resulted in decreased

Received 16th May 2014 Accepted 12th July 2014

intestinal permeability in GO-exposed nematodes. Therefore, for the underlying mechanism, we hypothesize that both intestinal permeability and defecation behavior may have crucial roles in controlling the functions of the identified molecular signals. The molecular signals may further

DOI: 10.1039/c4nr02688h

contribute to the control of transgenerational toxic effects of GO. Our results provide an important

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insight into understanding the molecular basis for the in vivo translocation and toxicity of GO.

Introduction Graphene is a single- or few-layered sheet of sp2-bonded carbon atoms. Among the members of the graphene family, graphene oxide (GO) and its derivatives have great potential for electrical applications and biomedical uses, including bioimaging, diagnostics, and therapeutics, by taking advantage of their unique properties and 2-D structure.1–3 So far, GO has become a favored form of functionalization of graphene, because it can be adequately dispersed in water and allows further functionalization.1 However, a limited amount of data in the literature further indicate the possible adverse effects of GO on health and the environment, such as cytotoxicity, immunotoxicity, and pulmonary toxicity.4–11 Recently, several studies have been performed to examine the possible molecular mechanism for GO toxicity. The interaction of GO with toll-like receptor 4 may be the predominant molecular mechanism for GO induced macrophagic necrosis.12 A proteome analysis was performed to identify 30 differentially

Key Laboratory of Environmental Medicine Engineering in Ministry of Education, Medical School of Southeast University, Nanjing 210009, China. E-mail: dayongw@ seu.edu.cn; Tel: +86-25-83272510 † Electronic supplementary 10.1039/c4nr02688h

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expressed proteins involved in metabolic pathways, redox regulation, cytoskeleton formation, and cell growth in GOexposed hepatoma HepG2 cells.13 We recently examined the microRNA (miRNA) response to in vitro GO exposure and found that the dysregulated miRNAs may activate both a death receptor pathway and a mitochondrial pathway in GO-exposed GLC-82 cells.14 Translocation is one of the key reasons for GO-induced toxicity, and GO can be translocated or distributed into different organs or tissues in animals.5,7,15 In mice, GO mainly accumulates in the liver, lung, spleen, or kidney.5,7,15 The accumulated GO was difficult to clean by the kidneys of mice.7 Nevertheless, the molecular mechanism for the translocation and toxicity of GO is still largely unclear. Nematode Caenorhabditis elegans, a model organism having a simple and well-dened anatomy, short life cycle, and ease in handling,16 has been employed as a benchmark system for toxicological studies because of its high sensitivity to various stresses or toxicants.17–19 C. elegans can be successfully used for toxicological studies of carbon engineered nanomaterials (ENMs), such as nanodiamond, fullerene, carbon nanotubes, graphite, and graphene oxide.20–28 C. elegans has also been used for the study of translocation of carbon ENMs.20,23,25,26,28 In C. elegans, prolonged exposure to GO can induce adverse effects on nematodes, which was largely associated

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with altered permeability of the intestinal barrier and defecation behavior.28 The chemical mechanism of stressinduced GO toxicity has been further examined.23 In the present study, we attempted to examine the possible molecular basis for the translocation and toxicity of GO with the aid of a C. elegans assay system. We identied several genetic loci by screening the mutants of genes required for control of stress response or oxidative stress. Our data demonstrate the crucial roles of intestinal permeability and defecation behavior in regulating the functions of identied molecular signals to control the translocation and toxicity of GO. Our results provide an important insight into understanding the molecular basis for in vivo translocation, as well as toxicity formation of GO.

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Results and discussion Identication of genes required for control of GO translocation Considering the fact that both translocation and oxidative stress are the key basis for toxicity formation of ENMs, we employed 20 C. elegans strains with mutations of genes required for stress response or oxidative stress (Table S1†) to screen the possible genetic loci affecting the translocation of GO. GO was labeled with the molecular probe Rho B.29 Aer prolonged exposure to 100 mg L1 of GO from L1-larvae to young adult, we found that mutations of the gst-5, gst-8, gst-24, hsp-16.1, hsp16.2, mtl-1, mtl-2, mev-1, sod-1, sod-4, sod-5, par-4, or xpa-1 genes did not obviously inuence the distribution and translocation

Distributions of GO-Rho B in wild-type and mutant nematodes. (a) Pictures showing the distributions of GO-Rho B in wild-type and mutant nematodes. (b) Comparison of relative fluorescence of GO-Rho B in the intestine between wild-type and mutant nematodes. (c) Comparison of relative fluorescence of GO-Rho B in the pharynx between wild-type and mutant nematodes. (d) Comparison of relative fluorescence of GO-Rho B in the spermatheca between wild-type and mutant nematodes. The arrowheads indicate the pharynx. The intestine (**) and the spermatheca (*) are also indicated. GO exposure was performed from L1-larvae to young adult. The exposure concentration of GO was 100 mg L1. Bars represent means  SEM. **P < 0.01 vs. N2. Fig. 1

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of GO in C. elegans (Fig. 1a and b). In contrast, mutation of the hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, or isp-1 genes noticeably altered the distribution and translocation of GO (Fig. 1a and b). Mutation of the hsp-16.48, gas-1, sod-2, sod-3, or aak-2 genes signicantly increased the distribution of GO in the intestine, pharynx, and spermatheca of nematodes compared with wild-type nematodes (Fig. 1). However, mutation of the clk1 or isp-1 genes caused a signicant decrease of GO distribution in the intestine, pharynx, and spermatheca of nematodes compared with wild-type nematodes (Fig. 1). Compared with the distribution of GO-Rho B in wild-type and mutant nematodes, exposure to Rho B caused the relatively equable distribution of uorescence in tissues of wild-type and the examined mutant nematodes (Fig. S1†). In the UV/Vis spectra, the absorption peak for Rho B adsorbed on GO was red-shied by 20 nm compared with that of Rho B (Fig. S2†), which suggests the binding of Rho B to GO. In C. elegans, the hsp-16.48 gene encodes a heat-shock protein, the gas-1 gene encodes a subunit of mitochondrial complex I, the sod-2 and sod-3 genes encode manganesesuperoxide dismutase (Mn-SODs), the aak-2 gene encodes a catalytic alpha subunit of AMP-activated protein kinase, the clk1 gene encodes a ubiquinone biosynthesis protein COQ7, and the isp-1 gene encodes a “Rieske” iron–sulfur protein (Table S1†). These data imply that the hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, and isp-1 genes may regulate the translocation and toxicity formation of GO in nematodes.

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prolonged exposure to 100 mg L1 of GO, signicant intestinal ROS production was induced in wild-type nematodes.28 Aer prolonged exposure to 100 mg L1 of GO, the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 mutants exhibited a more severe induction of intestinal ROS production compared with that of wild-type nematodes, whereas the clk-1 and isp-1 mutants showed a signicantly decreased induction of intestinal ROS production compared with that of wild-type nematodes (Fig. 2). These data imply that the hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, and isp-1 genes may regulate the effects of GO on the function of the primary targeted organs in nematodes. Effects of GO on lifespan in wild-type and mutant nematodes To evaluate the long-term toxic effects of GO, we investigated the effects of GO on lifespan in wild-type and mutant nematodes. Aer prolonged exposure to 100 mg L1 of GO, we found that the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 mutants had a more severely reduced lifespan compared with that of wild-type nematodes; however, the clk-1 or isp-1 mutants showed an obvious resistance to the effects of GO on the lifespan of animals compared with that of wild-type nematodes (Fig. S3†). These results imply that the hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, and isp-1 genes may regulate the toxicity of GO induced by ROS overproduction in nematodes. Effects of GO on the function of secondary targeted organs in wild-type and mutant nematodes

Effects of GO on the function of primary targeted organs in wild-type and mutant nematodes In C. elegans, the intestine is an important primary targeted organ for ENMs.19,28 We used the endpoint of intestinal reactive oxygen species (ROS) production to assess the functional state of the intestine in the wild-type and candidate mutant nematodes. Similar to what was observed in wild-type nematodes, the hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, and isp-1 mutants had no obvious induction of intestinal ROS production (Fig. 2). Aer

In C. elegans, the reproductive organs and neurons are secondary targeted organs for ENMs.19 Our previous study indicated that prolonged exposure to 100 mg L1 of GO reduced the brood size and decreased the locomotion behavior in wildtype nematodes.28 We used the endpoints of brood size and locomotion behavior to evaluate the functional state of secondary targeted organs in wild-type and the candidate mutant nematodes. The hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk1, and isp-1 mutants had similar brood size, as well as

Fig. 2 Comparison of induction of intestinal ROS production between wild-type and mutant nematodes exposed to GO. GO exposure was performed from L1-larvae to young adult. The exposure concentration of GO was 100 mg L1. Bars represent means  SEM. **P < 0.01 vs. N2.

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GO, the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 mutant nematodes had a greater accumulation of Nile Red in the body compared with the wild-type nematodes; however, the clk-1 and isp-1 mutant nematodes showed a signicantly decreased accumulation of Nile Red in the body compared with the wildtype nematodes (Fig. 4a and b). Considering the fact that Nile Red is also a lipophilic uorescent dye used to label fat storage in nematodes,30 we also examined the triglyceride content in wild-type and mutant nematodes. Prolonged exposure to 100 mg L1 of GO did not alter the triglyceride content of the nematodes.28 The hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, and isp-1 mutant nematodes had similar triglyceride content to that of wild-type nematodes (Fig. 4c). The GO-exposed hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, and isp-1 mutant nematodes also had similar triglyceride content to that of GO-exposed wild-type nematodes (Fig. 4c). Based on these data, we conclude that the GO-exposed hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, and isp-1 mutant nematodes may have altered permeability at the intestinal barrier rather than altered lipid accumulation. Comparison of defecation behavior in GO-exposed wild-type and mutant nematodes

Comparison of brood size and locomotion behavior between wild-type and mutant nematodes exposed to GO. Brood size, number of progeny. Locomotion behavior was assessed by endpoints of head thrash frequency and body bend frequency. GO exposure was performed from L1-larvae to young adult. The exposure concentration of GO was 100 mg L1. Bars represent means  SEM. **P < 0.01 vs. N2.

Fig. 3

locomotion behavior to those of wild-type nematodes (Fig. 3). Aer prolonged exposure to 100 mg L1 of GO, the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 mutants showed a greater reduction in brood size and a greater reduction in locomotion behavior compared with those in wild-type nematodes; however, the clk-1 and isp-1 mutants exhibited a signicantly increased brood size and locomotion behavior compared with those of wild-type nematodes (Fig. 3). These results demonstrate that the hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, and isp1 genes may regulate the toxicity of GO on the function of both the primary and the secondary targeted organs in nematodes.

Another possible inuence on the translocation of ENMs is the defecation behavior in nematodes.25,26 The endpoint of mean defecation cycle length was used to assess the physiological state of defecation behavior.28 In C. elegans, mutations of the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 genes did not obviously inuence the mean defecation cycle length (Fig. 5). In contrast, mutations of clk-1 and isp-1 signicantly increased the mean defecation cycle length (Fig. 5). Previous study has demonstrated that prolonged exposure to 100 mg L1 of GO can signicantly increase the mean defecation cycle length of nematodes.28 Aer prolonged exposure to 100 mg L1 of GO, we observed a greater increase in the mean defecation cycle length in hsp-16.48, gas-1, sod-2, sod-3, and aak-2 mutant nematodes compared with that of wild-type nematodes (Fig. 5). The prolonged exposure to 100 mg L1 of GO did not noticeably inuence the mean defecation cycle length in clk-1 or isp-1 mutant nematodes (data not shown). Therefore, both intestinal permeability and defecation behavior may serve as important contributors to the altered translocation and toxicity of GO in the examined mutants.

Comparison of intestinal permeability in GO-exposed wildtype and mutant nematodes

Toxicity comparison in progeny of GO-exposed wild-type and mutant nematodes

One of the key factors in the altered translocation of ENMs is the change of intestinal permeability in nematodes.19,28 We employed the molecular probe Nile Red to evaluate the physiological state of intestinal permeability. The hsp-16.48, gas-1, sod2, sod-3, aak-2, clk-1, and isp-1 mutant nematodes had similar Nile Red staining results to those observed in wild-type nematodes and exhibited a weak accumulation of Nile Red in the body of the nematodes (Fig. 4a and b). Previous study has indicated that prolonged exposure to 100 mg L1 of GO signicantly increased the relative uorescent intensity of Nile Red in the body of nematodes.28 Aer prolonged exposure to 100 mg L1 of

In C. elegans, the altered intestinal permeability and translocation of toxic ENMs may affect the development of the progeny of exposed nematodes.19,28 Using body length as the endpoint, our previous study has suggested that the adverse effects of GO could still be detected in the progeny of nematodes exposed to 100 mg L1 of GO.28 We further investigated the possible toxicity in the progeny of wild-type and the candidate mutant nematodes exposed to 100 mg L1 of GO. Compared with the control, the progeny of GO-exposed wild-type nematodes still had a signicant induction of intestinal ROS production (Fig. S4†). Moreover, we found that the progeny of

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Fig. 4 Comparison of intestinal permeability between wild-type and mutant nematodes exposed to GO. (a) Pictures showing the Nile Red staining results in wild-type and mutant nematodes exposed to GO. (b) Comparison of relative fluorescence of Nile Red staining between wildtype and mutant nematodes exposed to GO. (c) Comparison of triglyceride amount between wild-type and mutant nematodes exposed to GO. GO exposure was performed from L1-larvae to young adult. The exposure concentration of GO was 100 mg L1. Bars represent means  SEM. **P < 0.01 vs. N2.

Fig. 5 Comparison of mean defecation cycle length between wild-type and mutant nematodes. GO exposure was performed from L1-larvae to young adult. The exposure concentration of GO was 100 mg L1. Bars represent means  SEM. **P < 0.01 vs. N2.

GO-exposed hsp-16.48, gas-1, sod-2, sod-3, and aak-2 mutant nematodes showed a more severe induction of intestinal ROS production compared with that of the progeny of GO-exposed wild-type nematodes; however, the progeny of GO-exposed clk-1 and isp-1 mutant nematodes exhibited a signicantly decreased induction of intestinal ROS production compared with that of the progeny of GO-exposed wild-type nematodes (Fig. 6a). In C. elegans, under our experimental conditions, the brood size of wild-type nematodes was approximately 281, the head thrash of wild-type nematodes was approximately 140, and the body bend of wild-type nematodes was approximately 14.2 (Fig. 3). Aer prolonged exposure to 100 mg L1 of GO, we observed a noticeable reduction of brood size and decrease in both head thrash frequency and body bend frequency in exposed wild-type nematodes (Fig. 6b and c). Furthermore, we found that the progeny of GO-exposed hsp-16.48, gas-1, sod-2, sod-3, and aak-2 mutant nematodes had a more severely decreased head thrash and body bend compared with those of the progeny of GO-exposed wild-type nematodes; however, the

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progeny of GO-exposed clk-1 and isp-1 mutant nematodes showed a signicantly increased head thrash and body bend compared with those of the progeny of GO exposed wild-type nematodes (Fig. 6b). Similarly, the progeny of GO-exposed hsp16.48, gas-1, sod-2, sod-3, and aak-2 mutant nematodes exhibited a more severely reduced brood size compared with the progeny of GO exposed wild-type nematodes, whereas the progeny of GO exposed clk-1 and isp-1 mutant nematodes showed a signicantly increased brood size compared with the progeny of GO exposed wild-type nematodes (Fig. 6c). Therefore, the hsp-16.48, gas-1, sod-2, sod-3, aak-2, clk-1, and isp-1 genes may not only regulate the effects of toxicity on GO exposed nematodes, but also inuence the formation of toxic effects on the progeny of GO exposed nematodes. The translocation and toxicity of GO in biological systems are important fundamental issues that require signicant attention.11 In the present study, we employed C. elegans as an in vivo assay system to examine the possible molecular basis for GO translocation and toxicity formation. As a model animal,

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Toxicity comparison in progeny of wild-type and mutant nematodes exposed to GO. (a) Comparison of induction of intestinal ROS production in progeny of wild-type and mutant nematodes exposed to GO. (b) Comparison of locomotion behavior in progeny of wildtype and mutant nematodes exposed to GO. (c) Comparison of brood size in progeny of wild-type and mutant nematodes exposed to GO. GO exposure was performed from L1-larvae to young adult. The exposure concentration of GO was 100 mg L1. Bars represent means  SEM. **P < 0.01 vs. N2. Fig. 6

C. elegans has many genetic resources, which are available for researchers. Considering the key role of oxidative stress or stress response in inducing the toxicity of ENMs,19 we selected the candidate mutants from mutants with mutations of genes required for the control of oxidative stress or stress response (Table S1†). Because the L1-larvae are relatively more sensitive than L4-larvae or young adults,31,32 we prolonged the exposure to assess the translocation and toxicity of GO. Aer prolonged exposure to 100 mg L1 of GO, we identied 5 mutants (hsp16.48, gas-1, sod-2, sod-3, aak-2) with greater accumulation of GO and 2 mutants (isp-1 and clk-1) with noticeably decreased accumulation of GO compared with GO-exposed wild-type nematodes (Fig. 1). These data further highlight the possible involvement of genes required for the control of oxidative stress or stress response in the regulation of translocation of ENMs. Our data also imply that the oxidative stress may act as an important inducer for the altered in vivo translocation of ENMs. Besides involvement in the regulation of GO translocation, our data also provide evidence to demonstrate the important functions of identied molecular signals in controlling the toxicity formation of GO in nematodes. Mutations of the hsp16.48, gas-1, sod-2, sod-3, and aak-2 genes strengthened the adverse effects of GO on the function of the primary targeted organ of the intestine as indicated by the observed alteration in the induction of intestinal ROS production (Fig. 2). Mutations

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of the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 genes also increased the adverse effects of GO on the functions of the secondary targeted organs of reproductive organs and neurons as indicated by the observed alterations in brood size and locomotion behavior (Fig. 3). In contrast, mutations of the isp-1 and clk-1 genes were resistant to the toxic effects of GO on functions of both the primary and the secondary targeted organs (Fig. 2 and 3). These data are largely consistent with the reported responses of mutations of these genes to other stresses or toxicants. In C. elegans, mutation of the hsp-16.48 gene increased susceptibility to the toxicity of TiO2-nanoparticles (NPs).33 Mutation of the gas-1 gene increased sensitivity to oxidative stress.34 Mutations of the sod-2 and sod-3 genes increased susceptibility to the toxicity of heavy metals or Fe2O3NPs.35,36 Mutation of the aak-2 gene increased susceptibility to the toxicity of clenbuterol or ractopamine.37 Mutations of the isp-1 and clk-1 genes increased resistance to oxidative stress.34 For the underlying mechanisms, we rst hypothesize that intestinal permeability may be crucial for the functions by which the identied molecular signals regulate GO toxicity. Mutations of the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 genes caused severely increased intestinal permeability in GO exposed nematodes, whereas mutations of the isp-1 and clk-1 genes resulted in signicantly decreased intestinal permeability in GO-exposed nematodes (Fig. 4). The altered intestinal

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permeability may contribute greatly to the formation of different translocation patterns and adverse effects of GO in hsp-16.48, gas-1, sod-2, sod-3, aak-2, isp-1, or clk-1 mutants compared to wild-type nematodes. Moreover, the changed induction of intestinal ROS production may directly induce the altered intestinal permeability in GO exposed hsp-16.48, gas-1, sod-2, sod-3, aak-2, isp-1, and clk-1 mutant nematodes compared with wild-type nematodes. Previous in vitro and in vivo studies have demonstrated that GO exposure can cause a large increase in intracellular ROS production, which contributes to the toxicity of GO.12 For the underlying mechanisms, we further hypothesize that the defecation behavior may also affect the functions by which the identied molecular signals regulate GO toxicity. Mutations of the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 genes caused a more prolonged mean defecation cycle length in GO-exposed nematodes (Fig. 5), implying that GO may be more difficult to be excreted out of the body in hsp-16.48, gas-1, sod-2, sod-3, and aak-2 mutant nematodes compared to wild-type nematodes. In contrast, the defecation behavior may not be the key reason for isp-1 and clk-1 mutants to be resistant to the GO toxicity, because the isp-1 and clk-1 mutants had a prolonged mean defecation cycle length compared with wild-type nematodes (Fig. 5). Using body length as the endpoint, our previous study has indicated that the toxicity of GO can be transferred from exposed nematodes to their progeny.28 Our data in the current study further support this notion. In the progeny of nematodes exposed to 100 mg L1 of GO, we observed decits in brood size and locomotion behavior and the induction of intestinal ROS production (Fig. 6). Moreover, we found that toxicity in the progeny of GO-exposed nematodes was also regulated by the hsp-16.48, gas-1, sod-2, sod-3, aak-2, isp-1 and clk-1 genes. The adverse effects on the progeny of GO exposed nematodes were strengthened by mutations of the hsp-16.48, gas-1, sod-2, sod-3, and aak-2 genes and suppressed by mutations of the isp-1 and clk-1 genes (Fig. 6). Therefore, the differences in transgenerational toxic effects of GO in hsp-16.48, gas-1, sod-2, sod-3, aak-2, isp-1 and clk-1 mutants may be closely associated with the altered GO translocation and intestinal permeability in these mutants.

Conclusion In the present study, we identied several molecular signals having functions in the regulation of translocation and toxicity of GO. Both positive and negative molecular signals were examined here for the regulation of GO translocation and toxicity. Our data imply that the molecular signals required for oxidative stress control may be the key inducers for the regulation of translocation of GO into the secondary targeted organs through the barrier of primary targeted organs. For the underlying mechanism, we hypothesize that both the intestinal permeability and the defecation behavior may have crucial roles in controlling the ability of the identied molecular signals to regulate the translocation and toxicity of GO. The altered translocation and intestinal permeability in GO exposed

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nematodes may further contribute to the formation of transgenerational toxic effects of GO, which was also under the control of the hsp-16.48, gas-1, sod-2, sod-3, aak-2, isp-1 and clk-1 genes. Our results provide an important molecular basis for the in vivo translocation and toxicity of GO, which may further provide important clues for our understanding of the molecular mechanism for translocation and toxicity formation of other ENMs. Considering the conserved property of molecular signaling pathways in C. elegans,17–19 the molecular mechanism for toxicity and translocation of GO in nematodes discussed in this study may also be shared by other animal models and in vitro models. Moreover, considering the fact that specic surface modications, such as PEGylated functionalization can be used to reduce the toxicity of GO and to carry specic drugs,3,38–40 the data provided here will be helpful for future chemical design to effectively reduce the toxicity of GO.

Experimental Characterization of prepared GO GO was prepared from natural graphite powder by the modied Hummers method.41 The detailed characterization information for the prepared GO including the assays of transmission electron microscopy (TEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy has been described in our previous reports.28 Aer sonication, the morphology and size distribution of GO in Kmedium (50 mmol L1 NaCl, 30 mmol L1 KCl, 10 mmol L1 NaOAc, pH 5.5)42 were examined by TEM (Fig. S5†). The TEM images were collected on a eld emission JEM-200CX transmission electron microscope, equipped with a CCD camera. A few drops of GO suspension solution were deposited on a TEM grid, dried, and evacuated before analysis. Zeta potential was analyzed by the Nano Zetasizer using a dynamic light scattering technique, and the zeta potential of GO in K medium was 22.4  1.6 mV. All the other chemicals were obtained from SigmaAldrich (St. Louis, MO, USA). Strain preparation Nematodes used in the present study were wild-type N2, and mutants for genes required for stress response or oxidative stress (Table S1†) [gst-5(ok2726), gst-8(ok3111), gst-24(ok2980), hsp-16.1(ok3622), hsp-16.2(ok1077), hsp-16.48(ok577), mtl1(tm1770), mtl-2(gk125), isp-1(qm150), gas-1(fc21), clk-1(e2519), mev-1(kn1), sod-1(tm776), sod-2(ok1030), sod-3(gk235), sod4(gk101), sod-5(tm1146), aak-2(ok524), par-4(it47), and xpa1(ok698)]. They were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20  C as described.16 Age synchronous populations of L1-larvae were obtained as previously described.43 Because a prolonged exposure C. elegans assay system can be used to assess the toxicity of ENMs at predicted environmentally relevant concentrations,44–46 prolonged exposure to GO from L1-larvae to young adult (approximately 3.5 days) was performed in 12-well sterile tissue culture plates at 20  C in the presence of food. To assess the possible adverse effects of GO on the progeny of exposed

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nematodes, eggs were obtained from animals exposed to GO by treating them with bleaching mixture, and then transferred to a new NGM plate without the addition of GO.

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of the diluted solution was used for Nile Red staining. Twenty nematodes were used for each Sudan Black staining. Analysis of triglyceride content

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Distribution of GO in nematodes To investigate the translocation and distribution of GO in nematodes, Rho B was loaded on GO by mixing Rho B solution (1 mg mL1, 0.3 mL) with an aqueous suspension of GO (0.1 mg mL1, 5 mL) as previously described.29 Unbound Rho B was removed by dialysis against distilled water over 72 h. The resulting GO-Rho B was stored at 4  C. The examined nematodes were incubated with GO-Rho B for 3 h and washed with M9 buffer. Nematodes were then observed under a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany). The UV/Vis spectral measurements were taken on a Perkin Elmer Lambda 25 spectrophotometer to examine the binding of Rho B with GO. Reproduction and locomotion behavior assays The methods were performed as described previously.33,47 To assay brood size, the number of offspring at all stages beyond the egg was counted. Twenty nematodes were examined per treatment. The locomotion behavior was evaluated by the endpoints of head thrash and body bend. To assay the locomotion behavior, the examined nematodes were washed with K medium and transferred into a microtiter well containing 60 mL of K medium on top of the agar. A head thrash was dened as a change in the direction of bending at the mid body. A body bend was counted as a change in the direction of the part of the nematode corresponding to the posterior bulb of the pharynx along the y axis, assuming that nematode was traveling along the x axis. Twenty nematodes were examined per treatment. ROS production The method was performed as previously described.48 To examine the ROS production, the nematodes were transferred to 1 mL of M9 buffer containing 1 mmol L1 50 ,60 -chloromethyl20 ,70 dichlorodihydro-uorescein diacetate (CM-H2DCFDA; Molecular Probes) in 12-well sterile tissue culture plates to incubate for 3 h at 20  C. CM-H2DCFDA can specially detect the presence of various intracellularly produced ROS species. The nematodes were then mounted on 2% agar pads for examination with a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany) with an excitation wavelength of 488 nm and an emission lter of 510 nm. Relative uorescent intensities in intestines were measured using ImageJ Soware (NIH Image). The semiquantied ROS was expressed as relative uorescent units (RFU). Fiy nematodes were examined per treatment. Nile Red staining The methods were performed as previously described.28 Nile Red (Molecular Probes, Eugene, OR) was dissolved in acetone to produce a 0.5 mg mL1 stock solution and stored at 4  C. Stock solution was freshly diluted in 1  PBS to 1 mg mL1, and 150 mL Nanoscale

Lipid was extracted from the nematodes by a previously described method.49 The triglyceride content was measured using an enzymatic kit (Wako Triglyceride E-test, Wako Pure Chemical Ltd., Osaka, Japan). Fiy replicates were performed. Defecation behavior assay The method was performed as described previously.50 To assay mean defecation cycle length, each individual animal was examined for a xed number of cycles, and a cycle period was dened as the interval between initiations of two successive posterior body-wall muscle contraction steps. Thirty nematodes were used for each mean defecation cycle length assay. Lifespan assay The lifespan assay was performed as previously described.51-52 In this test, the hermaphrodites were transferred daily for the rst 4 days of adulthood at 20  C. Further, the nematodes were checked every 2 days and would be scored as dead when they did not move even aer repeated taps with a pick. Four nematodes were examined per treatment. For lifespan, graphs are representative of at least three trials. Statistical analysis All data were presented as means  standard error of the mean (S.E.M.). Graphs were generated using Microso Excel (Microso Corp., Redmond, WA). Statistical analysis was performed using SPSS 12.0 (SPSS Inc., Chicage, USA). Differences between groups were determined using analysis of variance (ANOVA). Probability levels of 0.05 and 0.01 were considered statistically signicant.

Acknowledgements Strains used in this study were provided by the Caenorhabditis Genetics Center (funded by the NIH National Center for Research Resource, USA). This work was supported by the grants from National Basic Research Program of China (no. 2011CB33404), National Natural Science Foundation of China (no. 81172698, 81202233), Jiangsu Province Ordinary University Graduate Research and Innovation Program (no. CXZZ13_0136), Southeast University Outstanding Doctoral Foundation, and Fundamental Research Funds for the Central Universities.

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Nanoscale

Molecular signals regulating translocation and toxicity of graphene oxide in the nematode Caenorhabditis elegans.

Both in vitro and in vivo studies have demonstrated the toxic effects of graphene oxide (GO). However, the molecular basis for the translocation and t...
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