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. 2018 Jul 2;7(6):1061–1070. doi: 10.1039/c8tx00136g

Deficit in the epidermal barrier induces toxicity and translocation of PEG modified graphene oxide in nematodes†

Li Zhao a, Jingting Kong a, Natalia Krasteva b, Dayong Wang a,✉
PMCID: PMC6220715  PMID: 30510679

graphic file with name c8tx00136g-ga.jpgOur data provide the molecular basis for the role of epidermal barrier against toxicity and translocation of nanomaterials in organisms.

Abstract

The developmental basis for the epidermal barrier against the translocation of nanomaterials is still largely unclear in organisms. We here investigated the effect of deficits in the epidermal barrier on the translocation and toxicity of PEG modified graphene oxide (GO-PEG) in Caenorhabditis elegans. In wild-type or NR222 nematodes, GO-PEG exposure did not cause toxicity and affect the expression of epidermal-development related genes. However, GO-PEG exposure resulted in toxicity in mlt-7(RNAi) nematodes with deficit in the function of epidermal barrier. Epidermal RNAi knockdown of mlt-7 allowed GO-PEG accumulation and translocation into targeted organs through the epidermal barrier. Epidermal-development related proteins of BLI-1 and IFB-1 were identified as targets for MLT-7 in the regulation of GO-PEG toxicity and accounted for MLT-7 function in maintaining the epidermal barrier. AAK-2, a catalytic α subunit of AMP-activated protein kinase, was identified as another target for MLT-7 in the regulation of GO-PEG toxicity. AAK-2 functioned synergistically with BLI-1 or IFB-1 in the regulation of GO-PEG toxicity. Our data provide the molecular basis for the role of epidermal barrier against the toxicity and translocation of nanomaterials in organisms.

Introduction

Graphene and its derivatives are a novel class of carbon engineered nanomaterials (ENMs) with a single layer of sp2-bonded carbon atoms.1 Graphene oxide (GO), one of the important graphene derivatives, has chemical stability, a high coefficient of thermal conduction, amphipathicity, a large surface area, and ease of functionalization.2 Due to these unique physical and chemical properties, GO has the potential to be at least used in drug delivery, cancer therapy, bioimaging, and biosensing.3 However, some in vitro studies have suggested the cytotoxicity of GO in inducing oxidative stress, cell division inhibition, apoptosis, and mutagenicity.4–8 Moreover, some in vivo studies in mammals have suggested the potential of GO in inducing pulmonary toxicity or reproductive toxicity.9–12 To reduce the GO toxicity, certain chemical modifications have been developed.13–15 One of the surface chemical modifications is to prepare PEG modified GO (GO-PEG). GO-PEG exhibited good biocompatibility in mice fibroblast cells.15

Caenorhabditis elegans is a classic non-mammalian model animal.16 Meanwhile, because of its sensitivity to environmental exposure, C. elegans has been widely used for both toxicity assessment and toxicological study of different environmental toxicants, including carbon-based ENMs.17–22 It has been shown that the exposure to GO or multi-walled carbon nanotubes (MWCNTs) could result in toxicity in both primary targeted organs, such as intestines, and secondary targeted organs, such as neurons and reproductive organs.23–33 After exposure, GO could be translocated into both the primary targeted organs and the secondary targeted organs.18,34,35 Moreover, PEG modification can effectively reduce GO toxicity on both primary and secondary targeted organs in wild-type nematodes.35 However, the biological effects of GO-PEG on nematodes with certain deficits in the biological barrier are still largely unclear.

In C. elegans, the epidermal barrier is an extremely flexible and resilient exoskeleton that confers the function of protection against environmental toxicants.36 The normal epidermal structure can protect nematodes against the damage from environmental toxicants.36 The epidermis is a collagenous extracellular matrix (ECM), which is synthesized by the underlying ectodermal cell layer termed the hypodermis surrounding the body in nematodes.36,37 In C. elegans, MLT-7, a heme peroxidase, is predominantly expressed in the hypodermis.37,38 MLT-7 is required for proper cuticle molting and re-synthesis, and mutation of mlt-7 resulted in impaired function of the epidermal barrier.38 In this study, we investigated the effects of GO-PEG on nematodes with the deficit in function of the epidermal barrier and the underlying mechanisms. Our results demonstrated that RNAi knockdown of mlt-7-induced deficit in the function of epidermal barrier can lead to the translocation of GO-PEG into targeted organs through the epidermal barrier and induce GO-PEG toxicity in nematodes. Our data provide an important molecular basis for the role of epidermal barrier against the translocation of ENMs in animals.

Materials and methods

Preparation and characterization of GO-PEG

GO was prepared from natural graphite powder according to the modified Hummers’ method.39 GO was finally obtained by ultrasonication of the as-made graphite oxide in water. GO was functionalized with PEG to prepare GO-PEG as described previously.40 The chemical structure of PEG-amine was mPEG-CH2CH2-NH2HCl.35 The PEG content in GO-PEG was measured by thermogravimetric analysis (TGA).35 The molecular weight of PEG-amine was 20 000, and the weight% of PEG in GO-PEG was 61.4%.35

GO-PEG was characterized by atomic force microscopy (AFM, SPM-9600, Shimadzu, Japan), Raman spectroscopy, and zeta potential analysis. The GO-PEG thickness was approximately 1.0 nm in the topographic height (Fig. 1A). The GO-PEG sizes in K-medium after sonication (40 kHz, 100 W, 30 min) were mainly in the range of 30–40 nm (Fig. 1B). Raman spectroscopy was performed using 632 nm wavelength excitation (Renishaw Invia Plus laser Raman spectrometer, Renishaw, UK). The Raman spectrum of GO-PEG showed both the G band at 1583 cm–1 and the D band at 1349 cm–1 (Fig. 1C). Zeta potential was measured by the dynamic light scattering (DLS) technique using a Nano Zetasizer (Malvern Instrument Ltd, UK). The zeta potential of GO-PEG in K-medium was –10.5 ± 2.2 mV. Our previous X-ray photoelectron spectroscopy (XPS) assay has indicated that the oxygen content in GO-PEG was as high as 29.6%.35 The intense peaks that correspond to the covalent bonds between carbon and oxygen atoms could be detected in GO-PEG, which may largely be due to the formation of high levels of oxygen-rich ether groups in PEG.35

Fig. 1. Physiochemical properties of GO-PEG. (A) AFM analysis of GO-PEG after sonication. (B) Size distribution of GO-PEG after sonication. (C) Raman spectrum of GO-PEG.

Fig. 1

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C. elegans strains and culture

Nematodes used in this study were wild-type N2 and the transgenic strain of NR222/rde-1(ne219);kzIs9. To confirm the function of epidermal MLT-7 in the regulation of GO-PEG toxicity, we employed the NR222 strain to perform the epidermal-specific RNAi knockdown of mlt-7.41 Nematodes were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 as a food source at 20 °C.16 Gravid nematodes were lysed with a bleaching mixture (0.45 mol L–1 NaOH, 2% HOCl) to separate eggs and worms. The collected eggs were then used to prepare the age synchronous L1-larvae or L2-larvae populations.

Exposure and toxicity assessment

A stock solution of GO-PEG (1 mg mL–1) was prepared in K medium by sonication for 30 min (40 kHz, 100 W). GO-PEG at the working concentrations (1, 10, and 100 mg L–1) was prepared by diluting the stock solution with K medium. Prolonged exposure to GO-PEG was performed from L1-larvae or L2-larvae to adult day-1 in liquid at 20 °C in the presence of food (OP50).

We used the endpoint of intestinal reactive oxygen species (ROS) production to reflect the functional state of the primarily targeted organ, the intestine.42 ROS production was analyzed as described previously.43,44 After GO-PEG exposure, we incubated the examined nematodes with 1 μmol L–1 5′,6′-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CM-H2DCFDA; Molecular Probes) solution for 3 h in the dark. After the labeling, the nematodes mounted on a 2% agar pad were observed and examined at 488 nm of excitation wavelength and 510 nm of emission filter under a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany). The relative fluorescence intensity of the intestinal ROS signals was semi-quantified. Thirty nematodes were examined.

Distribution and translocation of GO-PEG in the body of nematodes

To investigate the distribution and translocation of GO-PEG in nematodes, Rhodamine B (Rho B) was loaded on GO-PEG by mixing the Rho B solution (1 mg mL–1, 0.3 mL) with an aqueous suspension of GO-PEG (0.1 mg mL–1, 5 mL) as previously described.35,45 The unbound Rho B was removed by dialysis against distilled water over 72 h. Nematodes were incubated with GO-PEG/Rho B for 3 h and washed with M9 buffer. Nematodes were observed under a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany). UV/Vis spectral measurements were obtained on a PerkinElmer Lambda 25 spectrophotometer to examine the binding of Rho B with GO-PEG. Rho B treatment alone was used as a control.

Reverse-transcription and quantitative real-time polymerase chain reaction (qRT-PCR) assay

Total RNAs of nematodes were extracted using the RNeasy Mini kit (Qiagen). The prepared total RNAs were reverse transcribed using the PrimeScript ™ RT reagent kit (Takara, Otsu, Shiga, Japan). After the cDNA synthesis, real-time PCR was performed using SYBR Premix Ex Taq™ (Takara) for the amplification of PCR products. Real-time PCR was run at the optimized annealing temperature of 58 °C. The relative quantification of targeted genes in comparison with the reference tba-1 gene encoding a tubulin was performed. The final results were expressed as the relative expression ratio between the targeted gene and the reference gene. All reactions were performed in triplicate. The related primer information for qRT-PCR is shown in Table S1.†

RNA interference (RNAi) assay

RNAi assay was performed by feeding nematodes with E. coli strain HT115 (DE3) expressing double-stranded RNA that is homologous to a target gene as described.46,47 E. coli HT115 (DE3) grown in LB broth containing ampicillin (100 μg mL–1) at 37 °C overnight was plated onto NGM containing ampicillin (100 μg mL–1) and isopropyl 1-thio-β-d-galactopyranoside (IPTG, 5 mmol L–1). L2 larvae were placed on RNAi plates for 2 days at 20 °C until the nematodes became gravid. Gravid adults were transferred to fresh RNAi-expressing bacterial lawns to lay eggs to obtain the second generation of RNAi population. The RNAi efficiency was confirmed by qRT-PCR (data not shown).

DNA constructs and germline transformation

To generate an entry vector carrying promoter sequence, the promoter region for mlt-7 specially expressed in the hypodermis was amplified by PCR from wild-type C. elegans genomic DNA. This promoter fragment was inserted into the pPD95_77 vector in the sense orientation. mlt-7 cDNA was amplified by PCR and inserted into the corresponding entry vector carrying the mlt-7 promoter sequence. Germline transformation was performed as described by coinjecting a testing DNA at a concentration of 10–40 μg mL–1 and a marker DNA of Pdop-1::rfp at a concentration of 60 μg mL–1 into the gonad.48 The related primer information for DNA constructs is shown in Table S2.†

Biological permeability assay

The method was performed basically as described.49,50 The examined nematodes were suspended in the blue food dye of erioglaucine disodium (5.0% wt/vol in water) in the presence of OP50 for 3 h. After that, the nematodes were transferred onto normal NGM plates seeded with OP50 to analyze the distribution of blue food dye in the body using a microscope. Twenty nematodes were examined per treatment.

Statistical analysis

Data in this article were expressed as means ± standard deviation (SD). Statistical analysis was performed using SPSS 12.0 software (SPSS Inc., Chicago, USA). Differences between groups were determined using the analysis of variance (ANOVA), and probability levels of 0.05 and 0.01 were considered statistically significant. Graphs were generated using Microsoft Excel software (Microsoft Corp., Redmond, WA).

Results

Effect of GO-PEG exposure on MLT-7 expression

We first investigated the effect of GO-PEG exposure on the transcriptional expression of mlt-7. After prolonged exposure, we found that GO-PEG at all the examined concentrations (1–100 mg L–1) did not significantly affect the transcriptional expression of mlt-7 (Fig. S1A†). To further investigate the effect of GO-PEG exposure on MLT-7 expression, we generated the MLT-7::GFP transgenic strain. MLT-7 is expressed in the nuclei of hypodermal cells in adult nematodes.38 After prolonged exposure, GO-PEG at a concentration of 10 mg L–1 also did not affect the expression of MLT-7::GFP (Fig. S1B†).

Epidermal-specific RNAi knockdown of mlt-7 induced the GO-PEG toxicity

In the NR222 strain, we did not observe the significant induction of intestinal ROS production (Fig. 2A). Additionally, GO-PEG exposure (10 mg L–1) did not result in the significant induction of intestinal ROS production in NR222 nematodes (Fig. 2A). In contrast, GO-PEG exposure (10 mg L–1) caused the significant induction of intestinal ROS production in nematodes with epidermal-specific RNAi knockdown of mlt-7 (Fig. 2A).

Fig. 2. Effect of epidermal-specific RNAi knockdown of mlt-7 on GO-PEG toxicity in inducing intestinal ROS production (A) and epidermal permeability (B). Arrowheads indicate the body cavity. The asterisk indicates the intestinal cells. Prolonged exposure was performed from L2-larvae to adult day-1. GO-PEG exposure concentration was 10 mg L–1. Bars represent means ± SD. **P < 0.01 vs. NR222.

Fig. 2

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Under normal conditions, the blue dye is distributed in the intestinal lumen in NR222 nematodes (Fig. 2B). In contrast, epidermal-specific RNAi knockdown of mlt-7 resulted in the translocation of the blue dye into the body cavity through the epidermal barrier under normal conditions (Fig. 2B). These results confirm that MLT-7 acts in the epidermis to regulate the GO-PEG toxicity induction and epidermal permeability in nematodes.

Epidermal-specific RNAi knockdown of mlt-7 induced the accumulation of GO-PEG in the body of nematodes

To determine the accumulation and translocation of GO-PEG in the body of nematodes, we used the Rho B to label GO-PEG to prepare GO-PEG/Rho B. In NR222 nematodes, we did not observe the obvious accumulation of GO-PEG/Rho B in the body, and only weak signals were detected in the intestinal lumen (Fig. S2A†). However, in contrast, we observed the severe accumulation of GO-PEG/Rho B in the body of mlt-7(RNAi) nematodes (Fig. S2A†). In mlt-7(RNAi) nematodes, we not only observed the accumulation of GO-PEG/Rho B in the pharynx and the intestine, but also detected the accumulation of GO-PEG/Rho B in reproductive organs, such as the spermatheca (Fig. S2A†). For the prepared GO-PEG/Rho B, the absorption peaks for Rho B, GO-PEG, and GO-PEG/Rho B in the UV/Vis spectra suggest the binding of Rho B to GO-PEG (Fig. S2B†). In NR222 or mlt-7(RNAi) nematodes, Rho B was relatively equally distributed in different tissues (Fig. S3†).

Identification of AAK-2 as a potential target for MLT-7 in the regulation of GO-PEG toxicity

To elucidate the underlying molecular mechanisms for MLT-7 in the regulation of GO-PEG toxicity, we tried to identify the potential targets for MLT-7 in the regulation of GO-PEG toxicity. AAK-1 and AAK-2 are important targets for MLT-7 during the development.51 In C. elegans, aak-1 and aak-2 encode two catalytic α subunits of AMP-activated protein kinase (AMPK). GO-PEG exposure (10 mg L–1) did not affect the expressions of aak-1 and aak-2 in NR222 nematodes (Fig. 3A). Under normal conditions, RNAi knockdown of mlt-7 decreased the expressions of both aak-1 and aak-2 (Fig. 3A). However, after GO-PEG exposure, only the expression of aak-2 was more severely decreased by RNAi knockdown of mlt-7 compared with that without the GO-PEG exposure (Fig. 3A), implying that the AAK-2 is the potential target for MLT-7 in the regulation of GO-PEG toxicity.

Fig. 3. Identification of AAK-2 as the potential target for MLT-7 in the regulation of GO-PEG toxicity in nematodes. (A) Effects of GO-PEG exposure (10 mg L–1) on the expressions of aak-1 and aak-2. Bars represent means ± SD. **P < 0.01 vs. NR222 (control) (if not specially indicated). (B) Effect of epidermal-specific RNAi knockdown of aak-2 on the translocation of GO-PEG/Rho B. GO-PEG/Rho B exposure concentration was 10 mg L–1. (C) Effect of epidermal-specific RNAi knockdown of aak-2 on GO-PEG toxicity in inducing intestinal ROS production. GO-PEG exposure concentration was 10 mg L–1. Bars represent means ± SD. Prolonged exposure was performed from L2-larvae to adult day-1.

Fig. 3

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In C. elegans, aak-2 is expressed in almost all somatic tissues, including the hypodermis.52 We next examined the effects of epidermal-specific RNAi knockdown of aak-2 on the translocation and toxicity of GO-PEG. Using NR222 as an epidermal-specific RNAi tool, we found that the epidermal-specific RNAi knockdown of aak-2 did not cause the severe accumulation of GO-PEG/Rho B in the body of GO-PEG/Rho B exposed nematodes (Fig. 3B). In NR222 and nematodes with epidermal-specific RNAi knockdown of aak-2, Rho B was relatively equally distributed in different tissues (Fig. S3†).

Epidermal-specific RNAi knockdown of aak-2 could not cause the significant induction of intestinal ROS production under normal conditions (Fig. 3C). Meanwhile, epidermal-specific RNAi knockdown of aak-2 also did not result in the obvious toxicity of GO-PEG to nematodes as indicated by the endpoint of intestinal ROS production (Fig. 3C).

Identification of epidermal-development related proteins as potential targets for MLT-7 in the regulation of GO-PEG toxicity

In C. elegans, some proteins have been identified to be required for the developmental control of epidermis in different aspects.53–59 Besides AAK-2, we further studied whether the epidermal-development related proteins can also act as potential targets for MLT-7 in the regulation of GO-PEG toxicity. GO-PEG exposure (10 mg L–1) could not affect the expressions of the examined epidermal-development related genes (bli-1, mup-4, ifb-1, let-805, sma-1, zyg-9, unc-52, and vab-10a) in NR222 nematodes (Fig. 4A). Among the examined epidermal-development related genes, RNAi knockdown of mlt-7 significantly decreased the expressions of bli-1 and ifb-1 under normal conditions (Fig. 4A). Moreover, RNAi knockdown of mlt-7 caused a more severe decrease in the expressions of bli-1 and ifb-1 after the GO-PEG exposure (Fig. 4A).

Fig. 4. Identification of epidermal-development related proteins as potential targets for MLT-7 in the regulation of GO-PEG toxicity in nematodes. (A) Effects of GO-PEG exposure (10 mg L–1) on the expressions of bli-1, mup-4, ifb-1, let-805, sma-1, zyg-9, unc-52, and vab-10a. Bars represent means ± SD. **P < 0.01 vs. NR222 (control) (if not specially indicated). (B) Effect of epidermal-specific RNAi knockdown of bli-1 or ifb-1 on the translocation of GO-PEG/Rho B. Asterisks indicate the pharynx in the head and the intestine in the mid-region, respectively. Arrowheads indicate the spermatheca. GO-PEG/Rho B exposure concentration was 10 mg L–1. (C) Effect of epidermal-specific RNAi knockdown of bli-1 or ifb-1 on GO-PEG toxicity in inducing intestinal ROS production. GO-PEG exposure concentration was 10 mg L–1. Bars represent means ± SD. **P < 0.01 vs. NR222. Prolonged exposure was performed from L2-larvae to adult day-1.

Fig. 4

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In C. elegans, bli-1 is expressed in the hypodermis and the seam cells,60 and ifb-1 is expressed in various tissues including the hypodermis.61 We also investigated the effects of the epidermal-specific RNAi knockdown of bli-1 or ifb-1 on the translocation and toxicity of GO-PEG. Using NR222 as an epidermal-specific RNAi tool, we observed that the epidermal-specific RNAi knockdown of bli-1 or ifb-1 resulted in severe GO-PEG/Rho B accumulation in the body of GO-PEG/Rho B exposed nematodes (Fig. 4B). In nematodes with epidermal-specific RNAi knockdown of bli-1 or ifb-1, Rho B was also relatively equally distributed in different tissues (Fig. S3†).

Under normal conditions, epidermal-specific RNAi knockdown of bli-1 or ifb-1 did not induce significant intestinal ROS production (Fig. 4C). However, epidermal-specific RNAi knockdown of bli-1 or ifb-1 led to the significant induction of intestinal ROS production in GO-PEG exposed nematodes (Fig. 4C).

Combinational effect of AAK-2 and BLI-1 or IFB-1 in the regulation of the translocation and toxicity of GO-PEG

Moreover, we investigated the potential combinational effect of AAK-2 and BLI-1 or IFB-1 in the regulation of translocation and toxicity of GO-PEG. After GO-PEG/Rho B exposure, we observed a more severe accumulation of GO-PEG/Rho B in the body of GO-PEG/Rho B exposed nematodes with epidermal-specific RNAi knockdown of both aak-2 and bli-1 compared with that in the body of GO-PEG/Rho B exposed nematodes with epidermal-specific RNAi knockdown of bli-1 only (Fig. 5A). Similarly, a more severe accumulation of GO-PEG/Rho B was observed in the body of GO-PEG/Rho B exposed nematodes with epidermal-specific RNAi knockdown of both aak-2 and ifb-1 compared with that in the body of GO-PEG/Rho B exposed nematodes with epidermal-specific RNAi knockdown of ifb-1 only (Fig. 5A). In nematodes with epidermal-specific RNAi knockdown of both aak-2 and bli-1 and nematodes with epidermal-specific RNAi knockdown of both aak-2 and ifb-1, Rho B was relatively equally distributed in different tissues (Fig. S3†).

Fig. 5. Combinational effect of AAK-2 and BLI-1 or IFB-1 in the regulation of translocation and toxicity of GO-PEG in nematodes. (A) Combinational effect of AAK-2 and BLI-1 or IFB-1 in the regulation of translocation of GO-PEG/Rho B. Asterisks indicate the pharynx in the head and the intestine in the mid-region, respectively. Arrowheads indicate the spermatheca. GO-PEG/Rho B exposure concentration was 10 mg L–1. (B) Combinational effect of AAK-2 and BLI-1 or IFB-1 in the regulation of GO-PEG toxicity in inducing intestinal ROS production. GO-PEG exposure concentration was 10 mg L–1. Bars represent means ± SD. **P < 0.01 vs. NR222 (if not specially indicated). Prolonged exposure was performed from L2-larvae to adult day-1.

Fig. 5

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Furthermore, after GO-PEG exposure, we detected a more significant induction of intestinal ROS production in GO-PEG exposed nematodes with epidermal-specific RNAi knockdown of both aak-2 and bli-1 compared with that in GO-PEG exposed nematodes with epidermal-specific RNAi knockdown of bli-1 only (Fig. 5B). Similarly, a more significant induction of intestinal ROS production was found in GO-PEG exposed nematodes with epidermal-specific RNAi knockdown of both aak-2 and ifb-1 compared with that in GO-PEG exposed nematodes with epidermal-specific RNAi knockdown of ifb-1 only (Fig. 5B).

Effect of epidermal-specific RNAi knockdown of aak-2, bli-1, or ifb-1 on the function of epidermal barrier

Compared with NR222 strain, epidermal-specific RNAi knockdown of aak-2 did not obviously affect the function of epidermal barrier, and the blue dye was distributed in the intestinal lumen of nematodes with the epidermal-specific RNAi knockdown of aak-2 (Fig. 6). In contrast, epidermal-specific RNAi knockdown of bli-1 or ifb-1 could cause the translocation of blue dye into the body cavity through the epidermal barrier under normal conditions (Fig. 6).

Fig. 6. Epidermal permeability assay in nematodes with epidermal-specific RNAi knockdown of aak-2, bli-1, or ifb-1 under normal conditions. Arrowheads indicate the body cavity. The asterisk indicates the intestinal cells.

Fig. 6

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Discussion

In C. elegans, it is normally considered that the epidermal barrier can effectively help the animals against toxicity from environmental toxicants.59,62 MLT-7 is required for the maintenance of normal functions of the epidermal barrier.38 In wild-type nematodes, we found that both transcriptional and translational expressions of mlt-7 were not significantly affected by GO-PEG exposure at the examined concentrations (Fig. S1†). Moreover, we observed that GO-PEG exposure at a concentration of 10 mg L–1 did not affect the expressions of some epidermal-development related genes (bli-1, mup-4, ifb-1, let-805, sma-1, zyg-9, unc-52, and vab-10a) in NR222 nematodes (Fig. 4A). Our results imply that GO-PEG exposure may not be able to affect the development and function of the epidermal barrier in wild-type nematodes. Our data further confirm the crucial role of the epidermal barrier in nematodes against the adverse effects of environmental toxicants.

In wild-type nematodes, we did not detect the obvious toxicity of GO-PEG on animals.35 However, in contrast, we found that epidermal-specific RNAi knockdown of mlt-7 caused GO-PEG toxicity and induced intestinal ROS production (Fig. 2A). These results demonstrate that different from the effects in wild-type nematodes, prolonged exposure to GO-PEG may potentially cause adverse effects in nematodes with deficits in the function of epidermal barrier induced by the RNAi knockdown of mlt-7.

In this study, we further observed that RNAi knockdown of mlt-7 caused a severe accumulation of GO-PEG/Rho B at least in the pharynx, the intestines, and the spermatheca (Fig. S2†). Normally, the GO-PEG/Rho B cannot accumulate in the body of wild-type nematodes, since GO-PEG would not affect the intestinal permeability and the defecation behavior in wild-type nematodes.35,63 Therefore, the deficits in the epidermal barrier may potentially cause the translocation of GO-PEG into the targeted organs through the epidermal barrier, which in turn further leads to the toxicity in the targeted organs.

In C. elegans, bli-1 encodes a cuticular collagen, and ifb-1 encodes an intermediate filament protein. In this study, the epidermal-development related proteins of BLI-1 and IFB-1 were identified as the targets of MLT-7 in the regulation of GO-PEG toxicity. Both the BLI-1 and the IFB-1 were more severely decreased in GO-PEG exposed nematodes with epidermal-specific RNAi knockdown of mlt-7 compared with those without GO-PEG exposure (Fig. 4A). More importantly, we found that epidermal-specific RNAi knockdown of bli-1 or ifb-1 induced the severe accumulation of GO-PEG/Rho B and the significant induction of intestinal ROS production in GO-PEG exposed nematodes (Fig. 4B and C). Additionally, epidermal-specific RNAi knockdown of mlt-7, bli-1 or ifb-1 disrupted the protection function of the epidermal barrier (Fig. 2B and 6). That is, epidermal-specific RNAi knockdown of bli-1 or ifb-1 induced similar phenotypes observed in GO-PEG exposed nematodes with the epidermal-specific RNAi knockdown of mlt-7, and the decreases in BLI-1 and IFB-1 may be helpful to explain the deficits in the function of the epidermal barrier and the induction of GO-PEG toxicity in nematodes with the epidermal-specific RNAi knockdown of mlt-7 (Fig. 7).

Fig. 7. A diagram showing the molecular mechanism of MLT-7 in the regulation of GO-PEG toxicity.

Fig. 7

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In C. elegans, AAK-2 is involved in the control of adult lifespan and environmental stress.32,64 Besides some of the epidermal-development related proteins, AAK-2 was also identified as the potential target of MLT-7 in the regulation of GO-PEG toxicity. Nevertheless, we found that epidermal-specific RNAi knockdown of aak-2 not only could not induce the severe accumulation of GO-PEG/Rho B in the body of nematodes, but also could not induce the obvious toxicity of GO-PEG on nematodes (Fig. 3B and C). These results imply that the observed GO-PEG toxicity induced by the epidermal-specific RNAi knockdown of mlt-7 may not be directly due to the decrease in aak-2 expression in nematodes with the epidermal-specific RNAi knockdown of mlt-7 (Fig. 7).

We further observed a more severe GO-PEG/Rho B accumulation and a more significant GO-PEG toxicity in the nematodes with the epidermal-specific RNAi knockdown of both aak-2 and bli-1 compared with those in the nematodes with epidermal-specific RNAi knockdown of bli-1 only (Fig. 5). Similarly, we also detected a more severe GO-PEG/Rho B accumulation and a more significant GO-PEG toxicity in the nematodes with the epidermal-specific RNAi knockdown of both aak-2 and ifb-1 compared with those in the nematodes with the epidermal-specific RNAi knockdown of ifb-1 only (Fig. 5). These results demonstrate the synergistic effects of AAK-2 and BLI-1 or IFB-1 on the regulation of GO-PEG toxicity. Our results imply that in the nematodes with epidermal-specific RNAi knockdown of mlt-7, the decreased BLI-1 and IFB-1 may initiate the effect of AAK-2 suppression in inducing the susceptibility to GO-PEG by allowing the translocation of GO-PEG into the targeted organs through the epidermal barrier (Fig. 7). The AAK-2 suppression may further in turn enhance the role of decreased BLI-1 and IFB-1 in inducing the translocation of GO-PEG into the targeted organs and the GO-PEG toxicity (Fig. 7).

Conclusions

In conclusion, in this study, we determined the developmental basis for the epidermal barrier against the toxicity and translocation of GO-PEG and the underlying mechanisms. Although GO-PEG exposure could not induce toxicity in wild-type nematodes, GO-PEG exposure caused toxicity in mlt-7(RNAi) nematodes with the deficit in the function of the epidermal barrier. One of the important reasons for the induction of GO-PEG toxicity in nematodes with epidermal-specific RNAi knockdown of mlt-7 is the translocation of GO-PEG into the targeted organs through the epidermal barrier. Epidermal-development related proteins of BLI-1 and IFB-1 were identified as potential targets for MLT-7 in the regulation of GO-PEG toxicity, and BLI-1 and IFB-1 showed a similar function to MLT-7 in the regulation of the translocation and toxicity of GO-PEG. Besides BLI-1 and IFB-1, AAK-2 was further identified as another potential target for MLT-7 in the regulation of GO-PEG toxicity. AAK-2 functions synergistically with BLI-1 or IFB-1 in the regulation of the translocation and toxicity of GO-PEG. Our data imply the potential toxicity of GO-PEG in animals with deficits in the function of the epidermal barrier.

Conflicts of interest

None of the authors have any conflicting interests.

Supplementary Material

Click here for additional data file. (365.7KB, pdf)

Footnotes

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c8tx00136g

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