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Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans
STOTEN-135623; No of Pages 7 Science of the Total Environment xxx (xxxx) xxx
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Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans Yingyue Zhao a,b, Dan Li b, Qi Rui a,⁎, Dayong Wang b,⁎ a b
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Environmental Medicine Engineering in Ministry of Education, Medical School, Southeast University, Nanjing 210009, China
H I G H L I G H T S
G R A P H I C A L
• We examined the effects of nanopolystyrene (30 nm) on microgravity treated nematodes. • Nanopolystyrene enhanced toxicity of microgravity stress in wild-type nematodes. • This toxicity enhancement was associated with oxidative stress and mt UPR activation. • This toxicity enhancement could be further strengthened by sod-3 mutation. • Our data highlights the role of nanopolystyrene in enhancing the microgravity toxicity.
Our results highlighted the potential of nanopolystyrene exposure in enhancing the toxicity of microgravity stress in nematodes.
a r t i c l e
a b s t r a c t
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Article history: Received 20 September 2019 Received in revised form 14 November 2019 Accepted 17 November 2019 Available online xxxx Editor: Daqiang Yin Keywords: Nanopolystyrene Microgravity stress Toxicity enhancement Caenorhabditis elegans
A B S T R A C T
Caenorhabditis elegans is a useful animal model for assessing adverse effects of environmental toxicants or stresses. C. elegans was used as an assay system to investigate the effects of exposure to nanopolystyrene (30 nm) on wild-type and sod-3 mutant animals under microgravity stress condition. Using brood size and locomotion behaviors as endpoints, we found that nanopolystyrene exposure enhanced the toxicity of microgravity stress on nematodes, and this toxicity enhancement could be further strengthened by mutation of sod-3 encoding a Mn-SOD protein. Induction of reactive oxygen species (ROS) production and activation of mitochondrial unfolded protein response (mt UPR) were associated with this toxicity enhancement. In sod-3 mutant nematodes, the enhancement in toxicity of microgravity stress by exposure to nanopolystyrene (10 μg/L) was detected. Our data will be helpful for understanding the potential effects of nanopolystyrene exposure on nematodes under the microgravity stress condition. © 2019 Published by Elsevier B.V.
⁎ Corresponding authors. E-mail addresses: [email protected] (Q. Rui), [email protected] (D. Wang).
https://doi.org/10.1016/j.scitotenv.2019.135623 0048-9697/© 2019 Published by Elsevier B.V.
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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1. Introduction In the recent years, the distribution, fate, and pollution of nanoplastics (plastic nanoparticles) in different ecosystems have gradually received the attention (da Costa et al., 2016; Wan et al., 2018; Chae and An, 2018; Alimi et al., 2018; Enfrin et al., 2019). The detection of nanoplastics in the environment and in the human food production chain has implied the possible exposure risk for human health (Bouwmeester et al., 2015; Lehner et al., 2019). Moreover, with nanopolystyrene as an example, nanoplastic exposure could cause multiple aspects of adverse effects on different organisms, such as Daphnia and zebrafish (Della Torre et al., 2014; Jeong et al., 2016; Rist et al., 2017; Chen et al., 2017). Nanopolystyrene was found to be further potentially transferred from exposed animals, such as zebrafish, to their offsprings (Zhao et al., 2017; Pitt et al., 2018). Caenorhabditis elegans is a classic model animal frequently employed to evaluate the potential toxicity of various engineered nanomaterials (ENMs) due to its sensitivity to environmental exposure (Wang, 2018; Yang et al., 2018; P.-D. Liu et al., 2019a; Zhao et al., 2019a; Starnes et al., 2019; Moon et al., 2019). Since 2017, C. elegans has been also used to assess the nanopolystyrene toxicity and to elucidate the underlying mechanisms (Zhao et al., 2017; Hanna et al., 2018; Qu et al., 2019a; Qu et al., 2019b; Kim et al., 2019; Yang et al., 2019). In nematodes, the toxicity of polystyrene particles was size-dependent (Qu et al., 2019c). The nanopolystyrene (such as 100 nm) showed the more severe toxicity than polystyrene particles with large sizes (such as 10 or 100 μm) (Qu et al., 2019c). Exposure to nanopolystyrene could at least cause the neurotoxicity, reproductive toxicity, and intestinal toxicity (Lei et al., 2018; Qu et al., 2019c; Qu et al., 2019d; Qu and Wang, 2019). Nevertheless, the nanopolystyrene exposures were mainly performed under the normal conditions in nematodes. Microgravity stress contributes greatly to the observed pathological changes during the spaceflight (Longnecker et al., 2004; Fitts et al., 2010). Recently, the adverse effects of simulated microgravity on nematodes have been detected, such as induction of reactive oxygen species (ROS) production and decrease in locomotion behavior (Tee et al., 2017; H.-L. Liu et al., 2019; Kong et al., 2019; Rui et al., 2019). In nematodes, mutation of sod-3 encoding a mitochondrial Mn-SOD could induce a susceptibility to the toxicity of environmental stresses or toxicants, such as ENMs (Wang, 2018; Wang, 2019a). The aim of this study was to examine the toxicity induction of nanopolystyrene (30 nm) under the microgravity stress condition in both wild-type and sod-3 mutant nematodes. 2. Materials and methods 2.1. Characterization of nanopolystyrene Physicochemical characterization of 30 nm nanopolystyrene (Janus New-Materials Co., Nanjing, China) was evaluated by Raman spectroscopy (Renishaw Invia Plus laser Raman spectrometer, Renishaw, UK), transmission electron microscopy (TEM, JEOL Ltd., Japan), and Zeta potential using dynamic light scattering (DLS) technique (Zetasizer NanoZS90, Malvern Instruments Ltd., UK). The TEM image showed morphology and size of nanopolystyrene particles (Fig. 1A). Raman spectrum data was provided in Fig. 1B. Based on DLS analysis, the size of nanopolystyrene was 29.8 ± 2.3 nm, and the zeta potential of nanopolystyrene was −20.5 ± 2.47 mV.
ROS production and decrease in locomotion behavior, but did not affect brood size (Li et al., 2018). Control young adult nematodes were maintained in liquid S medium without microgravity treatment (the normal condition). 2.3. Nematode strains and maintenance Wild-type N2, sod-3(gk235) mutant, and transgenic strain of SJ4100/ zcIs13[HSP-6::GFP]) were used in this study. The nematodes were fed with Escherichia coli OP50 on nematode growth medium (NGM) plates (Brenner, 1974). To prepare age-synchronous young adults, the mature gravid nematodes were treated with bleaching mixture solution (0.45 M NaOH and 2% HOCl) to release the eggs and let them further hatch on NGM plates. 2.4. Exposure and toxicity assessment Exposure to nanopolystyrene was performed from young adult stage for 24-h in liquid solutions under normal or simulated microgravity stress condition. After exposure, the nematodes were washed with M9 buffer for three times. After that, the nematodes were used for toxicity assessment using endpoints of brood size and locomotion behaviors. Reproduction was evaluated by the endpoints of brood size (Qu et al., 2019e). Brood size was used to reflect the reproductive capacity (Wang, 2019b). The brood size refers to the number of offspring at all stages beyond the egg. For each treatment, ten animals were examined. Each single examined nematode was transferred onto a NGM plate to count the number of its progeny. Locomotion behavior was used to reflect the functional state of motor neurons (Cheng et al., 2019). Head thrash and body bend were used to assess the possible neurotoxicity on locomotion behaviors (Zhao et al., 2019b). After washing with M9 buffer, the examined nematodes were transferred onto the NGM plates (without OP50 feeding) to recover for 1-min. After that, the numbers of head thrash and body bend were counted under a dissecting microcopy. A head thrash is considered as an alteration in bending direction at body mid-region, and a body bend is considered as an alteration in posterior bulb direction. For each treatment, thirty animals were examined. 2.5. ROS production To examine the activation of oxidative stress, ROS production was determined (Qu et al., 2019f). After exposure and the following washing with M9 buffer, the nematodes were labeled with CM-H2DCFDA (1 μM) in darkness for 3 h. After the further washing with M9 buffer for three times, the nematodes were examined for both excitation wavelength at 488 nm and emission filter at 510 nm under a laser scanning confocal microscope. ROS signals were examined by relative fluorescence units after normalization to autofluorescence. For each treatment, forty animals were examined. 2.6. Mitochondrial unfolded protein response (mt UPR) HSP-6 was employed as a marker for mt UPR (Haynes et al., 2007). Relative fluorescence of HSP-6::GFP signals was examined. For each treatment, forty animals were examined.
2.2. Simulated microgravity
2.7. RNA interference (RNAi)
To perform the simulated microgravity, the young adult nematodes were suspended in liquid S medium in chamber of Rotary System™ (Synthecon) (half filled) with the balancing sedimentation-induced gravity by centrifugation (horizontally at 30 rpm for 24 h) (Li et al., 2018). Simulated microgravity treatment for 24-h could induce the significant
E. coli HT115 (DE3) expressing double-stranded RNA corresponding to sod-3 was used to feed the nematodes (P.-D. Liu et al., 2019b). L1larvae of SJ4100 were cultured on RNAi plates. When they developed into gravid animals, they were transferred to a new plate to lay eggs in order to obtain the next generation. Negative control was the
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Fig. 1. Properties of nanopolystyrene. (A) TEM image of nanopolystyrene in K medium. (B) Raman spectrum of nanopolystyrene. The Raman spectroscopy analysis indicated that the nanopolystyrene showed the peaks at 1001.56 cm−1 (breathing vibration of benzene ring), at 1031.54 cm−1 (symmetric extension vibration of carbon atoms in benzene ring), at 1200.71 cm−1 (stretching vibration of carbon atoms between benzene ring and polyethylene group), at 1449.94 cm−1 (asymmetric bending vibration of carbon atoms and hydrogen atoms), and at 1602.25 cm−1 (asymmetric stretching vibration of benzene ring carbon atoms).
HT115 bacteria harboring empty vector L4440. RNAi knockdown efficiency of sod-3 was confirmed by qRT-PCR (Fig. S1). 2.8. Statistical analysis Statistical analysis was carried out using SPSS 12.0 software (SPSS Inc., Chicago, USA). Differences between groups were analyzed with one-way analysis of variance (ANOVA). Two-way ANOVA analysis was performed for multiple factor comparison. Probability level of 0.01 (**) was considered to be statistically significant. 3. Results 3.1. Toxicity assessment of nanopolystyrene on brood size and locomotion behavior under microgravity stress condition in wild-type nematodes We employed brood size and locomotion behavior as endpoints to assess the possible nanopolystyrene toxicity under microgravity stress condition in wild-type nematodes. Microgravity treatment for 24-h did not obviously affect the brood size, but could significantly decrease the locomotion behavior of head thrash and body bend in wild-type nematodes (Fig. 2). Under the microgravity stress condition, exposure to nanopolystyrene (1–100 μg/L) did not obviously influence the brood size in wild-type nematodes (Fig. 2A). In contrast, under the microgravity stress condition, we detected the significant reduction in brood size in wild-type nematodes exposed to 1000 μg/L nanopolystyrene (Fig. 2A). Under the microgravity stress condition, exposure to nanopolystyrene (1–10 μg/L) did not affect the toxicity of microgravity treatment on locomotion behavior in wild-type nematodes (Fig. 2B). However, under the microgravity stress condition, exposure to nanopolystyrene (100–1000 μg/L) could significantly enhance the toxicity of microgravity treatment in decreasing locomotion behavior in wild-type nematodes (Fig. 2B). 3.2. Toxicity assessment of nanopolystyrene in inducing ROS production under microgravity stress condition in wild-type nematodes Considering the fact that activation of oxidative stress is an important cellular contributor to the toxicity induction of ENMs (Wang, 2018), we employed the endpoint of ROS production to assess the nanopolystyrene toxicity in activating oxidative stress under microgravity stress condition in wild-type nematodes. Microgravity treatment for 24-h could induce the significant ROS production in wildtype nematodes (Fig. 3). Exposure to nanopolystyrene (1–10 μg/L) did not significantly affect the induction of ROS production induced by microgravity stress in wild-type nematodes (Fig. 3). Different from this, we observed that exposure to 100–1000 μg/L nanopolystyrene could
enhance the ROS production induced by microgravity stress in wildtype nematodes (Fig. 3). 3.3. Effect of nanopolystyrene exposure in activating mt UPR under microgravity stress condition In nematodes, mt UPR activation is another important cellular modulator for the toxicity induction of environmental toxicants (Wang, 2019a, 2019b). We employed the strain of SJ4100 to investigate the effect of nanopolystyrene exposure in activating mt UPR under microgravity stress condition. Using SJ4100, we found that microgravity treatment for 24-h induced the obvious mt UPR activation (Fig. 4). Although exposure to nanopolystyrene (1–10 μg/L) did not affect the activation of mt UPR activation induced by microgravity stress, exposure to 100–1000 μg/L nanopolystyrene enhanced the activation of mt UPR induced by microgravity stress (Fig. 4). 3.4. Toxicity assessment of nanopolystyrene under microgravity stress condition in sod-3 mutant nematodes We further employed the sod-3 mutant to assess the nanopolystyrene toxicity under microgravity stress condition. Under the normal condition (without microgravity treatment and nanopolystyrene exposure), mutation of sod-3 did not affect both brood size and locomotion behavior (Fig. 2). Microgravity treatment for 24-h also could not affect the brood size, but significantly decreased the locomotion behavior in sod-3 mutant nematodes (Fig. 2). Additionally, mutation of sod-3 caused the more severe decrease in locomotion behavior than that in wild-types under the microgravity stress condition (Fig. 2B). Under the microgravity stress condition, exposure to nanopolystyrene (1–100 μg/L) could not affect the brood size, whereas exposed to 1000 μg/L nanopolystyrene could significantly reduce the brood size in sod-3 mutant nematodes (Fig. 2A). Under the microgravity stress condition, exposure to 1 μg/L nanopolystyrene could not influence the toxicity of microgravity treatment on locomotion behavior; however, exposure to nanopolystyrene (10–1000 μg/L) significantly enhanced the toxicity of microgravity treatment on locomotion behavior in sod-3 mutant nematodes (Fig. 2B). Moreover, under the microgravity stress condition, we observed the more severe toxicity on brood size in sod-3 mutant nematodes than that in wild-type nematodes after exposure to 1000 μg/L nanopolystyrene (Fig. 2A). Additionally, under the microgravity stress condition, we further detected the more severe toxicity on locomotion behavior in sod-3 mutant nematodes than those in wild-type nematodes after exposure to 10–1000 μg/L nanopolystyrene (Fig. 2B). These observations suggested
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Fig. 2. Toxicity of nanopolystyrene on brood size (A) and locomotion behaviors (B) in wild-type or sod-3 mutant nematodes under microgravity stress condition. Nanopolystyrene exposure was performed from young adults for 24-h. “+”, addition and/or treatment; “−”, without addition and treatment. Control, without both nanopolystyrene exposure and microgravity treatment. Bars represent means ± SD. **P b 0.01 vs control (if not specially indicated).
the susceptibility of sod-3 mutant nematodes to nanopolystyrene toxicity under microgravity stress condition.
3.5. Toxicity assessment of nanopolystyrene in inducing ROS production under microgravity stress condition in sod-3 mutant nematodes Under the normal condition, mutation of sod-3 did not cause the obvious ROS production (Fig. 3). Under the microgravity stress condition, mutation of sod-3 resulted in more severe induction of ROS production than that in wild-type nematodes (Fig. 3). In sod-3 mutant nematodes, exposure to 1 μg/L nanopolystyrene did not affect the ROS production induced by microgravity stress; however, exposure to nanopolystyrene (10–1000 μg/L) could significantly enhance the ROS production induced by microgravity stress (Fig. 3).
Moreover, under the microgravity stress condition, we observed the more severe ROS production in sod-3 mutant nematodes than that in wild-type nematodes after exposure to 10–1000 μg/L nanopolystyrene (Fig. 3). This observation further supported the observed susceptibility of sod-3 mutant nematodes to nanopolystyrene toxicity under the microgravity stress condition.
3.6. Effect of nanopolystyrene exposure in activating mt UPR under microgravity stress condition in sod-3(RNAi) nematodes Under the normal condition, RNAi knockdown of sod-3 did not in- 285 duce the mt UPR activation (Fig. 4). Under the microgravity stress condition, RNAi knockdown of sod-3 caused the more severe activation of mt UPR (Fig. 4). In sod-3(RNAi) nematodes, exposure to 1 μg/L
Fig. 3. Toxicity of nanopolystyrene in inducing ROS production in wild-type or sod-3 mutant nematodes under microgravity stress condition. Nanopolystyrene exposure was performed from young adults for 24-h. “+”, addition and/or treatment; “−”, without addition and treatment. Control, without both nanopolystyrene exposure and microgravity treatment. Bars represent means ± SD. **P b 0.01 vs control (if not specially indicated).
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Fig. 4. Effect of nanopolystyrene in inducing mt UPR activation in nematodes under microgravity stress condition. Nanopolystyrene exposure was performed from young adults for 24-h. “+”, addition and/or treatment; “−”, without addition and treatment. Control, without both nanopolystyrene exposure and microgravity treatment. Bars represent means ± SD. **P b 0.01 vs control (if not specially indicated).
nanopolystyrene could not influence the mt UPR activation induced by microgravity stress; however, exposure to nanopolystyrene (10–1000 μg/L) significantly enhanced the mt UPR activation induced by microgravity stress (Fig. 4). Moreover, under the microgravity stress condition, we found the more severe mt UPR activation in sod-3(RNAi) nematodes than that in control/(L4440) nematodes after exposure to 10–1000 μg/L nanopolystyrene (Fig. 4). 3.7. Effect of nanopolystyrene exposure on wild-type or sod-3 mutant nematodes under normal condition Meanwhile, we also investigated the possible effect of nanopolystyrene exposure on wild-type or sod-3 mutant nematodes under normal condition. Under the normal condition, exposure to nanopolystyrene (1–1000 μg/L) for 24-h did not affect brood size (Fig. 5A), influence locomotion behavior (Fig. 5B), and induce obvious ROS production (Fig. 5C) in wild-type nematodes. Similarly, exposure to nanopolystyrene (1–100 μg/L) for 24-h also did not reduce brood size (Fig. 5A), decrease locomotion behavior (Fig. 5B), and induce obvious ROS production (Fig. 5C) in sod-3 mutant nematodes under the normal condition. We only detected the significant decrease in locomotion behavior (Fig. 5B) and induction of ROS production (Fig. 5C) in 1000 μg/L nanopolystyrene exposed sod-3 mutant nematodes under the normal condition. Nevertheless, exposure to 1000 μg/L nanopolystyrene also did not affect the brood size in sod-3 mutant nematodes under the normal condition (Fig. 5A). 4. Discussion In the recent years, the potential adverse effects of nanopolystyrene have been examined in various organisms, including the C. elegans (Della Torre et al., 2014; Jeong et al., 2016; Zhao et al., 2017; Pitt et al., 2018; Rist et al., 2017; Chen et al., 2017). Nevertheless, most of the nanopolystyrene toxicity assessments were carried out under the normal conditions. In this study, we employed C. elegans as an assay system to determine the toxicity induction of nanopolystyrene under the microgravity stress condition. Because the nanopolystyrene toxicity was sizedependent, we here selected the nanopolystyrene with a small size (30 nm). Additionally, considering the fact that the treatment in nematodes for 24-h is approximately comparable to 4.2-year in humans (Rui et al., 2013), we performed the simulated microgravity for 24-h. Using two sensitive endpoints (brood size and locomotion behavior) for nanoparticle toxicity assessment (Wang, 2018), we observed the roles of exposure to nanopolystyrene (1000 μg/L) in enhancing toxicity of microgravity
stress in reducing brood size and exposure to nanopolystyrene (100–1000 μg/L) in enhancing toxicity of microgravity stress in decreasing locomotion behavior in wild-type nematodes (Fig. 2). During this toxicity assessment, the endpoint of locomotion behavior (head thrash and body bend) was more sensitive than the endpoint of brood size. More recently, it has also been found that exposure to nanopolystyrene could enhance the toxicity of other environmental toxicants, such as microcystinLR, in nematodes (Qu et al., 2019a). Therefore, exposure to nanopolystyrene can potentially enhance the toxicity of both environmental toxicants and environmental stresses in organisms. We raised two possible cellular mechanisms for the observed enhancement in toxicity of microgravity stress by nanopolystyrene exposure. One cellular mechanism is that the induction of ROS production may contribute to the enhancement in toxicity of microgravity stress by nanopolystyrene exposure, since exposure to nanopolystyrene (100–1000 μg/L) could enhance the ROS production in microgravity treated wild-type nematodes (Fig. 3). Another cellular mechanism is that the mt UPR activation was associated with the enhancement in toxicity of microgravity stress by nanopolystyrene exposure, since the more pronounced mt UPR was activated by nanopolystyrene (100–1000 μg/L) in microgravity treated wild-type nematodes (Fig. 4). The mt UPR is a protective response for nematodes against the toxicity of environmental toxicants or stresses (Wang, 2019a, 2019b). The observed more pronounced mt UPR induced by nanopolystyrene (100–1000 μg/L) in microgravity treated wild-type nematodes implied the activation of stronger protective response of nematode against the toxicity. In this study, HSP-6 was employed as a marker for mt UPR. HSP-6 is an ortholog of human HSP70, and the simulated microgravity could upregulate the HSP70 expression in human bone stem cells (Cazzaniga et al., 2016). In this study, we further compared the toxicity of nanopolystyrene under the microgravity stress condition between wild-type and sod-3 mutant nematodes. We observed that exposure to nanopolystyrene (100 and/or 1000 μg/L) could cause the more severe enhancement in toxicity of microgravity stress in reducing brood size and in decreasing locomotion behavior in sod-3 mutant nematodes compared with those in wild-type nematodes (Fig. 2). In the range of 100–1000 μg/L, we further observed that exposure to nanopolystyrene resulted in the more severe enhancement in ROS production in microgravity treated sod-3 mutant nematodes compared with that in simulated treated wild-type nematodes (Fig. 3). These observations further support the susceptibility of sod-3 mutant nematodes to the toxicity of environmental toxicants or stresses in nematodes (Wang, 2019a, 2019b). Additionally, in the range of 100–1000 μg/L, exposure to nanopolystyrene caused the
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Fig. 5. Effect of nanopolystyrene exposure on brood size (A), locomotion behaviors (B), and induction of ROS production (C) in wild-type or sod-3 mutant nematodes under normal condition. Nanopolystyrene exposure was performed from young adults for 24-h. Bars represent means ± SD. **P b 0.01 vs control.
more severe enhancement in mt UPR in microgravity treated sod-3 (RNAi) nematodes compared with that in simulated treated control/ (L4440) nematodes (Fig. 4). That is, our data also suggested that the mt UPR activation can be controlled by certain oxidative stress related molecular signals, such as SOD-3. Microgravity is an important contributor for the toxicity induction during the spaceflight, and C. elegans was used for assessing adverse effects of spaceflight early in 2003 (Szewczyk et al., 2005; Higashitani et al., 2005; Adenle et al., 2009). Moreover, in the sod-3 mutant nematodes, we detected the enhancement in toxicity of microgravity stress in decreasing locomotion behavior by nanopolystyrene (10 μg/L) (Fig. 2B). Meanwhile, we also observed the enhancement in ROS production induced by nanopolystyrene (10 μg/L) in microgravity treated sod-3 mutant nematodes (Fig. 3). The predicted environmental concentrations of nanopolystyrene (≤50 nm) have been considered in the range ≤ 15 μg/L (Lenz et al., 2016; Al-Sid-Cheikh et al., 2018). Therefore, our data implies the potential adverse effects of exposure to nanopolystyrene at predicted environment on organisms with susceptible property during the spaceflight. In this study, we also examined the effects of nanopolystyrene exposure for 24-h on nematodes. Exposure to nanopolystyrene (1–1000 μg/L) for 24-h did not affect brood size and locomotion behavior and induce obvious ROS production in wild-type nematodes (Fig. 5), which further supports the observed enhancement in toxicity of microgravity stress by nanopolystyrene exposure in wild-type nematodes. Exposure to nanopolystyrene (1–100 μg/L) for 24-h could not affect brood size and locomotion behavior in sod-3 mutant nematodes
(Fig. 5A–B). Exposure to nanopolystyrene (1–100 μg/L) for 24-h also did not induce obvious ROS production in sod-3 mutant nematodes (Fig. 5C), and did not activate obvious mt UPR in sod-3(RNAi) nematodes (Fig. S2), which supports the detected enhancement in toxicity of microgravity stress by nanopolystyrene (10–100 μg/L) exposure in sod-3 mutant nematodes. Nevertheless, exposure to nanopolystyrene (1000 μg/L) could significantly decrease locomotion behavior in sod-3 mutant nematodes (Fig. 5B), induced significant ROS production in sod-3 mutant nematodes (Fig. 5C), and caused the obvious mt UPR in sod-3(RNAi) nematodes (Fig. S2). These observations suggested that the detected enhancement in toxicity of microgravity stress by exposure to nanopolystyrene (1000 μg/L) might be due to the observed toxicity of nanopolystyrene (1000 μg/L) to a certain degree. In conclusion, we investigated the possible effects of exposure to nanopolystyrene (30 nm) on nematode C. elegans under the microgravity stress condition. In wild-type nematodes, we observed the enhancement in toxicity of microgravity stress by nanopolystyrene exposure. Moreover, this toxicity enhancement was associated with the induction of ROS production and the activation of mt UPR. Mutation of sod-3 induced the more severe enhancement in toxicity of microgravity stress by nanopolystyrene exposure. In sod-3 mutant nematodes, we even found that exposure to nanopolystyrene (10 μg/L) could enhance the toxicity of microgravity stress on nematodes. Our results are helpful for our understanding the possible effects of nanopolystyrene exposure on organisms under the microgravity stress condition. Our results highlighted the potential of nanopolystyrene exposure in enhancing the toxicity of microgravity stress on organisms.
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans Yingyue Zhao a,b, Dan Li b, Qi Rui a,⁎, Dayong Wang b,⁎ a b
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China Key Laboratory of Environmental Medicine Engineering in Ministry of Education, Medical School, Southeast University, Nanjing 210009, China
H I G H L I G H T S
G R A P H I C A L
• We examined the effects of nanopolystyrene (30 nm) on microgravity treated nematodes. • Nanopolystyrene enhanced toxicity of microgravity stress in wild-type nematodes. • This toxicity enhancement was associated with oxidative stress and mt UPR activation. • This toxicity enhancement could be further strengthened by sod-3 mutation. • Our data highlights the role of nanopolystyrene in enhancing the microgravity toxicity.
Our results highlighted the potential of nanopolystyrene exposure in enhancing the toxicity of microgravity stress in nematodes.
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 20 September 2019 Received in revised form 14 November 2019 Accepted 17 November 2019 Available online xxxx Editor: Daqiang Yin Keywords: Nanopolystyrene Microgravity stress Toxicity enhancement Caenorhabditis elegans
A B S T R A C T
Caenorhabditis elegans is a useful animal model for assessing adverse effects of environmental toxicants or stresses. C. elegans was used as an assay system to investigate the effects of exposure to nanopolystyrene (30 nm) on wild-type and sod-3 mutant animals under microgravity stress condition. Using brood size and locomotion behaviors as endpoints, we found that nanopolystyrene exposure enhanced the toxicity of microgravity stress on nematodes, and this toxicity enhancement could be further strengthened by mutation of sod-3 encoding a Mn-SOD protein. Induction of reactive oxygen species (ROS) production and activation of mitochondrial unfolded protein response (mt UPR) were associated with this toxicity enhancement. In sod-3 mutant nematodes, the enhancement in toxicity of microgravity stress by exposure to nanopolystyrene (10 μg/L) was detected. Our data will be helpful for understanding the potential effects of nanopolystyrene exposure on nematodes under the microgravity stress condition. © 2019 Published by Elsevier B.V.
⁎ Corresponding authors. E-mail addresses: [email protected] (Q. Rui), [email protected] (D. Wang).
https://doi.org/10.1016/j.scitotenv.2019.135623 0048-9697/© 2019 Published by Elsevier B.V.
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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1. Introduction In the recent years, the distribution, fate, and pollution of nanoplastics (plastic nanoparticles) in different ecosystems have gradually received the attention (da Costa et al., 2016; Wan et al., 2018; Chae and An, 2018; Alimi et al., 2018; Enfrin et al., 2019). The detection of nanoplastics in the environment and in the human food production chain has implied the possible exposure risk for human health (Bouwmeester et al., 2015; Lehner et al., 2019). Moreover, with nanopolystyrene as an example, nanoplastic exposure could cause multiple aspects of adverse effects on different organisms, such as Daphnia and zebrafish (Della Torre et al., 2014; Jeong et al., 2016; Rist et al., 2017; Chen et al., 2017). Nanopolystyrene was found to be further potentially transferred from exposed animals, such as zebrafish, to their offsprings (Zhao et al., 2017; Pitt et al., 2018). Caenorhabditis elegans is a classic model animal frequently employed to evaluate the potential toxicity of various engineered nanomaterials (ENMs) due to its sensitivity to environmental exposure (Wang, 2018; Yang et al., 2018; P.-D. Liu et al., 2019a; Zhao et al., 2019a; Starnes et al., 2019; Moon et al., 2019). Since 2017, C. elegans has been also used to assess the nanopolystyrene toxicity and to elucidate the underlying mechanisms (Zhao et al., 2017; Hanna et al., 2018; Qu et al., 2019a; Qu et al., 2019b; Kim et al., 2019; Yang et al., 2019). In nematodes, the toxicity of polystyrene particles was size-dependent (Qu et al., 2019c). The nanopolystyrene (such as 100 nm) showed the more severe toxicity than polystyrene particles with large sizes (such as 10 or 100 μm) (Qu et al., 2019c). Exposure to nanopolystyrene could at least cause the neurotoxicity, reproductive toxicity, and intestinal toxicity (Lei et al., 2018; Qu et al., 2019c; Qu et al., 2019d; Qu and Wang, 2019). Nevertheless, the nanopolystyrene exposures were mainly performed under the normal conditions in nematodes. Microgravity stress contributes greatly to the observed pathological changes during the spaceflight (Longnecker et al., 2004; Fitts et al., 2010). Recently, the adverse effects of simulated microgravity on nematodes have been detected, such as induction of reactive oxygen species (ROS) production and decrease in locomotion behavior (Tee et al., 2017; H.-L. Liu et al., 2019; Kong et al., 2019; Rui et al., 2019). In nematodes, mutation of sod-3 encoding a mitochondrial Mn-SOD could induce a susceptibility to the toxicity of environmental stresses or toxicants, such as ENMs (Wang, 2018; Wang, 2019a). The aim of this study was to examine the toxicity induction of nanopolystyrene (30 nm) under the microgravity stress condition in both wild-type and sod-3 mutant nematodes. 2. Materials and methods 2.1. Characterization of nanopolystyrene Physicochemical characterization of 30 nm nanopolystyrene (Janus New-Materials Co., Nanjing, China) was evaluated by Raman spectroscopy (Renishaw Invia Plus laser Raman spectrometer, Renishaw, UK), transmission electron microscopy (TEM, JEOL Ltd., Japan), and Zeta potential using dynamic light scattering (DLS) technique (Zetasizer NanoZS90, Malvern Instruments Ltd., UK). The TEM image showed morphology and size of nanopolystyrene particles (Fig. 1A). Raman spectrum data was provided in Fig. 1B. Based on DLS analysis, the size of nanopolystyrene was 29.8 ± 2.3 nm, and the zeta potential of nanopolystyrene was −20.5 ± 2.47 mV.
ROS production and decrease in locomotion behavior, but did not affect brood size (Li et al., 2018). Control young adult nematodes were maintained in liquid S medium without microgravity treatment (the normal condition). 2.3. Nematode strains and maintenance Wild-type N2, sod-3(gk235) mutant, and transgenic strain of SJ4100/ zcIs13[HSP-6::GFP]) were used in this study. The nematodes were fed with Escherichia coli OP50 on nematode growth medium (NGM) plates (Brenner, 1974). To prepare age-synchronous young adults, the mature gravid nematodes were treated with bleaching mixture solution (0.45 M NaOH and 2% HOCl) to release the eggs and let them further hatch on NGM plates. 2.4. Exposure and toxicity assessment Exposure to nanopolystyrene was performed from young adult stage for 24-h in liquid solutions under normal or simulated microgravity stress condition. After exposure, the nematodes were washed with M9 buffer for three times. After that, the nematodes were used for toxicity assessment using endpoints of brood size and locomotion behaviors. Reproduction was evaluated by the endpoints of brood size (Qu et al., 2019e). Brood size was used to reflect the reproductive capacity (Wang, 2019b). The brood size refers to the number of offspring at all stages beyond the egg. For each treatment, ten animals were examined. Each single examined nematode was transferred onto a NGM plate to count the number of its progeny. Locomotion behavior was used to reflect the functional state of motor neurons (Cheng et al., 2019). Head thrash and body bend were used to assess the possible neurotoxicity on locomotion behaviors (Zhao et al., 2019b). After washing with M9 buffer, the examined nematodes were transferred onto the NGM plates (without OP50 feeding) to recover for 1-min. After that, the numbers of head thrash and body bend were counted under a dissecting microcopy. A head thrash is considered as an alteration in bending direction at body mid-region, and a body bend is considered as an alteration in posterior bulb direction. For each treatment, thirty animals were examined. 2.5. ROS production To examine the activation of oxidative stress, ROS production was determined (Qu et al., 2019f). After exposure and the following washing with M9 buffer, the nematodes were labeled with CM-H2DCFDA (1 μM) in darkness for 3 h. After the further washing with M9 buffer for three times, the nematodes were examined for both excitation wavelength at 488 nm and emission filter at 510 nm under a laser scanning confocal microscope. ROS signals were examined by relative fluorescence units after normalization to autofluorescence. For each treatment, forty animals were examined. 2.6. Mitochondrial unfolded protein response (mt UPR) HSP-6 was employed as a marker for mt UPR (Haynes et al., 2007). Relative fluorescence of HSP-6::GFP signals was examined. For each treatment, forty animals were examined.
2.2. Simulated microgravity
2.7. RNA interference (RNAi)
To perform the simulated microgravity, the young adult nematodes were suspended in liquid S medium in chamber of Rotary System™ (Synthecon) (half filled) with the balancing sedimentation-induced gravity by centrifugation (horizontally at 30 rpm for 24 h) (Li et al., 2018). Simulated microgravity treatment for 24-h could induce the significant
E. coli HT115 (DE3) expressing double-stranded RNA corresponding to sod-3 was used to feed the nematodes (P.-D. Liu et al., 2019b). L1larvae of SJ4100 were cultured on RNAi plates. When they developed into gravid animals, they were transferred to a new plate to lay eggs in order to obtain the next generation. Negative control was the
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Fig. 1. Properties of nanopolystyrene. (A) TEM image of nanopolystyrene in K medium. (B) Raman spectrum of nanopolystyrene. The Raman spectroscopy analysis indicated that the nanopolystyrene showed the peaks at 1001.56 cm−1 (breathing vibration of benzene ring), at 1031.54 cm−1 (symmetric extension vibration of carbon atoms in benzene ring), at 1200.71 cm−1 (stretching vibration of carbon atoms between benzene ring and polyethylene group), at 1449.94 cm−1 (asymmetric bending vibration of carbon atoms and hydrogen atoms), and at 1602.25 cm−1 (asymmetric stretching vibration of benzene ring carbon atoms).
HT115 bacteria harboring empty vector L4440. RNAi knockdown efficiency of sod-3 was confirmed by qRT-PCR (Fig. S1). 2.8. Statistical analysis Statistical analysis was carried out using SPSS 12.0 software (SPSS Inc., Chicago, USA). Differences between groups were analyzed with one-way analysis of variance (ANOVA). Two-way ANOVA analysis was performed for multiple factor comparison. Probability level of 0.01 (**) was considered to be statistically significant. 3. Results 3.1. Toxicity assessment of nanopolystyrene on brood size and locomotion behavior under microgravity stress condition in wild-type nematodes We employed brood size and locomotion behavior as endpoints to assess the possible nanopolystyrene toxicity under microgravity stress condition in wild-type nematodes. Microgravity treatment for 24-h did not obviously affect the brood size, but could significantly decrease the locomotion behavior of head thrash and body bend in wild-type nematodes (Fig. 2). Under the microgravity stress condition, exposure to nanopolystyrene (1–100 μg/L) did not obviously influence the brood size in wild-type nematodes (Fig. 2A). In contrast, under the microgravity stress condition, we detected the significant reduction in brood size in wild-type nematodes exposed to 1000 μg/L nanopolystyrene (Fig. 2A). Under the microgravity stress condition, exposure to nanopolystyrene (1–10 μg/L) did not affect the toxicity of microgravity treatment on locomotion behavior in wild-type nematodes (Fig. 2B). However, under the microgravity stress condition, exposure to nanopolystyrene (100–1000 μg/L) could significantly enhance the toxicity of microgravity treatment in decreasing locomotion behavior in wild-type nematodes (Fig. 2B). 3.2. Toxicity assessment of nanopolystyrene in inducing ROS production under microgravity stress condition in wild-type nematodes Considering the fact that activation of oxidative stress is an important cellular contributor to the toxicity induction of ENMs (Wang, 2018), we employed the endpoint of ROS production to assess the nanopolystyrene toxicity in activating oxidative stress under microgravity stress condition in wild-type nematodes. Microgravity treatment for 24-h could induce the significant ROS production in wildtype nematodes (Fig. 3). Exposure to nanopolystyrene (1–10 μg/L) did not significantly affect the induction of ROS production induced by microgravity stress in wild-type nematodes (Fig. 3). Different from this, we observed that exposure to 100–1000 μg/L nanopolystyrene could
enhance the ROS production induced by microgravity stress in wildtype nematodes (Fig. 3). 3.3. Effect of nanopolystyrene exposure in activating mt UPR under microgravity stress condition In nematodes, mt UPR activation is another important cellular modulator for the toxicity induction of environmental toxicants (Wang, 2019a, 2019b). We employed the strain of SJ4100 to investigate the effect of nanopolystyrene exposure in activating mt UPR under microgravity stress condition. Using SJ4100, we found that microgravity treatment for 24-h induced the obvious mt UPR activation (Fig. 4). Although exposure to nanopolystyrene (1–10 μg/L) did not affect the activation of mt UPR activation induced by microgravity stress, exposure to 100–1000 μg/L nanopolystyrene enhanced the activation of mt UPR induced by microgravity stress (Fig. 4). 3.4. Toxicity assessment of nanopolystyrene under microgravity stress condition in sod-3 mutant nematodes We further employed the sod-3 mutant to assess the nanopolystyrene toxicity under microgravity stress condition. Under the normal condition (without microgravity treatment and nanopolystyrene exposure), mutation of sod-3 did not affect both brood size and locomotion behavior (Fig. 2). Microgravity treatment for 24-h also could not affect the brood size, but significantly decreased the locomotion behavior in sod-3 mutant nematodes (Fig. 2). Additionally, mutation of sod-3 caused the more severe decrease in locomotion behavior than that in wild-types under the microgravity stress condition (Fig. 2B). Under the microgravity stress condition, exposure to nanopolystyrene (1–100 μg/L) could not affect the brood size, whereas exposed to 1000 μg/L nanopolystyrene could significantly reduce the brood size in sod-3 mutant nematodes (Fig. 2A). Under the microgravity stress condition, exposure to 1 μg/L nanopolystyrene could not influence the toxicity of microgravity treatment on locomotion behavior; however, exposure to nanopolystyrene (10–1000 μg/L) significantly enhanced the toxicity of microgravity treatment on locomotion behavior in sod-3 mutant nematodes (Fig. 2B). Moreover, under the microgravity stress condition, we observed the more severe toxicity on brood size in sod-3 mutant nematodes than that in wild-type nematodes after exposure to 1000 μg/L nanopolystyrene (Fig. 2A). Additionally, under the microgravity stress condition, we further detected the more severe toxicity on locomotion behavior in sod-3 mutant nematodes than those in wild-type nematodes after exposure to 10–1000 μg/L nanopolystyrene (Fig. 2B). These observations suggested
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Fig. 2. Toxicity of nanopolystyrene on brood size (A) and locomotion behaviors (B) in wild-type or sod-3 mutant nematodes under microgravity stress condition. Nanopolystyrene exposure was performed from young adults for 24-h. “+”, addition and/or treatment; “−”, without addition and treatment. Control, without both nanopolystyrene exposure and microgravity treatment. Bars represent means ± SD. **P b 0.01 vs control (if not specially indicated).
the susceptibility of sod-3 mutant nematodes to nanopolystyrene toxicity under microgravity stress condition.
3.5. Toxicity assessment of nanopolystyrene in inducing ROS production under microgravity stress condition in sod-3 mutant nematodes Under the normal condition, mutation of sod-3 did not cause the obvious ROS production (Fig. 3). Under the microgravity stress condition, mutation of sod-3 resulted in more severe induction of ROS production than that in wild-type nematodes (Fig. 3). In sod-3 mutant nematodes, exposure to 1 μg/L nanopolystyrene did not affect the ROS production induced by microgravity stress; however, exposure to nanopolystyrene (10–1000 μg/L) could significantly enhance the ROS production induced by microgravity stress (Fig. 3).
Moreover, under the microgravity stress condition, we observed the more severe ROS production in sod-3 mutant nematodes than that in wild-type nematodes after exposure to 10–1000 μg/L nanopolystyrene (Fig. 3). This observation further supported the observed susceptibility of sod-3 mutant nematodes to nanopolystyrene toxicity under the microgravity stress condition.
3.6. Effect of nanopolystyrene exposure in activating mt UPR under microgravity stress condition in sod-3(RNAi) nematodes Under the normal condition, RNAi knockdown of sod-3 did not in- 285 duce the mt UPR activation (Fig. 4). Under the microgravity stress condition, RNAi knockdown of sod-3 caused the more severe activation of mt UPR (Fig. 4). In sod-3(RNAi) nematodes, exposure to 1 μg/L
Fig. 3. Toxicity of nanopolystyrene in inducing ROS production in wild-type or sod-3 mutant nematodes under microgravity stress condition. Nanopolystyrene exposure was performed from young adults for 24-h. “+”, addition and/or treatment; “−”, without addition and treatment. Control, without both nanopolystyrene exposure and microgravity treatment. Bars represent means ± SD. **P b 0.01 vs control (if not specially indicated).
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Fig. 4. Effect of nanopolystyrene in inducing mt UPR activation in nematodes under microgravity stress condition. Nanopolystyrene exposure was performed from young adults for 24-h. “+”, addition and/or treatment; “−”, without addition and treatment. Control, without both nanopolystyrene exposure and microgravity treatment. Bars represent means ± SD. **P b 0.01 vs control (if not specially indicated).
nanopolystyrene could not influence the mt UPR activation induced by microgravity stress; however, exposure to nanopolystyrene (10–1000 μg/L) significantly enhanced the mt UPR activation induced by microgravity stress (Fig. 4). Moreover, under the microgravity stress condition, we found the more severe mt UPR activation in sod-3(RNAi) nematodes than that in control/(L4440) nematodes after exposure to 10–1000 μg/L nanopolystyrene (Fig. 4). 3.7. Effect of nanopolystyrene exposure on wild-type or sod-3 mutant nematodes under normal condition Meanwhile, we also investigated the possible effect of nanopolystyrene exposure on wild-type or sod-3 mutant nematodes under normal condition. Under the normal condition, exposure to nanopolystyrene (1–1000 μg/L) for 24-h did not affect brood size (Fig. 5A), influence locomotion behavior (Fig. 5B), and induce obvious ROS production (Fig. 5C) in wild-type nematodes. Similarly, exposure to nanopolystyrene (1–100 μg/L) for 24-h also did not reduce brood size (Fig. 5A), decrease locomotion behavior (Fig. 5B), and induce obvious ROS production (Fig. 5C) in sod-3 mutant nematodes under the normal condition. We only detected the significant decrease in locomotion behavior (Fig. 5B) and induction of ROS production (Fig. 5C) in 1000 μg/L nanopolystyrene exposed sod-3 mutant nematodes under the normal condition. Nevertheless, exposure to 1000 μg/L nanopolystyrene also did not affect the brood size in sod-3 mutant nematodes under the normal condition (Fig. 5A). 4. Discussion In the recent years, the potential adverse effects of nanopolystyrene have been examined in various organisms, including the C. elegans (Della Torre et al., 2014; Jeong et al., 2016; Zhao et al., 2017; Pitt et al., 2018; Rist et al., 2017; Chen et al., 2017). Nevertheless, most of the nanopolystyrene toxicity assessments were carried out under the normal conditions. In this study, we employed C. elegans as an assay system to determine the toxicity induction of nanopolystyrene under the microgravity stress condition. Because the nanopolystyrene toxicity was sizedependent, we here selected the nanopolystyrene with a small size (30 nm). Additionally, considering the fact that the treatment in nematodes for 24-h is approximately comparable to 4.2-year in humans (Rui et al., 2013), we performed the simulated microgravity for 24-h. Using two sensitive endpoints (brood size and locomotion behavior) for nanoparticle toxicity assessment (Wang, 2018), we observed the roles of exposure to nanopolystyrene (1000 μg/L) in enhancing toxicity of microgravity
stress in reducing brood size and exposure to nanopolystyrene (100–1000 μg/L) in enhancing toxicity of microgravity stress in decreasing locomotion behavior in wild-type nematodes (Fig. 2). During this toxicity assessment, the endpoint of locomotion behavior (head thrash and body bend) was more sensitive than the endpoint of brood size. More recently, it has also been found that exposure to nanopolystyrene could enhance the toxicity of other environmental toxicants, such as microcystinLR, in nematodes (Qu et al., 2019a). Therefore, exposure to nanopolystyrene can potentially enhance the toxicity of both environmental toxicants and environmental stresses in organisms. We raised two possible cellular mechanisms for the observed enhancement in toxicity of microgravity stress by nanopolystyrene exposure. One cellular mechanism is that the induction of ROS production may contribute to the enhancement in toxicity of microgravity stress by nanopolystyrene exposure, since exposure to nanopolystyrene (100–1000 μg/L) could enhance the ROS production in microgravity treated wild-type nematodes (Fig. 3). Another cellular mechanism is that the mt UPR activation was associated with the enhancement in toxicity of microgravity stress by nanopolystyrene exposure, since the more pronounced mt UPR was activated by nanopolystyrene (100–1000 μg/L) in microgravity treated wild-type nematodes (Fig. 4). The mt UPR is a protective response for nematodes against the toxicity of environmental toxicants or stresses (Wang, 2019a, 2019b). The observed more pronounced mt UPR induced by nanopolystyrene (100–1000 μg/L) in microgravity treated wild-type nematodes implied the activation of stronger protective response of nematode against the toxicity. In this study, HSP-6 was employed as a marker for mt UPR. HSP-6 is an ortholog of human HSP70, and the simulated microgravity could upregulate the HSP70 expression in human bone stem cells (Cazzaniga et al., 2016). In this study, we further compared the toxicity of nanopolystyrene under the microgravity stress condition between wild-type and sod-3 mutant nematodes. We observed that exposure to nanopolystyrene (100 and/or 1000 μg/L) could cause the more severe enhancement in toxicity of microgravity stress in reducing brood size and in decreasing locomotion behavior in sod-3 mutant nematodes compared with those in wild-type nematodes (Fig. 2). In the range of 100–1000 μg/L, we further observed that exposure to nanopolystyrene resulted in the more severe enhancement in ROS production in microgravity treated sod-3 mutant nematodes compared with that in simulated treated wild-type nematodes (Fig. 3). These observations further support the susceptibility of sod-3 mutant nematodes to the toxicity of environmental toxicants or stresses in nematodes (Wang, 2019a, 2019b). Additionally, in the range of 100–1000 μg/L, exposure to nanopolystyrene caused the
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Fig. 5. Effect of nanopolystyrene exposure on brood size (A), locomotion behaviors (B), and induction of ROS production (C) in wild-type or sod-3 mutant nematodes under normal condition. Nanopolystyrene exposure was performed from young adults for 24-h. Bars represent means ± SD. **P b 0.01 vs control.
more severe enhancement in mt UPR in microgravity treated sod-3 (RNAi) nematodes compared with that in simulated treated control/ (L4440) nematodes (Fig. 4). That is, our data also suggested that the mt UPR activation can be controlled by certain oxidative stress related molecular signals, such as SOD-3. Microgravity is an important contributor for the toxicity induction during the spaceflight, and C. elegans was used for assessing adverse effects of spaceflight early in 2003 (Szewczyk et al., 2005; Higashitani et al., 2005; Adenle et al., 2009). Moreover, in the sod-3 mutant nematodes, we detected the enhancement in toxicity of microgravity stress in decreasing locomotion behavior by nanopolystyrene (10 μg/L) (Fig. 2B). Meanwhile, we also observed the enhancement in ROS production induced by nanopolystyrene (10 μg/L) in microgravity treated sod-3 mutant nematodes (Fig. 3). The predicted environmental concentrations of nanopolystyrene (≤50 nm) have been considered in the range ≤ 15 μg/L (Lenz et al., 2016; Al-Sid-Cheikh et al., 2018). Therefore, our data implies the potential adverse effects of exposure to nanopolystyrene at predicted environment on organisms with susceptible property during the spaceflight. In this study, we also examined the effects of nanopolystyrene exposure for 24-h on nematodes. Exposure to nanopolystyrene (1–1000 μg/L) for 24-h did not affect brood size and locomotion behavior and induce obvious ROS production in wild-type nematodes (Fig. 5), which further supports the observed enhancement in toxicity of microgravity stress by nanopolystyrene exposure in wild-type nematodes. Exposure to nanopolystyrene (1–100 μg/L) for 24-h could not affect brood size and locomotion behavior in sod-3 mutant nematodes
(Fig. 5A–B). Exposure to nanopolystyrene (1–100 μg/L) for 24-h also did not induce obvious ROS production in sod-3 mutant nematodes (Fig. 5C), and did not activate obvious mt UPR in sod-3(RNAi) nematodes (Fig. S2), which supports the detected enhancement in toxicity of microgravity stress by nanopolystyrene (10–100 μg/L) exposure in sod-3 mutant nematodes. Nevertheless, exposure to nanopolystyrene (1000 μg/L) could significantly decrease locomotion behavior in sod-3 mutant nematodes (Fig. 5B), induced significant ROS production in sod-3 mutant nematodes (Fig. 5C), and caused the obvious mt UPR in sod-3(RNAi) nematodes (Fig. S2). These observations suggested that the detected enhancement in toxicity of microgravity stress by exposure to nanopolystyrene (1000 μg/L) might be due to the observed toxicity of nanopolystyrene (1000 μg/L) to a certain degree. In conclusion, we investigated the possible effects of exposure to nanopolystyrene (30 nm) on nematode C. elegans under the microgravity stress condition. In wild-type nematodes, we observed the enhancement in toxicity of microgravity stress by nanopolystyrene exposure. Moreover, this toxicity enhancement was associated with the induction of ROS production and the activation of mt UPR. Mutation of sod-3 induced the more severe enhancement in toxicity of microgravity stress by nanopolystyrene exposure. In sod-3 mutant nematodes, we even found that exposure to nanopolystyrene (10 μg/L) could enhance the toxicity of microgravity stress on nematodes. Our results are helpful for our understanding the possible effects of nanopolystyrene exposure on organisms under the microgravity stress condition. Our results highlighted the potential of nanopolystyrene exposure in enhancing the toxicity of microgravity stress on organisms.
Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623
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Please cite this article as: Y. Zhao, D. Li, Q. Rui, et al., Toxicity induction of nanopolystyrene under microgravity stress condition in Caenorhabditis elegans, Science of the Total Environment, https://doi.org/10.1016/j.scitotenv.2019.135623