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. 2016 Apr 18;5(4):1097–1105. doi: 10.1039/c6tx00022c

Metallothioneins act downstream of insulin signaling to regulate toxicity of outdoor fine particulate matter (PM2.5) during Spring Festival in Beijing in nematode Caenorhabditis elegans†

Ruilong Yang a,b, Qi Rui a,✉, Ling Kong c, Nan Zhang c, Yu Li c, Xinyu Wang c, Jing Tao c, Peiyao Tian c, Yan Ma c, Jianrong Wei c, Guojun Li c,d,✉, Dayong Wang b,✉
PMCID: PMC6060693  PMID: 30090415

graphic file with name c6tx00022c-ga.jpg mtl-1 and mtl-2 encoded metallothioneins act downstream of insulin signaling to regulate toxicity of outdoor PM2.5 during Spring Festival in Beijing.

Abstract

In this study, we performed the toxicological assessment of outdoor PM2.5 collected from Beijing during Spring Festival using the in vivo assay system of Caenorhabditis elegans. Acute exposure to outdoor PM2.5 at a concentration of 10 mg L–1 and prolonged exposure to outdoor PM2.5 at concentrations of 0.1–10 mg L–1 decreased locomotion behavior and caused significant induction of intestinal ROS production. Meanwhile, outdoor PM2.5 exposure induced significant expression of gene (mtl-1 and mtl-2) encoded metallothioneins in the intestine. Mutation of the mtl-1 or mtl-2 gene resulted in a susceptible property of nematodes to outdoor PM2.5 toxicity. Genetic assays suggested that mtl-1 and mtl-2 genes acted downstream of the daf-16 gene encoding a FOXO transcriptional factor and daf-2 gene encoding an insulin receptor in the insulin signaling pathway to regulate outdoor PM2.5 toxicity. DAF-2 further acted upstream of DAF-16 and suppressed the function of DAF-16 to regulate outdoor PM2.5 toxicity. Therefore, we identified a signaling cascade of DAF-2-DAF-16-MTL-1/2 in the control of outdoor PM2.5 toxicity in nematodes. Our study provides an important molecular basis for the potential toxicity of outdoor PM2.5 during Spring Festival in Beijing in nematodes. Especially, our study will highlight the potential adverse effects of outdoor PM2.5 during Spring Festival on environmental organisms.

Introduction

Now, many of the urban regions in the world are facing heavy air pollution. Fine particulate matter (PM), especially PM2.5, has been identified as one of the crucial contributors to the formation of air pollution in urban regions.1 So far, both experimental and epidemiological studies have demonstrated that PM2.5 exposure can cause toxicity to human health and environmental organisms by inducing oxidative stress and/or dysregulating the functions of certain organs such as cardiac or pulmonary functions in mammals.2–5

Beijing, the capital of China, is such an urban region suffering from air pollution. In recent years, a series of reports have been published to pay attention to the identification of components and contributors to PM2.5 formed in urban regions in Beijing.6–8 Certain amounts of heavy metals such as Cd, Pb, Zn, and Cu and organic toxicants such as polycyclic aromatic hydrocarbons were detected in outdoor PM2.5 collected from certain regions in Beijing.7–10 During Spring Festival in Beijing, the fireworks and crackers may potentially contribute to the air pollution at least due to the released heavy metals. PM2.5 may not only contribute to the air pollution, but also be potentially deposited into the water or soil. However, the possible toxicity of outdoor PM2.5 during Spring Festival in Beijing on environmental organisms and the underlying mechanisms are still largely unclear.

Caenorhabditis elegans has been recognized as an important non-mammalian alternative assay model for toxicological study.11,12 C. elegans has been used in toxicological assessments of different toxicants such as heavy metals, organic compounds, and engineered nanomaterials with the aid of both lethal and sub-lethal endpoints.13–17 C. elegans has the conserved properties for basic biological processes including the stress response compared with those in mammals and humans.18 Recently, the in vivo assay system of C. elegans has been further used for the toxicity assessment of PM2.5, such as traffic-related PM2.5 or coal combustion-related PM2.5.19–21

In this study, we used the in vivo assay system of C. elegans to assess the possible adverse effects of outdoor PM2.5 during Spring Festival in Beijing on environmental organisms. Moreover, we examined the important role of mtl-1 and mtl-2 genes encoding metallothioneins in the control of outdoor PM2.5 toxicity and the underlying molecular mechanism. Our results imply the potential toxicity of outdoor PM2.5 on nematodes, and further provide the molecular basis for the toxicity of outdoor PM2.5 in nematodes.

Materials and methods

Outdoor PM2.5 sample collection

Outdoor PM2.5 samples were collected from Beijing during Spring Festival in February 2013.22 The sampling site was located at Beijing Research Center for Prevention Medicine, the urban region between the 2nd and 3rd ring road. No industrial sources and heavy traffic are nearby the sampling site. At the sampling site, intelligent flow samplers (KC-120H) with a PM2.5–100 particulate cutter were used synchronously to collect samples for 24 h in 1 day for a total of 1 month. The particulate samples were collected onto glass fiber membranes, and the flow rate of the sampler was 100 L min–1. Samplers were installed on the 1.5 m platform. Filters were pre-heated before sampling at 600 °C for 2 h, and the loaded filters were stored in petri dishes in a refrigerator in the dark at about 4 °C after collection. The control outdoor PM2.5 sample was collected from Beijing in September, 2012, when it was fine weather.

Elemental analysis on the prepared outdoor PM2.5 sample

For the elemental analysis, the sample filters were digested with 3 mL concentrated HNO3, 1 mL concentrated HCl, and 1 mL concentrated solution containing HF-complexed elements. The solutions were then dried, and diluted to 10 mL with deionized water. The major elements were analyzed by inductively-coupled plasma atomic emission spectroscopy (ICP, GE Co., USA). A blank membrane was used for the blank. The experiments were repeated three times.

The elemental analysis data for outdoor PM2.5 showed that Al and Ca were the most abundant elements, followed by Ba, Pb, and Zn. The examined outdoor PM2.5 also contained moderate amounts of Cu, Mn, and As. The detailed concentrations of each element are shown in Table 1. In the control outdoor PM2.5, these heavy metals were nearly undetectable (data not shown).

Table 1. Major element concentrations in outdoor PM2.5 (n = 3).

  PM2.5 (ng M–3)
Al 2787 ± 18.5
Ca 2498 ± 14.5
Ba 460 ± 19.4
Pb 135 ± 12.4
Zn 120 ± 9.8
Cu 54.1 ± 5.4
Mn 53.5 ± 2.5
As 10.7 ± 1.2
Se 6.09 ± 0.3
La 1.65 ± 0.2
Cd 1.60 ± 0.1
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C. elegans strains

Nematodes used in the present study were wild-type N2, mutants of daf-2(e1370), daf-16(mu86), daf-16(mu86);daf-2(e1370), mtl-1(tm1770), and mtl-2(gk125), and transgenic strains of Ex(mtl-1::GFP)23 and dvIs15[mtl-2::GFP]. Some strains were from Caenorhabditis Genetics Center (funded by NIH Office of Research Infrastructure Programs (P40 OD010440)). Nematodes were maintained on nematode growth medium (NGM) plates, which were seeded with Escherichia coli OP50 at 20 °C as described.24 Gravid nematodes were lysed with a bleaching mixture (0.45 mol L–1 NaOH, 2% HOCl), and age synchronous populations of L1-larvae or young adults were prepared based on their developmental duration from the embryos as described.25

Exposure and toxicity assessment

Acute exposure was performed from young adults for 24 h, and prolonged exposure was performed from L1-larvae to young adults (approximately 4 days) in 12-well sterile tissue culture plates at 20 °C in the presence of food (OP50). Nematodes were used for toxicity assessment using endpoints of locomotion behavior and reactive oxygen species (ROS) production. Stock PM2.5 suspension solution (1 g L–1) was prepared with K-medium (51 mmol L–1 NaCl, and 32 mmol L–1 KCl), and PM2.5 at the exposure concentrations (0.1–10 mg L–1) were prepared by diluting the stock solution with K-medium. Under our experimental conditions, the outdoor PM2.5 particles were normally stable in K-medium for at least 24 h. We renewed the PM2.5 exposure solutions each 24 h.

After exposure to outdoor PM2.5, the examined nematodes were used for locomotion behavior assay with the endpoints of head thrash and body bend as described previously.26,27 Locomotion behavior was used to reflect the functional state of neurons.26 Locomotion behavior was analyzed under a dissecting microscope by eye. Head thrash was defined as the change in the direction of bending at the mid body. Body bend was counted as the 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 travelling along the x-axis. Twenty nematodes were examined per treatment, and ten replicates were performed.

After exposure to outdoor PM2.5, the examined nematodes were also used for intestinal ROS production assay as described previously.28,29 Intestinal ROS production was used to reflect the functional state of the intestine. The examined nematodes were transferred to 1 μmol L–1 of 5′,6′-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CM-H2DCFDA; Molecular Probes) to incubate for 3 h at 20 °C in the dark without addition of food. After that, nematodes mounted on 2% agar pads were examined at 488 nm excitation wavelength and with 510 nm of emission filter with a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany). The relative fluorescence intensity of the intestine was semi-quantified, and expressed as relative fluorescence units (RFU) by normalizing to the autofluorescence. Twenty nematodes were examined per treatment, and ten replicates were performed.

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

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

RNA interference (RNAi)

RNAi was performed by feeding nematodes with E. coli strain HT115 (DE3) expressing double-stranded RNA that is homologous to a target gene as described.30E. coli HT115 (DE3) grown in LB (Luria-Bertani) 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 nematodes became gravid. Gravid adults were transferred to fresh RNAi-expressing bacterial lawns to lay eggs for 2 h so as to obtain the second generation of RNAi population. Eggs were then allowed to develop at 20 °C to young adults for the subsequent assays.

Statistical analysis

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

Results

Effects of acute exposure to outdoor PM2.5 on wild-type nematodes

Using endpoints of locomotion behavior and intestinal ROS production, we first investigated the effects of acute exposure to outdoor PM2.5 on wild-type nematodes. After acute exposure, outdoor PM2.5 at concentrations of 0.1–1 mg L–1 did not significantly alter both the head thrash and the body bend in wild-type nematodes (Fig. 1a). In contrast, acute exposure to outdoor PM2.5 at a concentration of 10 mg L–1 significantly decreased both the head thrash and body bend in wild-type nematodes (Fig. 1a). Similarly, after acute exposure, we observed that outdoor PM2.5 at concentrations of 0.1–1 mg L–1 did not result in the significant induction of intestinal ROS production in wild-type nematodes; however, acute exposure to outdoor PM2.5 at a concentration of 10 mg L–1 caused significant induction of intestinal ROS production in wild-type nematodes (Fig. 1b). These results suggest that acute exposure to outdoor PM2.5 at high concentrations such as 10 mg L–1 may induce toxic effects on wild-type nematodes. Acute exposure to control outdoor PM2.5 at a concentration of 10 mg L–1 did not cause adverse effects on the locomotion behavior and induce significant intestinal ROS production in wild-type nematodes (data not shown).

Fig. 1. Effects of acute exposure to outdoor PM2.5 on wild-type nematodes. (a) Effects of acute exposure to outdoor PM2.5 on locomotion behavior in wild-type nematodes. (b) Effects of acute exposure to outdoor PM2.5 on induction of intestinal ROS production in wild-type nematodes. Acute exposure was performed from young adults for 24 h. Bars represent mean ± SEM. **P < 0.01 vs. control.

Fig. 1

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Effects of prolonged exposure to outdoor PM2.5 on wild-type nematodes

We also used the endpoints of locomotion behavior and intestinal ROS production to investigate the effects of prolonged exposure to outdoor PM2.5 on wild-type nematodes. After prolonged exposure, outdoor PM2.5 at concentrations of 0.1–10 mg L–1 significantly decreased both the head thrash and the body bend in wild-type nematodes compared with that of the control (Fig. 2a). Similarly, after prolonged exposure, we found that outdoor PM2.5 at concentrations of 0.1–10 mg L–1 led to significant induction of intestinal ROS production in wild-type nematodes compared with that of the control (Fig. 2b). These results suggest that prolonged exposure to outdoor PM2.5 at all the examined concentrations may potentially exert toxic effects on wild-type nematodes. Prolonged exposure to control outdoor PM2.5 at a concentration of 10 mg L–1 also did not cause adverse effects on the locomotion behavior and induce significant intestinal ROS production in wild-type nematodes (data not shown).

Fig. 2. Effects of prolonged exposure to outdoor PM2.5 on wild-type nematodes. (a) Effects of prolonged exposure to outdoor PM2.5 on locomotion behavior in wild-type nematodes. (b) Effects of prolonged exposure to outdoor PM2.5 on induction of intestinal ROS production in wild-type nematodes. Prolonged exposure to GO was performed from L1-larvae to young adults in the presence of food (OP50). Bars represent mean ± SEM. **P < 0.01 vs. control.

Fig. 2

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Effects of outdoor PM2.5 on expression of genes encoding metallothioneins in nematodes

In C. elegans, metallothioneins are involved in the control of stress response to heavy metals.23,31 Considering the fact that usually the collected PM2.5 from urban regions in Beijing contains a certain amount of heavy metals,7–10 we further determined the possible role of metallothioneins in the control of outdoor PM2.5 toxicity. After acute exposure, we found that outdoor PM2.5 at a concentration of 10 mg L–1 significantly increased transcriptional expression of both mtl-1 (2.93-fold of the control) and mtl-2 (2.92-fold of the control) genes in wild-type nematodes (Fig. 3a). Under normal physiological conditions, expression of mtl-1::GFP or mtl-2::GFP is nearly undetectable (Fig. 3b). However, after exposure to heavy metals such as cadmium, expression of mtl-1::GFP or mtl-2::GFP in the intestine can be noticeably activated in nematodes.23,31 Moreover, we observed that acute exposure to outdoor PM2.5 at a concentration of 10 mg L–1 induced significant expression of mtl-1::GFP or mtl-2::GFP in the intestines in nematodes (Fig. 3b). Therefore, our results imply the possible involvement of genes encoding metallothioneins in the control of outdoor PM2.5 toxicity in nematodes.

Fig. 3. Effects of outdoor PM2.5 exposure on expression of genes encoding metallothioneins in nematodes. (a) Effects of outdoor PM2.5 exposure on transcriptional expression of mtl-1 and mtl-2 genes in wild-type nematodes. (b) Effects of outdoor PM2.5 exposure on mtl-1::GFP or mtl-2::GFP expression in nematodes. Arrowheads indicate the intestine. Twenty nematodes were examined per treatment, and ten replicates were performed. Acute exposure was performed from young adults for 24 h. Bars represent mean ± SEM. **P < 0.01 vs. control.

Fig. 3

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Mutation of mtl-1 or mtl-2 gene affected toxicity formation of outdoor PM2.5 in nematodes

Mutation of the mtl-1 or mtl-2 gene does not alter locomotion behavior or induce significant intestinal ROS production in nematodes (Fig. 4). However, after acute exposure, a more severe decrease in locomotion behavior and a more significant induction of intestinal ROS production were observed in mtl-1(tm1770) or mtl-2(gk125) mutants exposed to outdoor PM2.5 (10 mg L–1) compared with those in wild-type nematodes exposed to outdoor PM2.5 (10 mg L–1) (Fig. 4). These results confirm the important function of genes encoding metallothioneins in regulating outdoor PM2.5 toxicity in nematodes. Acute exposure to control outdoor PM2.5 at a concentration of 10 mg L–1 also did not lead to adverse effects on the locomotion behavior and induce significant intestinal ROS production in mtl-1(tm1770) or mtl-2(gk125) nematodes (data not shown).

Fig. 4. Effects of mtl-1 or mtl-2 mutation on toxicity of outdoor PM2.5 exposure on nematodes. (a) Effects of mtl-1 or mtl-2 mutation on toxicity of outdoor PM2.5 exposure on locomotion behavior in nematodes. (b) Effects of mtl-1 or mtl-2 mutation on toxicity of outdoor PM2.5 exposure in inducing intestinal ROS production in nematodes. Exposure concentration of outdoor PM2.5 was 10 mg L–1. Acute exposure was performed from young adults for 24 h. Bars represent mean ± SEM. **P < 0.01 vs. wild-type.

Fig. 4

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Genetic interaction between daf-16 and genes encoding metallothioneins in regulating outdoor PM2.5 toxicity

In C. elegans, mtl-1 and mtl-2 act as the direct targeted genes for daf-16 gene encoding transcriptional factor DAF-16/FOXO in the insulin signaling pathway.32,33 We employed the endpoint of locomotion behavior to investigate the genetic interaction between daf-16 and genes encoding metallothioneins in regulating outdoor PM2.5 toxicity. Mutation of daf-16 gene, as well as RNAi knock-down of the mtl-1 or mtl-2 gene, did not obviously influence both the head thrash and the body bend in nematodes (Fig. 5a). After acute exposure, we found that mutation of the daf-16 gene, as well as RNAi knock-down of the mtl-1 or mtl-2 gene, induced a susceptible property for nematodes to outdoor PM2.5 toxicity on locomotion behavior, and the head thrash and body bend in the daf-16(mu86) mutant exposed to outdoor PM2.5 were similar to those in mtl-1(RNAi) or mtl-2(RNAi) nematodes exposed to outdoor PM2.5 (Fig. 5a). The double mutants of daf-16(mu86);mtl-1(RNAi) and daf-16(mu86);mtl-2(RNAi) exhibited the similar locomotion behavior to that of the wild-type nematodes (Fig. 5a). Moreover, after PM2.5 exposure, we observed that the head thrash and body bend in the double mutant of daf-16(mu86);mtl-1(RNAi) were similar to those in daf-16(mu86) and mtl-1(RNAi) nematodes (Fig. 5a). Similarly, after PM2.5 exposure, the head thrash and body bend in the double mutant of daf-16(mu86);mtl-2(RNAi) were similar to those in daf-16(mu86) and mtl-2(RNAi) nematodes (Fig. 5a). Therefore, our results suggest that mtl-1 or mtl-2 may act in the same genetic pathway with daf-16 in regulating outdoor PM2.5 toxicity in nematodes.

Fig. 5. Genetic interaction between daf-16 or daf-2 and genes encoding metallothioneins in regulating outdoor PM2.5 toxicity on locomotion behavior. (a) Genetic interaction between daf-16 and mtl-1 or mtl-2 in regulating outdoor PM2.5 toxicity on locomotion behavior. (b) Genetic interaction between daf-2 and mtl-1 or mtl-2 in regulating outdoor PM2.5 toxicity on locomotion behavior. Exposure concentration of outdoor PM2.5 was 10 mg L–1. Acute exposure was performed from young adults for 24 h. Bars represent mean ± SEM. **P < 0.01 vs. wild-type.

Fig. 5

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Genetic interaction between daf-2 and genes encoding metallothioneins in regulating outdoor PM2.5 toxicity

In C. elegans, the daf-2 gene encodes the insulin receptor in the insulin signaling pathway. Using the endpoint of locomotion behavior, we further investigated the genetic interaction between daf-2 and genes encoding metallothioneins in regulating outdoor PM2.5 toxicity. Mutation of the daf-2 gene also did not noticeably affect the head thrash and body bend in nematodes (Fig. 5b). Different from the phenotypes observed in the daf-16(mu86) mutant, mutation of the daf-2 gene induced a resistant property for nematodes to outdoor PM2.5 toxicity on locomotion behavior, and the head thrash and body bend in the daf-2(e1370) mutant exposed to outdoor PM2.5 were similar to those in the wild-type nematodes without outdoor PM2.5 exposure (Fig. 5b). The double mutants of daf-2(e1370);mtl-1(RNAi) and daf-2(e1370);mtl-2(RNAi) showed a similar locomotion behavior to that of the wild-type nematodes (Fig. 5b). Moreover, we found that the outdoor PM2.5 exposed daf-2(e1370);mtl-1(RNAi) mutant exhibited a similar head thrash and body bend to those in outdoor PM2.5 exposed mtl-1(RNAi) nematodes, and the outdoor PM2.5 exposed daf-2(e1370);mtl-2(RNAi) mutant exhibited a similar head thrash and body bend to those in outdoor PM2.5 exposed mtl-2(RNAi) nematodes (Fig. 5b). That is, RNAi knock-down of the mtl-1 or mtl-2 gene suppressed the resistant property of the daf-2(e1370) mutant to outdoor PM2.5 toxicity. These results suggest that mtl-1 and mtl-2 genes may act downstream of the insulin signaling pathway to regulate outdoor PM2.5 toxicity in nematodes.

Genetic interaction between daf-2 and daf-16 in regulating outdoor PM2.5 toxicity

In C. elegans, DAF-2 can inactivate DAF-16/FOXO transcriptional factor by activating some tyrosine kinases, thereby blocking the transcription of the target genes of daf-16.34 We finally investigated the genetic interaction between daf-16 and daf-2 in regulating outdoor PM2.5 toxicity. The double mutant of daf-16(mu86);daf-2(e1370) had normal locomotion behavior (Fig. 6a). After acute exposure, we found that the head thrash and body bend in the daf-16(mu86);daf-2(e1370) mutant exposed to outdoor PM2.5 were similar to those in the single mutant of daf-16(mu86) exposed to outdoor PM2.5 (Fig. 6a). Therefore, in the insulin signaling pathway, mutation of the daf-16 gene suppressed the resistant property of the daf-2(e1370) mutant to outdoor PM2.5 toxicity in nematodes.

Fig. 6. Genetic interaction between daf-2 and daf-16 in regulating outdoor PM2.5 toxicity. (a) Genetic interaction between daf-2 and daf-16 in regulating outdoor PM2.5 toxicity on locomotion behavior. Exposure concentration of outdoor PM2.5 was 10 mg L–1. Acute exposure was performed from young adults for 24 h. Bars represent mean ± SEM. **P < 0.01 vs. wild-type. (b) A diagram showing the role of metallothioneins and insulin signaling in regulating outdoor PM2.5 toxicity in nematodes.

Fig. 6

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Discussion

In this study, using the in vivo assay system of C. elegans, we found that acute exposure to outdoor PM2.5 at concentrations such as 10 mg L–1 could cause adverse effects on the functions of neurons and the intestine (Fig. 1). In contrast, prolonged exposure to outdoor PM2.5 even at a concentration of 0.1 mg L–1 could result in adverse effects on the functions of neurons and the intestine in nematodes (Fig. 2). That is, long-term exposure to outdoor PM2.5 in the range of μg L–1 may potentially induce adverse effects on environmental organisms. These results imply a possible risk of long-term exposure to outdoor PM2.5 collected from regions with heavy air pollution on environmental organisms.

In nematodes, it has been shown that the expression of genes encoding metallothioneins is an important indicator for the existence of heavy metals in a certain examined sample.31,32,35 In this study, we found that acute exposure to outdoor PM2.5 (10 mg L–1) induced a significant increase in the expression of mtl-1 and mtl-2 genes (Fig. 3), implying that the examined sample of outdoor PM2.5 may contain a certain amount of heavy metals. Heavy metals are usually considered as one of the important contributors to the toxicity formation of PM2.5 on organisms.20 Nevertheless, not all the heavy metals can induce an increase in the expression of genes encoding metallothioneins in nematodes.36 In C. elegans, exposure to cadmium, methylmercury or zinc could induce increased expression of the mtl-1 or mtl-2 gene.23,36,37 In the examined outdoor PM2.5, we at least detected the existence of a high dose of Zn (Table 1).

Metallothioneins are small cysteine-rich proteins that have a high affinity binding property for various heavy metals.38 In the present study, the increase in expression of the mtl-1 or mtl-2 gene may further imply the activation of protection mechanisms in nematodes. In the daf-2 mutant with a resistant property to cadmium toxicity, expression of both the mtl-1 and mtl-2 genes was significantly induced.32 In addition, mutation of the mtl-1 or mtl-2 genes induced a susceptible property of nematodes to outdoor PM2.5 toxicity on the functions of neurons and the intestine (Fig. 4). Moreover, we observed that exposure to outdoor PM2.5 induced noticeable expression of mtl-1::GFP and mtl-2::GFP in the intestine (Fig. 3b), which further implies that metallothioneins may encode a protection mechanism in the intestinal barrier in outdoor PM2.5 exposed nematodes. Previous studies have suggested the crucial role of the intestinal barrier against toxicity from toxicants such as toxic nanomaterials in nematodes.39–41 Together, our results imply the potential key role of metallothioneins for nematodes against possible toxicity from outdoor PM2.5.

In this study, we further identified the signaling cascade of DAF-2-DAF-16-MTL-1/2 in the control of outdoor PM2.5 toxicity (Fig. 6b). In this signaling cascade, MTL-1 and MTL-2 act as the targets for the transcriptional factor of DAF-16.32,33 Genetic interaction assay confirmed that MTL-1 or MTL-2 functioned in the same genetic pathway with DAF-16 in regulating outdoor PM2.5 toxicity (Fig. 5a). Genetic mutation of the mtl-1 or mtl-2 gene suppressed the resistant property of the daf-2 mutant to outdoor PM2.5 toxicity (Fig. 5b), which further suggests that MTL-1 and MTL-2 may function downstream of insulin signaling to regulate outdoor PM2.5 toxicity. Genetic mutation of the daf-16 gene suppressed the resistant property of the daf-2 mutant to outdoor PM2.5 toxicity (Fig. 6a), which suggests that DAF-2 acts upstream of DAF-16 and inhibits the function of DAF-16 in regulating outdoor PM2.5 toxicity. A previous study has also indicated that insulin signaling might regulate cadmium toxicity through the functions of metallothioneins in nematodes.32 Therefore, DAF-2-DAF-16-MTL-1/2 may be a conserved signaling cascade for organisms against the potential toxicity of environmental samples containing heavy metals.

Conclusions

In this study, we performed a toxicological assessment of outdoor PM2.5 collected during Spring Festival in Beijing using the in vivo assay system of C. elegans. Our data suggest that prolonged exposure to outdoor PM2.5 in the range of μg L–1 could cause adverse effects on the functions of neurons and the intestine. Meanwhile, exposure to outdoor PM2.5 induced significant expression of genes encoding metallothioneins in the intestine, implying that metallothioneins may encode a protection mechanism for nematodes against outdoor PM2 toxicity. Moreover, we identified a signaling cascade of DAF-2-DAF-16-MTL-1/2 in the control of outdoor PM2.5 toxicity. Our results highlight the crucial role of insulin signaling and metallothioneins in the control of outdoor PM2.5 toxicity in nematodes.

Conflict of interest

None of the authors have any conflicting interest.

Funding

This work was supported by the grants from Key Project of Science and Technology of Beijing (no. Z121100000312007), and Beijing Natural Science Foundation of China (no. 7152024).

Supplementary Material

Click here for additional data file. (93.6KB, pdf)

Footnotes

†Electronic supplementary information (ESI) available. See DOI: 10.1039/c6tx00022c

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