Ming-Hsien Tsai1, How-Ran Chao This email address is being protected from spambots. You need JavaScript enabled to view it.2,3,4, Jheng-Jie Jiang5,6, Yu-Hsieh Su2, Mariene-syne P. Cortez7, Lemmuel L. Tayo7, I-Cheng Lu2, Hao Hsieh2Chih-Chung Lin2, Sheng-Lun Lin8,9, Wan Nurdiyana Wan Mansor10,11, Ching-Kai Su12, Sen-Ting Huang13, Wen-Li Hsu4,13

1 Department of Child Care, National Pingtung University of Science and Technology, Neipu, Pingtung 91201, Taiwan
2 Department of Environmental Science and Engineering, National Pingtung University of Science and Technology, Neipu, Pingtung 91201, Taiwan
3 Institute of Food Safety Management, College of Agriculture, National Pingtung University of Science and Technology, Neipu, Pingtung 91201, Taiwan
4 Emerging Compounds Research Center, General Research Service Center, National Pingtung University of Science and Technology, Neipu, Pingtung 91201, Taiwan
5 Department of Environmental Engineering, Chung Yuan Christian University, Taoyuan 320314, Taiwan
6 Center for Environmental Risk Management, Chung Yuan Christian University, Taoyuan 320314, Taiwan
7 School of Chemical, Biological and Materials Engineering and Sciences, Mapúa University, Muralla St., Intramuros, Manila 1002, Philippines
8 School of Mechanical Engineering, Beijing Institute of Technology, Beijing 100081, China
9 Center for Environmental Toxin and Emerging-contaminant Research, Cheng Shiu University, Kaohsiung 83347, Taiwan
10 Faculty of Ocean Engineering Technology & Informatics, Universiti Malaysia Terengganu, 21300, Malaysia
11 Air Quality and Environment Research Group, Universiti Malaysia Terengganu, 21300, K. Nerus, Malaysia
12 Department of Internal Medicine, Kaohsiung Veterans General Hospital Pingtung Branch, Neipu, Pingtung 91245, Taiwan
13 Research Institute for Life Support Innovation, Research Organization for Nano and Life Innovation, Waseda University, Shinjuku, Tokyo 162-8480, Japan


Received: September 13, 2020
Revised: November 3, 2020
Accepted: December 6, 2020

 Copyright The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cited.


Download Citation: ||https://doi.org/10.4209/aaqr.200559  

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Cite this article:

Tsai, M.H., Chao, H.R., Jiang, J.J., Su, Y.H., Cortez, M.P., Tayo, L.L., Lu, I.C., Hsieh, H., Lin, C.C., Lin, S.L., Wan Mansor, W.N., Su, C.K., Huang, S.T., Hsu, W.L. (2021). Toxicity of Low-dose Graphene Oxide Nanoparticles in an in-vivo Wild Type of Caenorhabditis elegans Model. Aerosol Air Qual. Res. 21, 200559. https://doi.org/10.4209/aaqr.200559


HIGHLIGHTS

  • GO NP exposure reduced the brood size of nematodes.
  • GO NP exposure delayed head thrash and body bend in nematodes.
  • GO NPs shortened the longevity of nematodes.
  • GO NP exposure caused the oxidative stress in nematodes.
 

ABSTRACT


Carbon-based engineered nanomaterials, such as graphene oxide nanoparticles (GO NPs), are widely available for application, but their potentially adverse health effects on humans still require investigation. In this study, the environmental levels of GO NPs are addressed to examine whether GO leads to adverse effects on an in-vivo model of Caenorhabditis elegans (C. elegans). Nematodes with prolonged exposure (L1 larvae to young adult) to GO NPs at 0.00100, 0.0100, 0.100, and 1.00 mg L–1 were used to evaluate the potential toxic effects, including lethality (acute toxicity), reproductive (brood size) and neurological (locomotion including head thrash and body bend) responses, longevity (lifespan), and oxidative stress (gene expression of sod-1, sod-3, and clt-2). Prolonged exposure to GO NPs was not found to induce lethality at the selective levels. In the brood-size and head-thrash tests, the biological responses in nematodes were significantly reduced at 0.0100-1.00 ng L–1 GO NP exposure as compared with the untreated control. The nematodes exposure to GO NPs at 0.00100–1.00 ng L–1 exhibited significant delays in body bending behavior compared with the control. In the examination of the longevity of nematodes, it was found that the lifespan of all GO NP-exposed worms was significantly shortened as compared to the untreated worms. Gene expression of sod-1, sod-3, and ctl-2 presented significantly higher induction folds in the exposed worms compared with the controls. Consequently, prolonged exposure to the low-dose GO NPs might be associated with disruption of reproduction and locomotion, attenuation of longevity, and induction of oxidative stress in nematodes.


Keywords: Graphene oxide, Caenorhabditis elegans, Reproductive toxicity, Neurobehavioral toxicity, Oxidative stress


1 INTRODUCTION


Graphene oxide nanoparticles (GO NPs), one of the most promising derivatives of graphene, comprise a monolayered engineered nanomaterial (ENM) with high oxygen-containing functional groups such as carboxyl, epoxy, carbonyl, and hydroxyl (Bianco et al., 2013; Li et al., 2015). Graphene oxide (GO) is known to have excellent dispersibility in many solvents, chemical reactivity, and the capacity for chemical functionalization (Konios et al., 2014; De Marchi et al., 2018). The adsorption capacity of GO NPs has been taken advantage of for environmental remediation applications such as in GO-based membranes, which remove gaseous contaminants such as sulfur dioxide and hydrogen sulfide (Fakhri, 2017). In addition, they also act as adsorbents for elimination of various aqueous contaminants owing to their high content of functionalized oxygen groups available to interact with metal ions (Li et al., 2019). Furthermore, GO NPs can be applied in nanoelectronics, catalysis, nanocomposites, sensor technology, water purification and desalination, and drug delivery (Zhang et al., 2011; Pan et al., 2012; Giust et al., 2018; Prasad et al., 2020). According to studies from Yang et al. (2015) and Maharubin et al. (2016), GO can be utilized for hydrogen storage (anode, cathode, and lithium sulfur batteries) and supercapacitor management. Furthermore, GO composites are used as antimicrobial agents for water disinfection to remove organic molecules and waterborne pathogens (Upadhyay et al., 2014). Several studies used the environmental levels at µg L–1 to mg L–1 of GO NP contamination to test the in-vivo models (He et al., 2017; Zhang et al., 2017; Li et al., 2019).

The global market for graphene-based products, such as GO, is increasing. The demand is expected to be $675 and 987 million by 2020 and 2022, respectively (Ahmed and Rodrigues, 2013). Due to their potential for both production and application, GO materials are expected to be released in the environment during their lifecycle and eventually be generated in landfills and wastewater treatment plants (Du et al., 2017; Suárez-Iglesias et al., 2017; Jamialahmadi et al., 2018). GO can be released into the water environment through the development of its composites as adsorbents for aqueous contamination, membranes for water filtration and purification, and catalysts for environmental decontamination (Zhao et al., 2014; Goodwin et al., 2018). Several studies have reported that the dispersion and long retention time of GO within microbial communities can lead to serious negative effects on wastewater microbial flora due to its hydrophility (Lyon and Alvarez, 2008; Kang et al., 2009; Rodrigues and Elimelech, 2010). The predicted environmental concentrations of GO can be correlated with those of multi-walled carbon nanotubes because both have relatively similar properties, such as nanometer size, a carbon-based structure, and applications in consumer electronic devices (Zhang et al., 2017).

Few epidemiological studies focused on human exposure to GO particularly for the highly exposed population. For the in-vivo models including rats, mice, zebra fish, nematodes, and daphnia, the animals could induce nanotoxicity including acute, developmental, neurological, reproductive, immunological, and neurobehavioral toxicity as well as shortened longevity after they were exposed to GO NPs (Sanchez et al., 2012; Patlolla et al., 2017; Qu et al., 2017; Souza et al., 2017; Kim et al., 2018; Qu et al., 2019; Kim et al., 2020). In the past years, in vivo and in vitro GO NPs toxic effects, including immunotoxicity, activation of inflammation, induction of reactive oxygen species (ROS), generation of oxidative stress, apoptosis, and potential GO exposure mechanisms have been investigated (Guo and Mei, 2014; Bengtson et al., 2017; Pelin et al., 2018; Tang et al., 2018). The accumulation of GO in the cytoplasm causes dramatic morphological alterations and reduces the ability of toll-like receptor 4 (TLR4) for phagocytosis (Qu et al., 2013a). However, an increase in intracellular ROS contributes to necrotic cell death in macrophages (Qu et al., 2013a). Previous studies have also reported that GO promotes cell growth inhibition, hatching delay, ROS generation, and damages the circulatory system of zebrafish embryos (Liu et al., 2014; Chen et al., 2016; Souza et al., 2017). In mice, GO can accumulate in organs such as the liver, lungs, spleen, and kidneys, which may induce organismal toxicity through intracellular oxidative stress caused by the accumulation of ROS (Qu et al., 2013b; Yang et al., 2013). GO can enter the human body through inhalation and may be deposited in regions of the respiratory tract. When deposited in alveolar regions, it may impair clearance, form granulomas, and possibly produce fibrosis (Sanchez et al., 2012).

The in vivo model used in this study was the transparent nematode, Caenorhabditis elegans (C. elegans), which has been successfully used in toxicological evaluation of various nanomaterials such as GO NPs (Zhang et al., 2012; Wu et al., 2013; Piechulek and von Mikecz, 2018). Advantages of C. elegans as an in-vivo model system were as the following: (1) simple anatomy, (2) transparent, (3) invariant cell lineage, (4) short life cycle with large brood size, (5) easily accessible embryos, (6) easy and cheap maintenance in lab, and (7) powerful experimental tool (Brenner, 1974; Hunt, 2017). The C. elegans model is not a mammal model to unavailable to examine several toxic endpoints like blood sugar and pressure, tissues in the skin, probiotic system in the intestine, heart and cardiovascular diseases. C. elegans is considered to be a novel tool for in-vivo techniques, and testing of C. elegans is known to be analogous to mammalian neurotoxin testing (Cole et al., 2004). Recently, scientists have focused on the disruption of biological effects from ROS, reproductive effects, gene expression, neurological development, and neurobehavior with treatment of GO NPs in C. elegans models (Wu et al., 2013; Qu et al., 2017; Kim et al., 2018; Rive et al., 2019; Kim et al., 2020; Zhao et al., 2020). Zhang et al. (2012) indicated no negative impact on longevity after exposing L4-larva-to-young-adult C. elegans to GO NPs at concentrations ranging from 5 to 20 mg L–1. Inversely, C. elegans with prolonged exposure (from L1 larva to young adult) to 0.5–100 mg L–1 of GO NPs presented adverse effects on primary (digestive organs such as the intestine) and secondary (neurological tissues such as neurons and reproductive organs) target organs (Wu et al., 2013). GO NPs possibly shortened lifespan by influencing the expression of the DAF-2-AGE-1-AKT-1/2-DAF-16 signaling cascade in the intestine of the nematodes (Zhao et al., 2016b). After GO NP exposure, expression of neuronal substances may decrease ROS generation and reduce locomotion behavior in nematodes (Zhao et al., 2020). GO NPs probably caused damage to the dopaminergic and glutamatergic neurons in C. elegans after chronic exposure to GO NPs for 6 days, from L1 larvae to the adult stage (Li et al., 2017). Liu et al. (2020) observed that GO NPs induced intestinal barrier dysfunction in C. elegans. Rive et al. (2019) proposed that worms chronically (or prolongedly) exposed to GO NPs (levels of 100 and 200 mg L–1) were significantly shortened in size and developed morphological abnormalities in the pharynx and intestine. Kim et al. (2018) found accumulation of GO NPs in the reproductive organs of C. elegans using Raman spectrometry. Also, GO NP exposure promoted reproductive toxicity by suppressing spermatogenesis of C. elegans during development, resulting in decreased sperm numbers and progeny numbers (Kim et al., 2018). This study was aimed toward evaluating the effect of environmentally-relevant concentrations of GO NPs in C. elegans by assessing toxicological endpoints including acute lethality, reproduction, locomotion, lifespan, and gene expression.

 
2 METHODS


 
2.1 Preparation of Graphene Oxide Nanoparticles

GO NPs were prepared from expandable graphite using a modified Hummers’ method (Yan et al., 2014). In brief, 1 g graphite power and 50 mL sulfuric acid (H2SO4, 98%) were poured into a 250 mL flask, followed by the addition of 0.5 g NaNO3. The mixture was mechanically agitated for 30 min in an ice bath. For further oxidation, 5 g of potassium permanganate (KMnO4) was added while slowly stirring the mixture for 4 h. Subsequently, H2O2 was added to MnO2 until the mixture became yellow. Afterward, 1% HCl was added, and the mixture was centrifuged at 8000 rpm for 5 min, followed by washing 3 times with distilled water to dilute the acid solution.

 
2.2 Reagents, Chemicals, and Nematode Cultivation

The GO NPs underwent sonication for 30 min (40 kHz and 100 W) to disperse them in K medium (50 mM, 30 mM KCl, 1.0 mg mL–1 and PH of 6.0) as the stock solution (200 mg L–1) following the methods in previous studies (Wu et al., 2013; Zhao et al., 2016b). The stock solution was diluted to various concentrations using K medium prior to exposure.

The wild-type N2 C. elegans strain was gifted from Dr. Chang-Shi Chen in the Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University (Tainan, Taiwan). C. elegans was maintained on nematode growth medium (NGM) seeded with OP50 Escherichia coli (E. coli) cultures from the Bioresources Collection and Research Center (Hsinchu, Taiwan), and Luria-Bertani broth was obtained from Sigma-Aldrich (St. Louis, MO, USA). The NGM plates contained bacteriological agar and bactopeptone, which were obtained from Laboratories Conda (S.A., Spain). The NaCl was obtained from Honeywell Fluka™ (New Jersey, USA). Age-synchronized worms were collected using a bleaching mixture that contained NaOCl obtained from J.T. Baker (Central Valley, PA) and KOH obtained from Duksan Pure Chemicals (Gyeonggi-do, South Korea). Supplemental reagents such as CaCl2, K2HPO4, and cholesterol were obtained from Sigma-Aldrich (St. Louis, MO, USA); MgSO4 was acquired from Avantor Performance Materials, ltd. (Gyeonggi-do, South Korea); the KH2PO4 used for the phosphate buffer was acquired from Avantor Performance Materials, LLC (Radnor, PA, USA), and the Na2HPO4 used for the M9 buffer was obtained from Honeywell Fluka™ (New Jersey, USA). All physiological observations were done under a dissecting microscope (Olympus, SZX10, Waltham MA, USA). The experimental protocols in the nematode model followed those used in our previous study (Chung et al., 2019, 2020). The lethality, growth, reproduction, locomotion behavior examinations followed the protocols previously published, with minor modifications (Chung et al., 2019, 2020).

 
2.3 Lethality and Lifespan Assay

The nematodes were exposed to GO NPs (control, 0.00100, 0.0100, 0.100, and 1.00 µg L–1) for 48 h (prolonged exposure) from L1-larvae to young adults incubated at 20°C. Prolonged exposure was performed in a fresh plate with an OP50 E. coli lawn. After treatment, the lethal toxicity of the samples was evaluated by softly poking them using a worm picker. The worms that did not respond were considered dead. Three biological replicates were performed, and a total of 150 worms were assayed.

The worms evaluated for the lifespan assay were exposed for a prolonged period of time to the different GO NP concentrations (the untreated control, 0.00100, 0.0100, 0.100, and 1.00 µg L–1) from L1 to the mature stage for the lifespan test. Fifty worms were transferred to fresh plates every other day for 4–5 days of egg-laying. Live and dead nematodes were evaluated daily by softly poking them with a worm picker. Three biological replicates were performed, and a total of 150 worms were evaluated. Several lifespan indicators (mean lifespan, day of 50th percentile death, day of 75th percentile death, day of 95th percentile death, and day of all death) were evaluated by following the Chung’s study (Chung et al., 2020).

 
2.4 Reproductive Assay and Locomotion Assay

The brood size (reproductive assay) of the L3 or young L4 nematodes was assessed for 4–5 days after prolonged exposure to different GO NP concentrations (control, 0.00100, 0.0100, 0.100, and 1.00 µg L–1) at 20°C. Each worm was transferred to a fresh plate and transferred again until the egg-laying period stopped. The plates with eggs were incubated until the progeny could be easily counted. A total of 30 worms was evaluated for the reproductive assay.

Locomotion behavior including head thrashing and body bending in the nematode models was expressed as the motor neuron function (Qu et al., 2019; Zhao et al., 2020). The body bending and head thrashing of the nematodes (locomotion assay) were evaluated after prolonged exposure to various concentrations of GO NP (control, 0.00100, 0.0100, 0.100, and 1.00 µg L–1). The body bending was evaluated by transferring the exposed worm onto a fresh plate. After one day, the body bending of the worms was counted for 20 secs. The head thrashing was evaluated by placing the exposed worm on a glass slide containing an adequate amount of K-media. The head thrashing of the worms was counted for 1 min. Three biological replicates were performed, where 60 worms were evaluated for body bending, and 30 worms were evaluated for head thrashing.

 
2.5 Gene Expression Tests

C. elegans in the different treatment groups (untreated control, 0.00100, 0.100, and 1.00 µg L–1) were collected from three replicates for RNA extraction after exposure. Trizol reagent (TIANGEN, China) was used to extract total RNA in accordance with the manufacturer's standard protocol. RNA concentrations were measured by the absorbance at 260 nm, and the RNA purity was evaluated based on the ratio of the optical densities from RNA samples measured at 260 and 280 nm. The first-strand cDNA synthesis reaction was conducted with 500 ng of purified RNA using a Fast Quant RT Kit (with gDNase) according to the manufacturer's protocol (TIANGEN, China). Specific superoxide dismutase genes, including (sod) 1 (sod-1), sod-3, and catalase 2 (ctl-2) were detected in the present study. The data were analyzed using the 2-ΔΔCt method, as previously reported (Zhou et al., 2016), and the mRNA expressions were normalized based on the act-1 mRNA. For each tested gene, a qRT-PCR analysis was conducted in triplicate (technical replicates).

 
2.6 Statistical Analysis

The Statistical Package for the Social Sciences (SPSS) version 12 software (International Business Machines Corp., New York, USA) was used to perform all statistical analyses. All data was checked to normality, and the Shapiro-Wilk test was used determined the normal and non-normal distribution. A one-way ANOVA was used to analyze the significance levels of the differences between treatments. The plots and figures were made using GraphPad Prism 6 (San Diego, California, USA).

 
3 RESULTS AND DISCUSSION


 
3.1 GO NPs Lethality

C. elegans was evaluated through a variety of toxicological endpoints, including lethality, reproduction, locomotion, lifespan, and oxidative stress (gene expression of sod-1, sod-3, and clt-2) in the present study. This study is the first time that low doses of GO NP (approximately at least 1000-fold lower compared to those used in the previous studies) has been used to examine nanotoxicity in a C. elegans model. Exposure from L1-larvae to young adult was performed to assess the effects of prolonged GO exposure on both larvae and adult nematodes. As shown in Fig. 1, the survival rate of the nematodes after prolonged exposure to GO did not indicate significant lethal effects. No significant between-group differences in mortality were observed in thecontrol group and in the group treated with concentrations ranging from 0.00100 to 1.00 μg L–1. Few studies have examined acute toxicity of GO NPs in N2 C. elegans models (Wu et al., 2013, 2014; Li et al., 2019). Wu et al. (2013) indicated that no lethality was observed at GO NP concentrations from 0.100 to 100 mg L–1 after the worms were acutely or prolongedly exposed to these carbon-based ENMs. On the contrary, a study examining acute toxicity with treatment of high doses of 5, 10, 50, and 100 mg L–1 of GO NPs in nematodes found that GO concentrations higher than 5 mg L–1 may cause lethality, where no worms survived at a dosage of 100 mg L–1 (Li et al., 2017). Wu et al. (2014) also examined low levels of GO NPs from 0.00100 to 1.00 mg L–1 and found no significant differences in lethality except in the case of the highest concentration of 1.00 mg L–1 after the nematodes were chronically exposed to these carbon-based ENMs from L1 larvae to adult-day 8. Most GO NP studies refer to Wu’s study (Wu et al., 2013) and use similar dosage levels (ppm levels) to examine neurological, reproductive, neurobehavioral, and immunological toxicity, and inflammatory responses.

Fig. 1. Survival rates of C. elegans after prolonged exposure to GO NPs at concentrations of 0.00100, 0.0100, 0.100, and 1.00 and the untreated control. Bars shown as mean ± SD. Significant differences were expressed as * p < 0.05, **p < 0.01, and ***p < 0.001.Fig. 1. Survival rates of C. elegans after prolonged exposure to GO NPs at concentrations of 0.00100, 0.0100, 0.100, and 1.00 and the untreated control. Bars shown as mean ± SD. Significant differences were expressed as * < 0.05, **< 0.01, and ***< 0.001.

 
3.2 Reproductive Toxicity of GO NPs

Reproduction in nematodes is a vital endpoint because it has been shown to be sensitive to lower concentrations of chemical stressors than those that impair the behavior and viability of nematodes (Wu et al., 2019). The results in Fig. 2 show that prolonged exposure to GO NPs in the nematodes reduced brood size production. A significant decrease was observed in the progeny number at concentrations of 0.0100 (= 0.036), 0.100 (= 0.008), and 1.00 (< 0.001) µg L–1 of GO NPs. The reduction rates in the brood size at these three concentrations compared to the control group were 15.2, 20.1 and 27.3%, respectively. The results showed that higher concentrations of GO NPs induced more reproductive toxicity based on our experiments on brood size number in nematodes. Our results were consistent with most GO NP studies reporting that GO exposure can cause adverse effects through damaging the fertility and egg ejection behavior of nematodes (Wu et al., 2013; Zhao et al., 2016a; Kim et al., 2018; Rive et al., 2019). Wu et al. (2013) showed that C. elegans with prolonged exposure to 1–100 mg L–1 exhibited significantly decreased brood size compared to the control, but there were no significant between-group differences at 0.1 and 0.5 mg L–1. A similar result was also found in a previous study (Rive et al., 2019), indicating that prolonged exposure to GO NPs at 100 and 200 mg L–1 significantly decreased egg-laying rates compared to the untreated control. Kim et al. (2018) revealed that accumulation of GO NPs (10 mg L–1) in the reproductive organs, which might be the direct cause of reproductive toxicity, could reduce brood size and sperm count by suppressing spermatogenesis of the hermaphrodite nematodes at the GO levels of 5 or 10 mg L–1. However, the negative impact of GO NP exposure on the reproductive function in the present and published studies (Wu et al., 2013; Zhao et al., 2016a; Kim et al., 2018; Rive et al., 2019), as well as our results, suggest that prolonged exposure to GO NPs at low doses from 0.0100 to 1.00 µg L–1 could decrease progeny number or fecundity in N2 C elegans models.

Fig. 2. Effects of the brood size in the C. elegans after prolonged exposure to GO NPs at concentrations of 0.00100, 0.0100, 0.100, and 1.00 and the untreated control. Bars shown as mean ± SD. Significant differences were expressed as *p < 0.05, ** p < 0.01, and ***p < 0.001.Fig. 2Effects of the brood size in the C. elegans after prolonged exposure to GO NPs at concentrations of 0.00100, 0.0100, 0.100, and 1.00 and the untreated control. Bars shown as mean ± SD. Significant differences were expressed as *< 0.05, ** < 0.01, and ***< 0.001.

 
3.3. GO NP Exposure Affects Locomotive Behavior

Locomotive behavior assays are well-established methods for studying nematode neurotoxicity. After prolonged exposure, GO induced obvious decreases in both head thrashing and body bending in nematodes (Fig. 3). In the head thrash examination, 0.0100, 0.100 and, 1.00 µg L–1 concentrations of GO NPs significantly decreased head thrashing by 12.0, 5.41, and 19.8%, respectively, compared to the untreated control. Furthermore, body bending was significantly reduced at 0.00100, 0.0100, 0.100 and 1.00 µg L–1 GO NPs by 8.78, 21.2, 31.5, and 40.8%, respectively, in comparison with the control groups. Our results were consistent with those in most published articles, implying that GO NP exposure damages the neurological functions and negatively disrupts head thrashing and body bending behavior (Wu et al., 2013, 2014; Zhao et al., 2015, 2016c; Chen et al., 2017; Li et al., 2017; Qu et al., 2017; Kim et al., 2018; Rive et al., 2019; Zhao et al., 2020). In Wu’s report (Wu et al., 2014), head thrash and body bend locomotion was significantly reduced at 0.0100, 0.100, and 1.00 mg L–1 levels compared with an untreated control. Li et al. (2017) indicated that prolonged exposure to GO NPs (5.00–100 mg L–1) significantly reduced body bending, head thrashing, pharynx pumping frequency, mean speed, bending angle-frequency, and the wavelength of the crawling movement of nematodes. GO NPs also induced damage to dopaminergic and glutamatergic neurons in nematodes (Li et al., 2017). Kim et al. (2020) also proposed that GO significantly accumulated in the head regions, generated ROS induction, reduced neurotransmitter substances in dopaminergic and glutamatergic neurons, and damaged AFD neurons, which are the main thermosensors in Celegans, after the nematodes were exposed to GO NPs (10 mg L–1). In a Korean study, Kim et al. (2018) also found that neurotransmitters, such as dopamine, γ-Aminobutyric acid (GABA), tyramine, tryptophan, and tyrosine, were reduced in nematodes exposed to GO NPs. According to the current data, including the present study (Wu et al., 2013, 2014; Zhao et al., 2015, 2016a; Chen et al., 2017; Li et al., 2017; Qu et al., 2017; Kim et al., 2018; Rive et al., 2019; Zhao et al., 2020), it has been concluded that GO NPs exposure causes adverse effects on the neurological system of C. elegans particularly in terms of damage to neurons, influences on neurotransmitter neurodisruptions, and delays in neurobehavioral development. In the present study, environmental levels (0.0100–1.00 µg L–1) of GO NP doses were used to treat the nematodes to determine the negative impact on their locomotion behavior.

Fig. 3. Effects of head thrashing and body bending in the nematodes after prolonged exposure to GO NPs at concentrations of 0.00100, 0.0100, 0.100, and 1.00 and the untreated control. Bars shown as mean ± SD. Significant differences were expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.Fig. 3. Effects of head thrashing and body bending in the nematodes after prolonged exposure to GO NPs at concentrations of 0.00100, 0.0100, 0.100, and 1.00 and the untreated control. Bars shown as mean ± SD. Significant differences were expressed as *< 0.05, **< 0.01, and ***< 0.001.

 
3.4 Effect of GO NPs on Lifespan

In C. elegans models, lifespan is an important endpoint for assessment of toxicants. After prolonged exposure in nematodes, GO NPs at concentrations of 0.00100–1.00 µg L–1 led to shorter lifespans than was the case for the untreated controls (Fig. 4). Several indicators of lifespan, including mean lifespan (Fig. 4(b)), mean day of median (50th percentile) death (Fig. 4(c)), mean day of 75th percentile death (Fig. 4(d)), mean day of 95th percentile death (Fig. 4(e)), and the day of all death (Fig. 4(a)) indicated significantly longer longevity in the untreated control as compared to in GO NP-exposed nematodes (p < 0.001). The mean lifespan and the day of all death were 13.9, 7.01, 6.23, 6.94, and 6.35 days in the untreated control and 0.00100, 0.0100, 0.100, and 1.00 µg L–1, respectively, and 20, 16, 14, 14, 16 days in the untreated control and 0.00100, 0.0100, 0.100, and 1.00 µg L–1, respectively. After 6 days, the percent survival rate of nematodes decreased to as much as 50% of the total population. It was also observed that the nematodes treated with GO NPs exhibited faster reductions in lifespan than the control group. In summary, Fig. 4 indicates that prolonged exposure to GO NPs reduces the lifespan of nematodes (p < 0.001). According to the current data from previous reports (Zhang et al., 2012; Zhao et al., 2016b, 2016c; Qu et al., 2017; Rive et al., 2019), contradictory results were obtained, where two studies indicated that GO NP exposure, including both acute and prolonged exposure didn’t have effects on longevity (Zhang et al., 2012; Rive et al., 2019), and other studies obtained different results indicating that the worms with prolonged exposure to GO NPs exhibited significantly reduced longevity (Zhao et al., 2016b, 2016c; Qu et al., 2017) at GO NP concentrations between 1.00 and 200 mg L–1. Two molecular mechanisms of intestinal insulin signaling may be involved in the shortened longevity of nematodes exposed to an GO NP concentration of 100 mg L–1 due to association with suppression of DAF-16 and sod-3 functions (Zhao et al., 2016b). Based on our results, the low dose of 0.00100 µg L–1 significantly reduced the nematodes’ longevity.

Fig. 4. Lifespan of C. elegans after prolonged exposure to GO NPs at levels of 0.00100, 0.0100, 0.100, and 1.00 µg L–1 and the control (a) nanotoxic assessment of worms with prolonged GO exposure for lifespan, (b) mean lifespan, (c) mean day of 50th percentile death, (d) mean day of 75th percentile death, and (e) mean day of 95th percentile death. Bars shown as mean ± SD. Significant differences were expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.Fig. 4. Lifespan of C. elegans after prolonged exposure to GO NPs at levels of 0.00100, 0.0100, 0.100, and 1.00 µg L–1 and the control (a) nanotoxic assessment of worms with prolonged GO exposure for lifespan, (b) mean lifespan, (c) mean day of 50th percentile death, (d) mean day of 75th percentile death, and (e) mean day of 95th percentile death. Bars shown as mean ± SD. Significant differences were expressed as *< 0.05, **< 0.01, and ***< 0.001.

 
3.5 Gene Expression after GO NPs Exposure

The sod genes encode superoxide dismutases (SODs), which comprise an antioxidant system for C. elegans against oxidative stress after GO NP exposure (Ren et al., 2018). SODs which exist in three isoforms of sod1, sod2, and sod-3 are a class of the antioxidant protein. The increased folds in expression from the induced sod-1, sod-3, and ctl-2 genes after C. elegans had undergone prolonged exposure to 0.00100, 0.100, and 1.00 µg L–1 GO NP compared with the untreated control are shown in Fig. 5. The activated expressions of sod-1, sod-3, and ctl-2 at the concentrations of 0.00100, 0.100, and 1.00 µg L–1 in the GO NP-exposed C. elegans were significantly higher than those in the untreated control. SOD is a key enzyme in the detoxification function of free radicals. It removed free radicals generated from GO NPs in extracellular sources in nematodes. Results similar to those found in the present study were also found in previous studies (Wu et al., 2013; Zhao et al., 2016b), which indicates that GO NPs could induce sod-1 or sod-3 activation. The findings from Wu’s study suggested that oxidative stress induced in the treated GO NP nematodes may be related to changes of SOD activities (Wu et al., 2013). Based on these findings, it can be inferred that oxidative stress is a possible mechanism causing adverse effects on neurodevelopment and neurobehavioral development after prolonged GO NP exposure, as suggested in previous reports (Wu et al., 2013; Zhao et al., 2016b), in combination with the results of induced SOD activation and neurotoxicity in the GO-exposed nematodes in the present study (Figs. 3 and 5). Furthermore, sod-1, sod-3, and ctl-2 activation may be associated with the shortened longevity in the GO-exposed worms, based on Figs. 4 and 5. In Zhou’s study (Zhou et al., 2016), C. elegans ctl-2 gene encoded peroxisomal catalase was linked to environmental oxidative stress after worms were exposed to bisphenol A. Few studies have addressed to link between ctl-2 expression and GO exposure in C. elegans. Although a positive association between ctl-2 expression and GO NP exposure was shown in the present study, the mechanism is still unclear.

Fig. 5. Gene expression in C. elegans with prolonged exposure to GO NPs at the levels of the untreated control, 0.00100, 0.100, and 1.00 µg L–1 (a) SOD-1 (C15F1.7), (b) SOD-3 (C08A9.1), and (c) ctl-2 (Y54G11A.5); Actin-1 (T04C12.6) as the internal control. Bars shown as mean ± SD. Significant differences were expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.Fig. 5. Gene expression in C. elegans with prolonged exposure to GO NPs at the levels of the untreated control, 0.00100, 0.100, and 1.00 µg L–1 (a) SOD-1 (C15F1.7), (b) SOD-3 (C08A9.1), and (c) ctl-2 (Y54G11A.5); Actin-1 (T04C12.6) as the internal control. Bars shown as mean ± SD. Significant differences were expressed as *< 0.05, **< 0.01, and ***< 0.001.

Finally, it was concluded in the present study that extremely low doses of GO NPs, compared with the dosages discussed in recent published articles, can cause reproductive and neurobehavioral toxicity and induce several-fold increases of sod-1, sod-3, and clt-2 gene expression. It is worth noting that in the present study, the potentially toxic effects of environmental levels of GO NPs in in-vivo C. elegans models were evaluated to show the negative impacts on reproduction, neurobehavioral development, and oxidative stress. It is thus reiterated that based on our findings, GO NPs at environmental levels may cause chronically toxic effects.

 
4 CONCLUSIONS


It is the first time to use the low dosage of GO NPs treating in the in-vivo model to find the adverse effects in nematodes. Based on our findings, prolonged exposure to GO NPs causes reproductive effects, generates neurotoxicity, shortens longevity, and induces oxidative stress in C. elegans. It is reiterated that low-dose GO NPs at environmental levels from 0.00100 to 1.00 µg L–1 caused significantly negative impacts on nematodes in contrast to the current published data. Thus, the adverse effects of low-level GO NPs on human health should be evaluated in the future.

 
ACKNOWLEDGMENTS


This study was supported by grants from the Ministry of Science and Technology (MOST 106-2221-E-020-001-MY3) and Kaohsiung Veterans General Hospital, Pingtung Branch (VHCL-108002). We acknowledge Ms. Danielle E. Que in the Department of Environmental engineering, National Cheng Kung University for assisting us to maintain, culture, and test C. elegans. We also want to thank Miss Yi-Jun Hsieh from Kaohsiung Medical University for assisting us in performing the C. elegans experiments. We would also like to thank Dr. Chang-Shi Chen at National Cheng Kung University for his advice and help in obtaining the C. elegans culture.

 
DISCLAIMER


The authors of this paper declare no conflict of interest.


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