Letter pubs.acs.org/journal/estlcu
Cite This: Environ. Sci. Technol. Lett. 2018, 5, 622−628
Toxicity of Graphene Oxide in Nematodes with a Deficit in the Epidermal Barrier Caused by RNA Interference Knockdown of unc-52 Xuecheng Ding,†,‡ Qi Rui,*,† Yunli Zhao,*,§ Huimin Shao,‡ Yiping Yin,‡ Qiuli Wu,‡ and Dayong Wang*,‡ †
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Environmental Medicine Engineering in Ministry of Education, Medical School, Southeast University, Nanjing 210009, China § Department of Preventive Medicine, Bengbu Medical College, Bengbu 233030, China Downloaded via UNIV OF SOUTH DAKOTA on November 13, 2018 at 10:56:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: The epidermal barrier is important for the defense of environmental organisms against the damage from engineered nanomaterials (ENMs). We employed Caenorhabditis elegans to examine the possible influence of a deficit in the epidermal barrier caused by RNA interference (RNAi) knockdown of unc-52 encoding a perlecan protein on the toxicity of graphene oxide (GO). Epidermal RNAi knockdown of unc-52 caused a functional deficit in the epidermal barrier and susceptibility to GO toxicity. Epidermal knockdown of unc-52 decreased the level of expression of f bl-1 encoding a membrane protein fibulin and sax-7 encoding a cell adhesion receptor, and epidermal knockdown of f bl-1 or sax-7 also resulted in a functional deficit in the epidermal barrier and susceptibility to GO toxicity. Additionally, epidermal knockdown of unc-52 inhibited expression of cnc-2 and prx-11 encoding two antimicrobial proteins, and epidermal knockdown of cnc-2 or prx-11 could strengthen the GO toxicity in f bl1(RNAi) or sax-7(RNAi) nematodes. Our data further highlight the important function of the epidermal barrier against toxicity of environmental ENMs.
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development in different aspects.37−43 Among these proteins, UNC-52, a perlecan protein, is a component of the ECM.44 In nematodes, UNC-52 is an important marker of the basement ECM.38,45 However, its potential role in mediating the role of the epidermis in protecting against adverse effects of environmental toxicants and the underlying mechanism are unclear. Therefore, in this study, we tried to determine the function and underlying mechanism of UNC-25 in affecting the epidermal barrier and GO toxicity induction in nematodes. Our data demonstrate the importance of UNC-52 for the epidermal barrier in protecting against the toxicity of environmental ENMs in nematodes.
INTRODUCTION With the increase in the number of potential applications of graphene oxide (GO) in drug delivery, biosensors, bioimaging, catalysis, and environmental decontamination,1−5 its availability to organisms has received attention only gradually. The possible cytotoxicity or toxicity to targeted organs, such as the lung, in mammals has been reported recently.6−14 Caenorhabditis elegans is an animal with properties that are typical of model animals.15 Because of its sensitivity to various types of exposure,16−19 C. elegans has been successfully and widely used to assess the toxicity of various engineered nanomaterials (ENMs).20−27 In nematodes, GO can potentially induce toxicity in different aspects of the function of intestine, neurons, and reproductive organs.28−32 Biological barriers, including the epidermal barrier, are very important for the defense of animals against the toxic effects of toxicants.33,34 The underlying mechanisms for the intestinal barrier against GO toxicity have already been determined in nematodes.33 Some genes required for the epidermal barrier against adverse effects of GO have also been identified in nematodes.30 Under normal conditions, the epidermal barrier in nematodes is an extremely resilient exoskeleton, which therefore can confer an environmental protection function.35 The epidermis of nematodes is organized by the collagenous extracellular matrix (ECM).35,36 In nematodes, some proteins have been proven to have functions in regulating epidermal © 2018 American Chemical Society
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MATERIALS AND METHODS Preparation and Characterization of GO. Using a modified Hummers method, we prepared GO from natural graphite powder.46 Its thickness is 1.0 nm as analyzed by atomic force microscopy, which suggests it has one layer.47 Most GO in K medium after sonication (40 kHz, 100 W, 30 min) was 40−50 nm in size (Figure S1). Raman spectroscopy analysis indicated the presence of a G band at 1592 cm−1 and a Received: Revised: Accepted: Published: 622
September 11, 2018 October 25, 2018 October 29, 2018 October 29, 2018 DOI: 10.1021/acs.estlett.8b00473 Environ. Sci. Technol. Lett. 2018, 5, 622−628
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Environmental Science & Technology Letters
Figure 1. Effect of epidermis RNAi knockdown of unc-52 on GO toxicity. (A) Effect of epidermis RNAi knockdown of unc-52 on epidermal permeability. Arrowheads indicate the body cavity. The intestinal lumen (one asterisk) and the intestinal cells (two asterisks) are denoted. Scale bars are 50 μm. (B) Effect of epidermis RNAi knockdown of unc-52 on GO toxicity in inducing ROS production. The GO exposure concentration was 10 mg/L. GO exposure was performed with L4 larvae for 48 h. Bars represent means ± the standard deviation. **P < 0.01 vs control (if not specially indicated).
D band at 1326 cm−1.47 Its ζ potential of (10 mg/L in K medium) was −22.3 ± 2.7 mV.47 C. elegans Strains and Culture. N2 is a wild type. NR222/rde-1(ne219);kzIs9 is a genetic tool for epidermal RNA interference (RNAi) with gene(s).48 Escherichia coli OP50 was used as a food source of nematodes. 15 Hermaphrodite adult animals were lysed using a solution (0.45 M NaOH and 2% HOCl) to separate eggs from adults, which allows us to obtain age synchronous L2 larvae. Exposure. A working concentration (10 mg/L) for GO was selected as described previously.47 The working solution was prepared by diluting a stock solution (1 mg/mL) with K medium. Nematodes were exposed to GO from L4 larvae for 48 h in the presence of OP50. Induction of Reactive Oxygen Species (ROS). This end point reflects activation of oxidative stress.49,50 Animals were incubated with CM-H2DCFDA (1 μM) for 3 h in darkness. After that, the animals were analyzed using a laser scanning confocal microscope (excitation wavelength, 488 nm; emission filter, 510 nm). ROS signals in the intestine was semiquantified in comparison to the autofluorescence. For each treatment, 60 animals were analyzed. Locomotion Behavior. The method was performed as described previously.47 A head thrash was recorded as an alteration in the bending direction at midbody. A body bend was recorded as an alteration in the posterior bulb direction (y axis), assuming that the x axis is the direction of travel. For each treatment, 40 animals were analyzed. Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction (PCR). After total RNA isolation using Trizol reagent and cDNA synthesis, relative
levels of gene expression were examined in an ABI 7500 realtime PCR system with Evagreen. Triplet repeats were performed for all reactions. Quantification was reflected by the expression ratio between targeted genes and reference gene (tba-1 encoding tubulin). Information for related primer is given in Table S1. RNAi. Animals were fed E. coli HT115 (DE3) expressing double-stranded RNA for certain genes from L2 larvae to L4 larvae..51 HT115 was plated on NGM plates containing ampicillin (100 μg/mL) and isopropyl 1-thio-β-D-galactopyranoside (5 mM). The RNAi efficiency of certain genes under different genetic backgrounds is provided in Figure S2. Biological Permeability Assay. Nematodes were incubated with 5 wt %/volume erioglaucine disodium, a blue dye, for 5 h.52,53 The nematodes were analyzed for the distribution of blue dye in the body. For each experiment, 20 animals were analyzed. Statistical Analysis. Group differences were analyzed by analysis of variance (ANOVA). Probability levels of 0.05 and 0.01 were considered to be significant.
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RESULTS AND DISCUSSION Epidermal RNAi Knockdown of an unc-52-Induced Functional Deficit in the Epidermal Barrier. Under normal conditions (without the GO exposure), the blue dye of erioglaucine disodium was mainly distributed in the intestinal lumen in NR222 (Figure 1A). Under normal conditions, the severe accumulation of blue dye in body cavity and even intestinal cells could be observed in unc-25(RNAi) nematodes (Figure 1A).
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DOI: 10.1021/acs.estlett.8b00473 Environ. Sci. Technol. Lett. 2018, 5, 622−628
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Environmental Science & Technology Letters
Figure 2. Identification of FBL-1 and SAX-7 as the potential targets for epidermal UNC-52 in the regulation of GO toxicity. (A) Effect of epidermis RNAi knockdown of unc-52 on expression of cki-1, f bl-1, sax-7, and egl-17. Bars represent the standard deviation. **P < 0.01 vs NR222. (B) Effect of GO exposure on expression of unc-52, f bl-1, and sax-7. Bars represent the standard deviation. (C) Effect of epidermis RNAi knockdown of f bl-1 or sax-7 on GO toxicity in inducing ROS production. Bars represent the standard deviation. **P < 0.01 vs control (if not specially indicated). The GO exposure concentration was 10 mg/L. GO exposure was performed with L4 larvae for 48 h.
Figure 3. Identification of CNC-2 and PRX-11 as the potential targets for epidermal UNC-52 in the regulation of GO toxicity. (A) Effect of epidermis RNAi knockdown of unc-52 on expression of nlp-27, nlp-29, nlp-30, nlp-31, cnc-2, cnc-4, and prx-11. Bars represent the standard deviation. **P < 0.01 vs NR222. (B) Effect of GO exposure on the expression of cnc-2 and prx-11. Bars represent the standard deviation. **P < 0.01 vs control. (C) Effect of epidermis RNAi knockdown of cnc-2 or prx-11 on GO toxicity in inducing ROS production. Bars represent the standard deviation. **P < 0.01 vs control. The GO exposure concentration was 10 mg/L. GO exposure was performed with L4 larvae for 48 h.
Epidermal RNAi Knockdown of unc-52 Led to Susceptibility to GO Toxicity. We selected ROS production as an end point to determine the effect of RNAi knockdown of unc-52 on GO toxicity. Under normal conditions, no obvious ROS production was observed in NR222 or unc-52(RNAi) nematodes (Figure 1B). After GO exposure, more pronounced ROS production could be detected in unc-52(RNAi) nematodes than in NR222 nematodes (Figure 1B). Therefore,
epidermal RNAi knockdown of unc-52 causes susceptibility to GO toxicity. Identification of FBL-1 and SAX-7 as the Potential Targets for Epidermal UNC-52 in Controlling GO Toxicity. cki-1, f bl-1, sax-7, egl-17, and egl-20 are potential downstream targets of unc-52 in regulating various biological events.54−57 Among these genes, egl-20 encodes a Wnt ligand, and mutation of egl-20 did not influence GO toxicity.58 Under 624
DOI: 10.1021/acs.estlett.8b00473 Environ. Sci. Technol. Lett. 2018, 5, 622−628
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Environmental Science & Technology Letters normal conditions, epidermal unc-52 knockdown could not affect the transcriptional expression of cki-1 and egl-17 (Figure 2A). In contrast, epidermal unc-52 knockdown decreased the levels of transcriptional expression of f bl-1 and sax-7 under normal conditions (Figure 2B). Nevertheless, GO (10 mg/L) exposure did not obviously influence transcriptional expression of unc-52, f bl-1, or sax-7 in wild-type nematodes (Figure 2B). In nematodes, FBL-1 and SAX-7 are expressed in many tissues, including the epidermis.59,60 Moreover, under normal conditions, no obvious ROS production was detected in NR222, fbl-1(RNAi), or sax-7(RNAi) nematodes (Figure 2C). After GO (10 mg/L) exposure, more pronounced ROS production could be detected in f bl-1(RNAi) or sax-7(RNAi) nematodes than in NR222 nematodes (Figure 2C), suggesting that RNAi knockdown of f bl-1 or sax-7 induces susceptibility to GO toxicity. f bl-1 encodes a basement membrane protein fibulin, and sax-7 encodes an ortholog of human cell adhesion receptor molecule L1CAM. These results imply that FBL-1 and SAX-7 are potential targets of UNC-52 in regulating GO toxicity. Identification of CNC-2 and PRX-11 as the Potential Targets for Epidermal UNC-52 in Controlling GO Toxicity. cnc-4, cnc-2, prx-11, nlp-31, nlp-30, nlp-29, and nlp27 encoding antimicrobial proteins are expressed in the epidermis.45,61,62 Under normal conditions, epidermal unc-52 knockdown could not affect the transcriptional expression of cnc-4, nlp-31, nlp-30, nlp-29, and nlp-27; however, epidermal RNAi knockdown of unc-52 decreased the levels of transcriptional expression of cnc-2 and prx-11 (Figure 3A). Additionally, we found significant increases in the levels of expression of cnc2 and prx-11 in nematodes exposed to wild-type GO (Figure 3B). Therefore, CNC-2 and PRX-11 are other potential targets of UNC-52 in regulating GO toxicity. Moreover, under normal conditions, we could not detect the obvious ROS production in NR222, cnc-2(RNAi), or prx11(RNAi) nematodes (Figure 3C). After GO (10 mg/L) exposure, a pronounced induction of ROS production was detected in NR222, cnc-2(RNAi), or prx-11(RNAi) nematodes (Figure 3C). Epidermal RNAi Knockdown of f bl-1 or sax-7 Also Induced a Functional Deficit in the Epidermal Barrier. Like the observation in NR222 nematodes, the blue dye of erioglaucine disodium was also mainly distributed in the intestinal lumen in cnc-2(RNAi) or prx-11(RNAi) nematodes (Figure S2). On the contrary, epidermal knockdown of f bl-1 or sax-7 caused accumulation of the blue dye in the body cavity and intestinal cells (Figure S2). Antimicrobial Proteins Acted Together with Epidermal FBL-1 or SAX-7 to Regulate GO Toxicity. Although we did not observe obvious differences between GO toxicity in cnc-2(RNAi) or prx-11(RNAi) nematodes and GO toxicity in NR222 nematodes, epidermal knockdown of cnc-2 or prx-11 could significantly strengthen GO toxicity in inducing ROS production in f bl-1(RNAi) nematodes (Figure 4). Similarly, epidermal knockdown of cnc-2 or prx-11 could significantly strengthen GO toxicity in inducing ROS production in sax7(RNAi) nematodes (Figure 4). These observations indicated formation of a synergistic effect between antimicrobial proteins (CNC-2 and PRX-11) and epidermal FBL-1 or SAX-7 in regulating GO toxicity. Effect of RNAi Knockdown of unc-52, f bl-1, or sax-7 on GO Toxicity in Transgenic Strain Ex(Pges-1-sod-2). In transgenic strain Ex(Pges-1-sod-2), it was found that over-
Figure 4. Antimicrobial proteins (CNC-2 and PRX-11) acted together with epidermal FBL-1 or SAX-7 to regulate the GO toxicity in inducing ROS production. The GO exposure concentration was 10 mg/L. GO exposure was performed with L4 larvae for 48 h. Bars represent the standard deviation. **P < 0.01 vs NR222 (if not specially indicated).
expression of sod-2 encoding a mitochondrial Mn-SOD in the intestine could effectively block the toxicity of carbon-based ENMs and the translocation of carbon-based ENMs through the intestinal barrier.19,62 To confirm the function of UNC-52, FBL-1, and SAX-7 in the epidermis to regulate GO toxicity, we further performed RNAi knockdown of unc-52, f bl-1, or sax-7 in transgenic strain Ex(Pges-1-sod-2). Intestinal overexpression of SOD-2 in wild-type nematodes inhibited GO toxicity (Figure S4). In contrast, RNAi knockdown of unc-52, f bl-1, or sax-7 could cause GO toxicity in decreasing locomotion behavior and in inducing ROS production in transgenic strain Ex(Pges-1-sod-2) (Figure S4). In nematodes, normally their epidermal barrier is strong enough to protect against toxicity and translocation of environmental toxicants into the body. However, the molecular basis of the protection function of the epidermal barrier is largely unknown. In this study, we tried to determine the UNC-52-mediated molecular basis of the epidermal barrier against environmental toxicants, such as GO. Epidermal knockdown of unc-52 encoding a perlecan protein in the ECM44 could cause a functional deficit in the epidermal barrier (Figure 1A). We further identify two important targets (FBL-1 and SAX-7) for UNC-52 to explain its underlying mechanism in regulating the function of the epidermal barrier (Figure S3). We did not observe the obvious differences in vulva 625
DOI: 10.1021/acs.estlett.8b00473 Environ. Sci. Technol. Lett. 2018, 5, 622−628
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Environmental Science & Technology Letters ORCID
morphology or egg laying between NR222 and unc-52(RNAi), f bl-1(RNAi), or sax-7(RNAi) nematodes (data not shown), which suggests that the observed alteration in the blue dye staining pattern in unc-52(RNAi), f bl-1(RNAi), or sax-7(RNAi) nematodes was not due to the change in vulva development. That is, UNC-52, a component of the ECM, may be involved in the maintenance of the function of the epidermal barrier by activating membrane protein fibulin EBL-1 and cell adhesion receptor molecule SAX-7 in the epidermis, which implies the important roles of cell membrane molecules in maintaining the function of the epidermal barrier. We provided the evidence to prove the protection function of epidermal UNC-52 and its two targets, FBL-1 and SAX-7, against GO toxicity. Epidermal knockdown of unc-52, f bl-1, or sax-7 caused susceptibility to GO toxicity (Figures 1B and 2C). More importantly, RNAi knockdown of unc-52, fbl-1, or sax-7 could still cause the GO toxicity in transgenic strain Ex(Pges-1sod-2) (Figure S4), which implies that the observed GO toxicity in NR222 with knockdown of unc-52, f bl-1, or sax-7 was mainly due to the deficit in their epidermal functions. These results further support the hypothesis that UNC-52 is required for the control of the epidermal barrier against GO toxicity by activating expression of FBL-1 and SAX-7. For the underlying mechanism for the protection function of the epidermal barrier, we further found unc-52 can act upstream of cnc-2 and prx-11 encoding antimicrobial proteins to regulate GO toxicity (Figure 3). Although CNC-2 and PRX11 are not necessary for the function of the epidermal barrier (Figure S3), these two antimicrobial proteins play an important function in strengthening the protective role of the epidermal barrier against GO exposure. Therefore, in the epidermis, UNC-52 can further maintain the protective role of the epidermal barrier against GO exposure by activating expression of antimicrobial proteins (CNC-2 and PRX-11). Together, we determined the UNC-52-mediated molecular basis of the epidermal barrier to GO exposure in nematodes. We identified two mechanisms for UNC-52 in maintaining the protective role of the epidermal barrier against GO exposure. One mechanism is that UNC-52 may be involved in the maintenance of the epidermal barrier by activating membrane protein fibulin FBL-1 and cell adhesion receptor molecule SAX-7. Another mechanism is that UNC-52 can activate the expression of antimicrobial proteins (CNC-2 and PRX-11). Our results demonstrate the enhancement of the toxicity of GO in exposed unc-52(RNAi) nematodes with a deficit in the epidermal barrier.
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Dayong Wang: 0000-0003-4656-7427 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the grants from National Natural Science Foundation of China (21707002 and 21577016), the Excellent Young Talents Fund Program of Higher Education Institutions of Anhui Province (gxyqZD2016162), and the Natural Science Foundation for Colleges and Universities of Anhui Province (KJ2017A227).
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.estlett.8b00473. Table of designed primers and figures showing the confirmation of RNAi efficiency and transgenic analysis (PDF)
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DOI: 10.1021/acs.estlett.8b00473 Environ. Sci. Technol. Lett. 2018, 5, 622−628
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