Pretreatment with paeonol prevents the adverse effects and alters the translocation of multi-walled carbon nanotubes in nematode Caenorhabditis elegans

Abstract

Paeonol is a natural compound isolated from the root bark of Moutan. In this study, we employed the in vivo assay system of Caenorhabditis elegans to investigate the possible beneficial effects of paeonol against the toxicity of multi-walled carbon nanotubes (MWCNTs). With the aid of lifespan, brood size, locomotion behavior, and intestinal reactive oxygen species (ROS) production as endpoints, we found that pretreatment with paeonol (300–500 mg L⁻¹) inhibited the adverse effects of MWCNTs (100 mg L⁻¹) on nematodes. Pretreatment with paeonol also suppressed the translocation of MWCNTs into the body through the intestinal barrier. Moreover, pretreatment with paeonol was helpful for maintaining normal intestinal permeability and prevented the formation of dysregulated expression patterns of genes required for intestinal development in MWCNT exposed nematodes. Pretreatment with paeonol further prevented the adverse effects of MWCNTs on defecation behavior and the development of AVL and DVB neurons controlling the defecation behavior in nematodes. Therefore, both the intestinal barrier and defecation behavior may contribute greatly to the formation of beneficial effects of paeonol against the toxicity of MWCNTs and the bioavailability of MWCNTs for nematodes. In addition, our data suggest that treatment with paeonol alone might have the lifespan-extending function in nematodes.

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Introduction

Multi-walled carbon nanotubes (MWCNTs) consist of single-walled carbon tubes stacked one inside the other.¹ MWCNTs have numerous useful properties, such as large length-to-diameter ratio and surface area, which provide the potential for MWCNTs to be used in electronics, drug delivery, biomedical engineering, gene therapy, and biosensor technology.^1,2 However, along with the increased production of MWCNTs, MWCNTs may be more bioavailable to both the human and the environmental organisms.^3–5 The in vivo studies have demonstrated that MWCNT exposure could cause a series of adverse effects, at least on animals, such as pulmonary toxicity, hepatotoxicity, and immune toxicity.^6–9

To reduce the possible toxicity of carbon nanotubes (CNTs), some surface chemical modifications have been developed.^10–12 For example, poly(ethylene glycol) (PEG)-modified CNTs have been tested in preclinical studies in the fields of oncology, neurology, vaccination, and imaging.¹² Nevertheless, based on the assay of long-term (2 month) effects, PEGylation could only partially, not substantially, improve the biocompatibility of MWCNTs in mice.¹³ Therefore, in addition to the chemical modifications, developing other prevention strategies is still very necessary to prepare against the toxicity of engineered nanomaterials (ENMs).

Due to the properties of the model animal, Caenorhabditis elegans has been proven to offer a system suitable for in vivo toxicological investigations at the organism level.^14–16 A series of sublethal endpoints, such as reproduction, behavior, intestinal development, and oxidative stress, have been used to assess the adverse effects of ENMs on both the primary targeted organs such as the intestine and the secondary targeted organs such as neurons and reproductive organs in nematodes.^17–22 C. elegans has been further used for the studies on translocation and toxicological mechanisms of carbon-based ENMs, including nanodiamonds, fullerene, graphite, graphene oxide (GO), and CNTs.^23–32 In C. elegans, our previous studies have demonstrated the adverse effects of MWCNTs on the functions of both the primary and secondary targeted organs.^33,34 The in vivo translocation pattern of MWCNTs has been well described in nematodes.^33,34 Some microRNAs were further identified to be involved in the control of the toxicity and translocation of MWCNTs in nematodes.³⁵

C. elegans is a useful model for pharmacological studies, and some drugs or compounds with beneficial effects have been investigated in nematodes.^36–38 Paeonol, a natural compound isolated from the root bark of Moutan, has various pharmacological activities such as antioxidation and neuroprotection.^39,40 In the present study, we employed the in vivo C. elegans assay system to investigate the effects of administration with paeonol on the toxicity and translocation of MWCNTs. Moreover, we discussed the cellular mechanism explaining the possible beneficial effects of paeonol against the toxicity of MWCNTs in nematodes. Our study provides a pharmaceutical strategy, which may be potentially used against the adverse effects of ENMs on organisms.

Results

Effects of paeonol at the examined concentrations on nematodes

First, we investigated the effects of paeonol treatment alone on nematodes. The paeonol treatments were applied from L1-larvae to young adult stage. Interestingly, we found that treatment with 100–500 mg L⁻¹ of paeonol significantly increased the lifespan of the nematodes compared with the control (Fig. 1a, Table S1†). In contrast, treatment with 300–500 mg L⁻¹ of paeonol did not result in any evident alterations in both the brood size and the locomotion, as reflected by the endpoints of head thrash and body bend (Fig. 1b and c). Moreover, treatment with 300–500 mg L⁻¹ of paeonol did not induce a significant induction of reactive oxygen species (ROS) production in the intestine of the nematodes compared with the control (Fig. 1d). Therefore, treatment with paeonol at the examined concentrations cannot cause the adverse effects on nematodes, based on a toxicity assessment with the aid of lifespan, brood size, locomotion, and intestinal ROS production as the endpoints.

Fig. 1 Effects of paeonol treatment on nematodes. (a) Effects of paeonol treatment on lifespan of nematodes. (b) Effects of paeonol treatment on brood size of nematodes. (c) Effects of paeonol treatment on head thrash and body bend of nematodes. (d) Effects of paeonol treatment on intestinal ROS production of nematodes. The left shows the pictures of intestinal ROS production in paeonol treated nematodes, and the right indicates the comparison of relative fluorescence intensity for signals labeling ROS production in intestine of nematodes. Treatment was performed from L1-larvae to young adult stage. Bars represent means ± S.E.M.

Effects of MWCNT exposure on nematodes

Our previous studies examined the possible adverse effects of MWCNT exposure on nematodes from the L1-larvae to young adult stage.^33,34 In this study, we further investigated the effects of MWCNT exposure on nematodes from the L4-larvae for 24 h. After exposure, 0.1 mg L⁻¹ of MWCNTs did not evidently affect the lifespan of the nematodes (Fig. 2a, Table S2†). In contrast, 1 mg L⁻¹ of MWCNTs moderately reduced the lifespan of the nematodes, and 10–100 mg L⁻¹ of MWCNTs noticeably decreased the lifespan of the nematodes (Fig. 2a, Table S2†). Moreover, we found that exposure to 1–100 mg L⁻¹ of MWCNTs significantly reduced the brood size of the nematodes (Fig. 2b). Similarly, exposure to 1–100 mg L⁻¹ of MWCNTs significantly decreased the locomotion, as reflected by the endpoints of head thrash and body bend in the nematodes (Fig. 2c).

Fig. 2 Toxicity assessment of MWCNTs in nematodes. (a) Effects of MWCNT exposure on the lifespan of nematodes. (b) Effects of MWCNT exposure on brood size of nematodes. (c) Effects of MWCNT exposure on head thrash and body bend of nematodes. (d) Effects of MWCNT exposure on intestinal ROS production of nematodes. The left shows the pictures of intestinal ROS production in MWCNT exposed nematodes, and the right indicates the comparison of relative fluorescence intensity for signals labeling ROS production in intestine of nematodes. Exposure was performed from L4-larvae stage for 24 h. Bars represent means ± S.E.M. *P < 0.05 vs. control, **P < 0.01 vs. control.

It has been shown that MWCNTs may exhibit their adverse effects through induction of oxidative stress.^6,41 We further observed that exposure to 1–100 mg L⁻¹ of MWCNTs caused a significant induction of ROS production in the intestines of nematodes (Fig. 2d). These results suggest that exposure to 1–100 mg L⁻¹ of MWCNTs from larvae for 24 h will result in adverse effects in nematodes.

Effects of paeonol pre-treatment on toxicity of MWCNTs in exposed nematodes

The nematodes were pre-treated with 100, 300, or 500 mg L⁻¹ of paeonol from the L1-larvae to L4 larvae. After pre-treatment with paeonol, the examined nematodes were exposed to 100 mg L⁻¹ of MWCNTs for 24 h. We found that pre-treatment with 100, 300, or 500 mg L⁻¹ of paeonol significantly inhibited the adverse effects of MWCNTs (100 mg L⁻¹) on the lifespan of the nematodes (Fig. 3a, Table S3†). Compared with the lifespan of MWCNT exposed nematodes without treatment, pre-treatment with 100, 300, or 500 mg L⁻¹ of paeonol significantly increased the lifespan of MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 3a, Table S3†). Moreover, we observed that pre-treatment with 300 or 500 mg L⁻¹ of paeonol significantly increased both the brood size and the locomotion, as reflected by endpoints of head thrash and body bend, of MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 3b and c). In contrast, pre-treatment with 100 mg L⁻¹ of paeonol did not significantly alter the brood size and locomotion of MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 3b and c). Therefore, pre-treatment with paeonol at certain concentrations can suppress the adverse effects of MWCNTs on nematodes.

Fig. 3 Effects of pre-treatment with paeonol on toxicity of MWCNTs in exposed nematodes. (a) Effects of pre-treatment with paeonol on lifespan in MWCNT exposed nematodes. (b) Effects of pre-treatment with paeonol on brood size in MWCNT exposed nematodes. (c) Effects of pre-treatment with paeonol on head thrash or body bend in MWCNT exposed nematodes. Nematodes were pre-treated with paeonol from L1-larvae to L4-larvae stage in the presence of food and then were exposed to MWCNTs from L4-larvae stage for 24 h. Bars represent means ± S.E.M. **P < 0.01 vs. control (if not specially indicated).

Effects of paeonol pre-treatment on translocation of MWCNTs in exposed nematodes

The translocation of ENMs is a crucial factor in influencing the toxicity formation in nematodes.¹⁶ After exposure, MWCNTs (100 mg L⁻¹) were distributed in both the pharynx and the intestine of the nematodes (Fig. 4a). Moreover, we found that MWCNTs could be distributed in the reproductive organs, such as the spermatheca and the gonad, of nematodes (Fig. 4a). In addition, we observed the strong MWCNT/Rhodamine B (Rho B) signals in the tail regions of the nematodes (Fig. 4a). In contrast, pre-treatment with paeonol (500 mg L⁻¹) resulted in the main distribution of MWCNTs in the pharynx and the intestine of the nematodes (Fig. 4a). After pre-treatment with paeonol, we did not observe MWCNT/Rho B signals in either the reproductive organs or the tail regions of the nematodes (Fig. 4a). Compared with the distribution of the MWCNT/Rho B, exposure to Rho B alone caused an equal distribution of fluorescent signals in the tissues of the nematodes (Fig. 4b).

Fig. 4 Effects of pre-treatment with paeonol on translocation of MWCNTs in nematodes. (a) Translocation and distribution of MWCNT/Rho B in nematodes. Arrowhead indicates the spermatheca, and asterisks indicate pharynx (*), intestine (**), and gonad (***). Nematodes were pre-treated with paeonol (500 mg L⁻¹) from L1-larvae to L4-larvae stage in the presence of food and then were exposed to MWCNT/Rho B (100 mg L⁻¹) from L4-larvae stage for 24 h period. (b) Distribution of Rho B (100 mg L⁻¹) in nematodes.

Effects of paeonol pre-treatment on ROS production induced by MWCNTs in exposed nematodes

Along with the effects of paeonol pre-treatment on the toxicity and translocation of MWCNTs in exposed nematodes, we further observed that pretreatment with 300 or 500 mg L⁻¹ of paeonol significantly decreased the induction of intestinal ROS production in MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 5). In contrast, pre-treatment with 100 mg L⁻¹ of paeonol did not evidently alter the induction of intestinal ROS production in MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 5). Therefore, paeonol pre-treatment may regulate the toxicity of MWCNTs in exposed nematodes by influencing the induction of oxidative stress.

Fig. 5 Effects of pre-treatment with paeonol on intestinal ROS production induced by MWCNTs in exposed nematodes. The left shows the pictures of intestinal ROS production in nematodes, and the right indicates the comparison of relative fluorescence intensity for signals labeling ROS production in intestine of nematodes. Nematodes were pre-treated with paeonol from L1-larvae to L4-larvae stage in the presence of food and then were exposed to MWCNTs from L4-larvae stage for 24 h. Bars represent means ± S.E.M. **P < 0.01 vs. control (if not specially indicated).

Effects of paeonol pre-treatment on intestinal permeability in MWCNT exposed nematodes

To examine the possible effects of paeonol pre-treatment on intestinal permeability in MWCNT exposed nematodes, we employed the lipophilic fluorescent dye, Nile Red, to stain the examined nematodes. After exposure, 100 mg L⁻¹ of MWCNTs induced a significant increase in the relative fluorescence intensity of Nile Red in the intestines of the nematodes (Fig. 6a). In contrast, pre-treatment with 500 mg L⁻¹ of paeonol significantly reduced the relative fluorescence intensity of Nile Red in the intestines of MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 6a). Considering the fact that Nile Red can be used to label the fat storage in nematodes, we also stained the examined nematodes with Sudan Black, a general lipid-staining probe, which requires the nematodes to be permeabilized.^28,42 The MWCNT (100 mg L⁻¹) exposed nematodes with or without paeonol (500 mg L⁻¹) pre-treatment showed a similar Sudan Black staining result to that in control nematodes (Fig. 6b). Therefore, the MWCNT exposed nematodes may have a hyper-permeable intestinal barrier rather than an increased lipid accumulation, and paeonol pre-treatment can inhibit the formation of a hyper-permeable state at intestinal barrier in MWCNT exposed nematodes.

Fig. 6 Effects of pre-treatment with paeonol on intestinal permeability in MWCNT exposed nematodes. (a) Comparison of Nile Red staining in nematodes. The left shows the pictures of Nile Red staining, and the left shows the comparison of relative fluorescence intensities of Nile Red in nematodes. (b) Comparison of Sudan Black staining in nematodes. (c) Comparison of expression patterns for genes required for the intestinal development in nematodes. The qRT-PCR results were expressed as the relative expression ratio between targeted gene and reference tba-1 gene. Nematodes were pre-treated with paeonol from L1-larvae to L4-larvae stage in the presence of food and then were exposed to MWCNTs from L4-larvae stage for 24 h. Bars represent means ± S.E.M. **P < 0.01 vs. control (if not specially indicated).

Moreover, we found that exposure to MWCNTs (100 mg L⁻¹) altered the expression patterns of some genes, required for intestinal development.⁴³ Exposure to MWCNTs (100 mg L⁻¹) significantly increased the expression level of the lin-7 gene and decreased the expression levels of the pgp-3, gem-4, par-3, pkc-3, ajm-1, inx-3, and abts-4 genes (Fig. 6c). In contrast, pre-treatment with paeonol (500 mg L⁻¹) significantly increased the expression levels of the pgp-3, gem-4, par-3, pkc-3, ajm-1, inx-3, and abts-4 genes and decreased the expression level of the lin-7 gene in MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 6c). In C. elegans, gem-4, par-6, pkc-3, and pgp-3 genes are required for the development of microvilli on intestinal cells, inx-3 and abts-4 genes are required for the development of the baso-lateral domain of the intestine, and ajm-1 and lin-7 genes are associated with the development of the apical junction of the intestine.⁴² These results further support the beneficial role of paeonol in inhibiting the increase in intestinal permeability in MWCNT exposed nematodes.

Effects of paeonol pre-treatment on defecation behavior in MWCNT exposed nematodes

In addition to the intestinal permeability, defecation behavior is another important contributor for regulating the toxicity of ENMs in nematodes.²⁸ We further found that exposure to MWCNTs (100 mg L⁻¹) significantly increased the mean defecation cycle length compared with the control (Fig. 7a). In contrast, pre-treatment with paeonol (500 mg L⁻¹) significantly reduced the mean defecation cycle length in MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 7a).

Fig. 7 Effects of pre-treatment with paeonol on defecation behavior in MWCNT exposed nematodes. (a) Effects of pre-treatment with paeonol on mean defecation cycle length in MWCNT exposed nematodes. (b) Effects of pre-treatment with paeonol on relative size of fluorescence puncta of AVL or DVB neuron in MWCNT exposed nematodes. (c) Effects of pre-treatment with paeonol on development of AVL or DVB neuron in MWCNT exposed nematodes. The neurons (*) were indicated. Nematodes were pre-treated with paeonol from L1-larvae to L4-larvae stage in the presence of food and then were exposed to MWCNTs from the L4-larvae for 24 h. Bars represent means ± S.E.M. **P < 0.01 vs. control (if not specially indicated).

Moreover, exposure to MWCNTs (100 mg L⁻¹) significantly reduced the relative size of fluorescent puncta for the cell body of AVL or DVB neurons compared with the control (Fig. 7b and c). In contrast, we observed that pre-treatment with paeonol (500 mg L⁻¹) significantly suppressed the decrease in relative size of fluorescent puncta for the cell body of AVL or DVB neurons in MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 7b and c). Therefore, treatment with paeonol may be helpful for the excretion of MWCNTs out of the body of nematodes.

Discussion

In C. elegans, our previous studies suggested that prolonged exposure (from L1-larvae to young adult) to MWCNTs in the range of μg L⁻¹ caused adverse effects on the nematodes.^33,34 In this study, with the aid of lifespan, brood size, head thrash, and body bend as endpoints, we found that acute exposure (from L4-larvae for 24 h) to MWCNTs in the range of mg L⁻¹ induces adverse effects on the nematodes (Fig. 2a–c). Moreover, acute exposure to MWCNTs in the range of mg L⁻¹ also induces significant intestinal ROS production (Fig. 2d), suggesting the activation of oxidative stress in exposed nematodes. These results further indicate that short-term exposure to carbon-based ENMs, such as MWCNTs or GO,²⁸ only at relatively high concentrations causes adverse effects on nematodes.

It has been shown that MWCNTs are bioavailable to organisms, including both the human and the environmental organisms.^3,5 To further prevent the possible adverse effects of MWCNTs on animals, we here investigated the possible beneficial effects of paeonol against the toxicity of MWCNTs using the in vivo assay system of C. elegans. With the aid of brood size, head thrash, and body bend as the endpoints, we found that pretreatment with paeonol (300–500 mg L⁻¹) could inhibit the adverse effects of MWCNTs (100 mg L⁻¹) on the reproduction and locomotion of nematodes (Fig. 3b and c). More interestingly, pretreatment with paeonol (100–500 mg L⁻¹) could further inhibit the adverse effects of MWCNTs (100 mg L⁻¹) on the lifespans of nematodes (Fig. 3a). Therefore, administration with certain concentrations of paeonol has the potential to be against the toxicity of MWCNTs. However, the extent of the beneficial effects of paeonol against the toxicity of MWCNTs may be different based on the toxicity assessments with the aid of different endpoints.

In addition to the function to be against the toxicity of the MWCNTs, we further found that pretreatment with paeonol (500 mg L⁻¹) prevented the translocation of MWCNTs (100 mg L⁻¹) into the secondary targeted organs such as the reproductive organs through the intestinal barrier in nematodes (Fig. 4). That is, pretreatment with paeonol can not only inhibit the toxicity of MWCNTs, but also suppress the bioavailability of MWCNTs to secondary targeted organs of nematodes.

Previous studies have demonstrated the beneficial effects, such as neuroprotection, hepatocellular protection, and anti-oxidation, of paeonol on animals.^9,44–46 In this study, we observed that pre-treatment with paeonol (300–500 mg L⁻¹) could effectively suppress the induction of significant intestinal ROS production caused by exposure to MWCNTs (100 mg L⁻¹) (Fig. 5). Our data support the function of paeonol to be against the oxidative stress in animals.^39,44,47 That is, pretreatment with paeonol may inhibit the damage of MWCNT exposure on intestinal development by suppressing the induction of ROS production in intestines of nematodes.

The investigations on the development and function of the intestine support the hypothesis raised above. We observed that pretreatment with paeonol (500 mg L⁻¹) inhibited the increase in relative fluorescence intensity of the Nile Red staining signal in the intestine caused by exposure to MWCNTs (100 mg L⁻¹) (Fig. 6a), suggesting the beneficial effects of paeonol in maintaining normal intestinal permeability in MWCNT exposed nematodes. Moreover, we found that pretreatment with paeonol (500 mg L⁻¹) could prevent the formation of dysregulated expression patterns of genes required for the intestinal development in MWCNT (100 mg L⁻¹) exposed nematodes (Fig. 6c), suggesting the beneficial effects of paeonol for maintaining the normal state of intestinal development in MWCNT exposed nematodes. The proteins encoded by dysregulated genes, including pgp-3, gem-4, inx-3, abts-4, par-3, ajm-1, lin-7, and pkc-3, may be the potential targets for paeonol in nematodes. Therefore, one of the cellular mechanisms for the beneficial effects of paeonol against the toxicity of MWCNTs is that pretreatment with paeonol can be helpful for maintaining the normal developmental or physiological state of the intestinal barrier in MWCNT exposed nematodes. The sustained normal intestinal barrier may in turn help the nematodes against the translocation of MWCNTs into the secondary targeted organs and the corresponding adverse effects.

In C. elegans, defecation behavior is a crucial regulator for the formation of adverse effects on nematodes caused by carbon-based ENMs such as GO.²⁸ Similarly, we observed that exposure to MWCNTs (100 mg L⁻¹) also resulted in the increase in mean defecation cycle length in nematodes (Fig. 7a). In addition, exposure to MWCNTs (100 mg L⁻¹) reduced the relative size of fluorescence puncta for the cell body of AVL and DVB neurons (Fig. 7b), which are involved in the control of defecation behavior in nematodes.⁴⁸ Moreover, we found that pretreatment with paeonol (500 mg L⁻¹) could effectively prevent the adverse effects of MWCNTs (100 mg L⁻¹) on defecation behavior and on the development of AVL and DVB neurons (Fig. 7). Therefore, another cellular mechanism for the beneficial effects of paeonol against the toxicity of MWCNTs is that pretreatment with paeonol can be helpful for maintaining the normal defecation state in MWCNT exposed nematodes. The sustained normal defecation behavior may cause the nematodes to excrete most of the MWCNTs out of the body.

In this study, we further observed that treatment with paeonol (100–500 mg L⁻¹) alone did not evidently affect the brood size, head thrash, and body bend of nematodes (Fig. 1b and c). Treatment with paeonol (100–500 mg L⁻¹) alone also did not induce the significant intestinal ROS production in nematodes (Fig. 1d). Therefore, paeonol at the examined concentrations may be unable to cause adverse effects on nematodes, at least in the several aspects examined. Moreover, we found that treatment with paeonol (100–500 mg L⁻¹) alone could evidently prolong the lifespan of nematodes (Fig. 1a). This observation may be helpful for understanding the beneficial effects of paeonol (100 mg L⁻¹) on lifespan in MWCNT (100 mg L⁻¹) exposed nematodes. In addition, our results further indicate that treatment with paeonol alone may have the potential to extend the lifespan of nematodes.

Conclusions

In this study, our data demonstrate that pretreatment with paeonol could effectively prevent the toxicity in MWCNT exposed nematodes. Pretreatment with paeonol could further inhibit the translocation of MWCNTs into the secondary targeted organs through the intestinal barrier. Moreover, pretreatment with paeonol could inhibit the induction of significant ROS production in MWCNT exposed nematodes. For the underlying cellular mechanisms, we hypothesize that pretreatment with paeonol might be helpful for maintaining the normal physiological states of the intestinal barrier and defecation behavior, which provides an important biological basis for the nematodes against the toxicity of MWCNTs. Our data here also suggest that it may be reasonable to integrate the strategy of pharmacological prevention with the strategy of chemical modification for the aim of being against the toxicity of ENMs.

Experimental

Chemicals

MWCNTs (diameter: 10–20 nm, length: 5–15 μm) were from Shenzhen Nanotech Port Co. Ltd (Shenzhen, China). Morphology of prepared MWCNTs in K-medium was examined by transmission electron microscopy (TEM) (JEM-200CX, JEOL, Japan) (Fig. S1a†). Our previous atomic force microscopy (AFM) data indicated that the diameter of the MWCNTs was 10–20 nm.³⁴ The physiochemical characterization of the MWCNTs is summarized in Fig. S1B.† Impurity of MWCNTs was determined by an elemental inductively coupled plasma mass spectrometer (ICP-MS). The data on the MWCNTs indicated the presence of Ni and Fe impurities at concentrations not more than 0.1% (Fig. S1b†). Zeta potential was analyzed by the Nano Zetasizer (Nano ZS90, Malvern Instrument, UK). The zeta potential of MWCNTs in K-medium was −33.2 ± 2.4 mV, and the zeta potential of MWCNTs in paeonol solution was −31.9 ± 2.8 mV (Fig. S1b†). MWCNTs were dispersed in K medium to prepare the stock solution (1 mg mL⁻¹). The stock solution was sonicated for 30 min (40 kHz, 100 W) and diluted to different concentrations with K medium just prior to exposure.

Paeonol (purity: 99.2%) was purchased from Nanjing Zelang Pharmaceutical Technology Co., Ltd. (Nanjing, China). Paeonol was dissolved in ethanol to prepare the stock solution (1 mg mL⁻¹) and then diluted into the used concentrations (100, 300, and 500 mg L⁻¹) with K medium before use. All the other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

C. elegans maintenance

The used nematodes were a wild-type N2 and transgenic strain of oxIs12[Is(Punc-47::GFP)] with green fluorescent protein (GFP) labeled AVL and DVB neurons, which were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 °C as described.⁴⁹ Gravid nematodes were washed off the plates into centrifuge tubes and lysed with a bleaching mixture (0.45 M NaOH, 2% HOCl). Age synchronous populations of L1- or L4-larvae were obtained as described.⁵⁰

MWCNT exposure

MWCNT exposures were performed from L4-larvae stage for 24 h in 12-well sterile tissue culture plates at 20 °C. MWCNT exposure concentrations were 0.1, 1, 10, and 100 mg L⁻¹. After exposure, nematodes were used for toxicity assessment with lifespan, reproduction, locomotion, and intestinal ROS production as the endpoints.

Pharmacological assay

Paeonol (100, 300, and 500 mg L⁻¹) was administrated from L1-larvae to L4-larvae or from L1-larvae to young adult stage in the nematodes. To examine the possible beneficial effects of paeonol against the toxicity of MWCNTs, paeonol was administrated from L1-larvae to L4-larvae stage in the presence of food. Again, the nematodes were further exposed to 100 mg L⁻¹ of MWCNTs for 24 h.

Toxicity assessment

Reproduction was assessed by the endpoint of brood size. The method was performed as described previously.^51,52 The number of offspring at all stages beyond the egg was counted. Twenty nematodes were examined per treatment. Three replications were performed.

Locomotion was evaluated by the endpoints of head thrash and body bend. The methods were performed as described previously.^53,54 A head thrash was defined 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 nematodes corresponding to the posterior bulb of the pharynx along the y axis, assuming that the nematode was traveling along the x axis. Twenty nematodes were examined per treatment. Three replications were performed.

To examine intestinal ROS production, nematodes were transferred to 1 μmol L⁻¹ of 5′,6′-chloromethyl-2′,7′dichlorodihydro-fluorescein diacetate (CM-H2DCFDA) in 12-well sterile tissue culture plates to incubate for 3 h at 20 °C. Nematodes were mounted on 2% agar pads for the examination with a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany) at 488 nm of excitation wavelength and 510 nm of emission filter. Relative fluorescence intensities of the intestines were semi-quantified. Fifty nematodes were examined per treatment. Three replications were performed.

Lifespan assay was performed as previously described.^55,56 Hermaphrodites were transferred daily for the first 4 days of adulthood. Nematodes were checked every day. Nematodes would be scored as dead if they did not move even after repeated taps with a pick. Forty nematodes were examined per treatment. For lifespan, graphs are representative of at least three trials.

Mean defecation cycle length assay

The method was performed as described previously.⁴⁸ Individual animal was examined for a fixed number of cycles. A cycle period was defined as the interval between the initiations of two successive posterior body-wall muscle contraction steps. Thirty nematodes were examined per treatment. Three replications were performed.

Fluorescent images of AVL and DVB neurons

The method was performed as described previously.⁴⁸ Using the transgenic strain of oxIs12, the fluorescent images of AVL and DVB neurons controlling the defecation behavior⁴⁸ were captured with a Zeiss Axiocam MRm camera with a Zeiss Axioplan 2 Imaging System using SlideBook software (Intelligent Imaging Innovations). Relative sizes of fluorescence puncta for cell bodies of AVL and DVB neurons were measured as the maximum radius for assayed fluorescence puncta. Thirty nematodes were examined per treatment. Three replications were performed.

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

Total RNA was isolated from nematodes using Trizol (Invitrogen, UK) according to the manufacturer's protocols. The purities and concentrations of RNA were evaluated by OD260/280 in a spectrophotometer. The cDNA synthesis was performed in a 12.5 μL reaction volume containing 625 ng of total RNA, 0.5 mmol L⁻¹ reverse-transcript primers, 50 mmol L⁻¹ Tris-HCl, 75 mmol L⁻¹ KCl, 3 mmol L⁻¹ MgCl₂, 10 mmol L⁻¹ dithiothreitol, 20 units of ribonuclease inhibitor and 100 U of reverse transcriptase (Takara, China). After cDNA synthesis, the relative expression levels were determined by real-time PCR in an ABI 7500 real-time PCR system with the aid of Evagreen (Biotium, USA). All reactions were performed in triplicate with the same cDNA samples. All reactions were performed in a 10 μL reaction volume. The relative quantification of targeted genes required for the intestinal development in comparison to the reference tba-1 gene was determined. The final results were expressed as the relative expression ratio between targeted gene and reference gene. The primer information is shown in Table S4.† The related information for the examined genes is shown in Table S5.†

Nile Red and Sudan Black staining

The method was performed as described previously.^28,42 Nile Red (Molecular Probes, Eugene, OR) was dissolved in acetone to produce a 0.5 mg mL⁻¹ stock solution and stored at 4 °C. Stock solution was freshly diluted in phosphate buffer saline (PBS) to 1 μg mL⁻¹, and 150 μL of the diluted solution was used to stain the examined nematodes. To perform Sudan Black staining, the examined nematodes were washed in M9 buffer and fixed in 1% paraformaldehyde in M9 buffer. Nematodes were subjected to 3 freeze–thaw cycles and dehydrated through an ethanol series. Nematodes were stained overnight in a 50% saturated solution of Sudan Black in 70% ethanol, rehydrated, and then photographed. Twenty nematodes were used for each Nile Red or Sudan Black staining. Three replications were performed.

Translocation and distribution of MWCNTs

To investigate the distribution and translocation of MWCNTs in nematodes, Rho B was loaded on MWCNTs by mixing Rho B solution (1 mg mL⁻¹, 0.3 mL) with an aqueous suspension of MWCNTs (0.1 mg mL⁻¹, 5 mL), as previously described.^33,57 Unbound Rho B was removed by dialysis against distilled water over 72 h. The resulting MWCNT/Rho B was stored at 4 °C. The examined nematodes were incubated with MWCNT/Rho B at the concentration of 1 mg L⁻¹ for 3 h and washed with M9 buffer. Nematodes were observed under a fluorescence microscopy. Rho B staining alone served as the control.

Statistical analysis

All data in this article were expressed as means ± standard error of the mean (S.E.M.). Graphs were generated using Microsoft Excel software (Microsoft Corp., Redmond, WA). Statistical analysis was performed using SPSS 12.0 software (SPSS Inc., Chicago, USA). Differences between groups were determined using analysis of variance (ANOVA). The probability levels of 0.05 and 0.01 were considered to be statistically significant.

Acknowledgements

This work was supported by grants from the National Basic Research Program of China (no. 2011CB933404) and the National Natural Science Foundation of China (no. 81202233, 81172698).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra14377a

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