Toxicity of Low-dose Graphene Oxide Nanoparticles in an in-vivo Wild Type of Caenorhabditis elegans Model

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 μg 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,


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8 1/2-DAF-16 signaling cascade in the intestine of the nematodes (Zhao et al., 2016c). After GO 133 NP exposure, expression of neuronal substances may decrease ROS generation and reduce 134 locomotion behavior in nematodes (Zhao et al., 2020). GO NPs probably caused damage to the 135 dopaminergic and glutamatergic neurons in C. elegans after chronic exposure to GO NPs for 6 136 days, from L1 larvae to the adult stage (Li et al., 2017). Liu et al. (2020) observed that GO NPs 137 induced intestinal barrier dysfunction in C. elegans. Rive et al. (2019) proposed that worms 138 chronically (or prolongedly) exposed to GO NPs (levels of 100 and 200 mg L -1 ) were 139 significantly shortened in size and developed morphological abnormalities in the pharynx and 140 intestine. Kim et al. (2018) found accumulation of GO NPs in the reproductive organs of C. 141 elegans using Raman spectrometry. Also, GO NP exposure promoted reproductive toxicity by 142 suppressing spermatogenesis of C. elegans during development, resulting in decreased sperm 143 numbers and progeny numbers (Kim et al., 2018). This study was aimed toward evaluating the 144 effect of environmentally-relevant concentrations of GO NPs in C. elegans by assessing 145 toxicological endpoints including acute lethality, reproduction, locomotion, lifespan, and gene 146 expression. 147

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9 GO NPs were prepared from expandable graphite using a modified Hummers' method (Yan 152 et al., 2014). In brief, 1 g graphite power and 50 mL sulfuric acid (H2SO4, 98%) were poured into 153 a 250 mL flask, followed by the addition of 0.5 g NaNO3. The mixture was mechanically agitated 154 for 30 min in an ice bath. For further oxidation, 5 g of potassium permanganate (KMnO4) was 155 added while slowly stirring the mixture for 4 h. Subsequently, H2O2 was added to MnO2 until the 156 mixture became yellow. Afterward, 1% HCl was added, and the mixture was centrifuged at 8000 157 rpm for 5 min, followed by washing 3 times with distilled water to dilute the acid solution. 158 159

Reagents, chemicals, and nematode cultivation 160
The GO NPs underwent sonication for 30 min (40 kHz and 100 W) to disperse them in K 161 medium (50 mM, 30 mM KCl, 1.0 mg mL -1 and PH of 6.0) as the stock solution (200 mg L -1 ) 162 following the methods in previous studies (Wu et al., 2013;Zhao et al., 2016). The stock solution 163 was diluted to various concentrations using K medium prior to exposure. 164 The wild-type N2 C. elegans strain was gifted from Dr. Chang-Shi Chen in the Department 165  (Chung et al., 2019;Chung et al., 2020). The lethality, growth, 181 reproduction, locomotion behavior examinations followed the protocols previously published, 182 with minor modifications (Chung et al., 2019;Chung et al., 2020). 183 184

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11 toxicity of the samples was evaluated by softly poking them using a worm picker. The worms 189 that did not respond were considered dead. Three biological replicates were performed, and a 190 total of 150 worms were assayed. 191 The worms evaluated for the lifespan assay were exposed for a prolonged period of time to 192 the different GO NP concentrations (the untreated control, 0.00100, 0.0100, 0.100, and 1.00 g L -193 1 ) from L1 to the mature stage for the lifespan test. Fifty worms were transferred to fresh plates 194 every other day for 4-5 days of egg-laying. Live and dead nematodes were evaluated daily by 195 softly poking them with a worm picker. Three biological replicates were performed, and a total of 196 150 worms were evaluated. Several lifespan indicators (mean lifespan, day of 50 th percentile 197 death, day of 75 th percentile death, day of 95 th percentile death, and day of all death) were 198 evaluated by following the Chung's study (Chung et al., 2020). 199 200

Reproductive assay and Locomotion assay 201
The brood size (reproductive assay) of the L3 or young L4 nematodes was assessed for 4-5 202 days after prolonged exposure to different GO NP concentrations (control, 0.00100, 0.0100, 203 0.100, and 1.00 g L -1 ) at 20℃. Each worm was transferred to a fresh plate and transferred again 204 until the egg-laying period stopped. The plates with eggs were incubated until the progeny could 205 be easily counted. A total of 30 worms was evaluated for the reproductive assay.

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12 Locomotion behavior including head thrashing and body bending in the nematode models 207 was expressed as the motor neuron function (Qu et al., 2019;Zhao et al., 2020). The body 208 bending and head thrashing of the nematodes (locomotion assay) were evaluated after prolonged 209 exposure to various concentrations of GO NP (control, 0.00100, 0.0100, 0.100, and 1.00 g L -1 ). 210 The body bending was evaluated by transferring the exposed worm onto a fresh plate. After one 211 day, the body bending of the worms was counted for 20 secs. The head thrashing was evaluated 212 by placing the exposed worm on a glass slide containing an adequate amount of K-media. The 213 head thrashing of the worms was counted for 1 min. Three biological replicates were performed, 214 where 60 worms were evaluated for body bending, and 30 worms were evaluated for head 215 thrashing. 216 217

Gene expression tests 218
C. elegans in the different treatment groups (untreated control, 0.00100, 0.100, and 1.00 g 219 L -1 ) were collected from three replicates for RNA extraction after exposure. Trizol reagent 220 (TIANGEN, China) was used to extract total RNA in accordance with the manufacturer's 221 standard protocol. RNA concentrations were measured by the absorbance at 260 nm, and the 222

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13 500 ng of purified RNA using a Fast Quant RT Kit (with gDNase) according to the 225 manufacturer's protocol (TIANGEN, China). Specific superoxide dismutase genes, including (sod) 226 1 (sod-1), sod-3, and catalase 2 (ctl-2) were detected in the present study. The data were analyzed 227 using the 2-△△Ct method, as previously reported (Zhou et al., 2016), and the mRNA 228 expressions were normalized based on the act-1 mRNA. For each tested gene, a qRT-PCR 229 analysis was conducted in triplicate (technical replicates). 230 231

Statistical Analysis 232
The Statistical Package for the Social Sciences (SPSS) version 12 software (International 233 Business Machines Corp., New York, USA) was used to perform all statistical analyses. All data 234 was checked to normality, and the Shapiro-Wilk test was used determined the normal and non-235 normal distribution. A one-way ANOVA was used to analyze the significance levels of the 236 differences between treatments. The plots and figures were made using GraphPad Prism 6 (San 237 Diego, California, USA). 238

RESULTS AND DISCUSSION
240 reproduction, locomotion, lifespan, and oxidative stress (gene expression of sod-1, sod-3, and clt-244 2) in the present study. This study is the first time that low doses of GO NP (approximately at 245 least 1000-fold lower compared to those used in the previous studies) has been used to examine 246 nanotoxicity in a C. elegans model. Exposure from L1-larvae to young adult was performed to 247 assess the effects of prolonged GO exposure on both larvae and adult nematodes. As shown in 248 NP concentrations from 0.100 to 100 mg L -1 after the worms were acutely or prolongedly 254 exposed to these carbon-based ENMs. On the contrary, a study examining acute toxicity with 255 treatment of high doses of 5, 10, 50, and 100 mg L -1 of GO NPs in nematodes found that GO 256 concentrations higher than 5 mg L -1 may cause lethality, where no worms survived at a dosage of 257 100 mg L -1 (Li et al., 2017). Wu et al. (2014) also examined low levels of GO NPs from 0.00100 258 to 1.00 mg L -1 and found no significant differences in lethality except in the case of the highest

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15 ENMs from L1 larvae to adult-day 8. Most GO NP studies refer to Wu's study (Wu et al., 2013) 261 and use similar dosage levels (ppm levels) to examine neurological, reproductive, 262 neurobehavioral, and immunological toxicity, and inflammatory responses. results, suggest that prolonged exposure to GO NPs at low doses from 0.0100 to 1.00 g L -1 287 could decrease progeny number or fecundity in N2 C elegans models. 288

GO NP exposure affects locomotive behavior 290
Locomotive behavior assays are well-established methods for studying nematode 291 neurotoxicity. After prolonged exposure, GO induced obvious decreases in both head thrashing 292 and body bending in nematodes (Fig. 3). In the head thrash examination, 0.0100, 0.100 and, 1.00 293 g L -1 concentrations of GO NPs significantly decreased head thrashing by 12. 0, 5.41, and 19.8%, 294 respectively, compared to the untreated control. Furthermore, body bending was significantly 295 reduced at 0.00100, 0.0100, 0.100 and 1.00 g/L GO NPs by 8.78, 21.2, 31.5, and 40.8%, 296 respectively, in comparison with the control groups. Our results were consistent with those in 297 most published articles, implying that GO NP exposure damages the neurological functions and 298 negatively disrupts head thrashing and body bending behavior (Chen et al., 2017;Kim et al., al., 2015;Zhao et al., 2016a;Zhao et al., 2020). In Wu's report (Wu et al., 2014), head thrash 301 and body bend locomotion was significantly reduced at 0.0100, 0.100, and 1.00 mg L -1 levels 302 compared with an untreated control. Li et al. (2017) indicated that prolonged exposure to GO 303 NPs (5.00-100 mg L -1 ) significantly reduced body bending, head thrashing, pharynx pumping 304 frequency, mean speed, bending angle-frequency, and the wavelength of the crawling movement 305 of nematodes. GO NPs also induced damage to dopaminergic and glutamatergic neurons in 306 nematodes (Li et al., 2017). Kim et al. (2020) also proposed that GO significantly accumulated in 307 the head regions, generated ROS induction, reduced neurotransmitter substances in dopaminergic 308 and glutamatergic neurons, and damaged AFD neurons, which are the main thermosensors 309 in C. elegans, after the nematodes were exposed to GO NPs (10 mg L -1 ). In a Korean study, Kim 310 et al. (2018) also found that neurotransmitters, such as dopamine, γ-Aminobutyric acid (GABA), 311 tyramine, tryptophan, and tyrosine, were reduced in nematodes exposed to GO NPs. According to 312 the current data, including the present study (Chen et al., 2017;Kim et al., 2018;Li et al., 2017;313 Qu et al., 2017;Rive et al., 2019;Wu et al., 2013;Wu et al., 2014;Zhao et al., 2015;Zhao et al., 314 2016a;Zhao et al., 2020), it has been concluded that GO NPs exposure causes adverse effects on 315 the neurological system of C. elegans particularly in terms of damage to neurons, influences on 316 neurotransmitter neurodisruptions, and delays in neurobehavioral development. In the present 317 study, environmental levels (0.0100-1.00 g L -1 ) of GO NP doses were used to treat the 318 nematodes to determine the negative impact on their locomotion behavior. 319 320

Effect of GO NPs on Lifespan 321
In C. elegans models, lifespan is an important endpoint for assessment of toxicants. After 322 prolonged exposure in nematodes, GO NPs at concentrations of 0.00100-1.00 g L -1 led to

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18 lifespan, including mean lifespan (Fig 4b), mean day of median (50 th percentile) death (Fig. 4c), 325 mean day of 75 th percentile death (Fig. 4d), mean day of 95 th percentile death (Fig. 4e), and the 326 day of all death (Fig. 4a) indicated significantly longer longevity in the untreated control as 327 compared to in GO NP-exposed nematodes (p < 0.001). The mean lifespan and the day of all 328 death were 13.9, 7.01, 6.23, 6.94, and 6.35 days in the untreated control and 0.00100, 0.0100, 329 0.100, and 1.00 g L -1 , respectively, and 20, 16, 14, 14, 16 days in the untreated control and 330 0.00100, 0.0100, 0.100, and 1.00 g L -1 , respectively. After 6 days, the percent survival rate of 331 nematodes decreased to as much as 50% of the total population. It was also observed that the 332 nematodes treated with GO NPs exhibited faster reductions in lifespan than the control group. In 333 summary, Fig. 4 indicates that prolonged exposure to GO NPs reduces the lifespan of nematodes 334 insulin signaling may be involved in the shortened longevity of nematodes exposed to an GO NP 342 concentration of 100 mg L -1 due to association with suppression of DAF-16 and sod-3 functions 343 (Zhao et al., 2016c). Based on our results, the low dose of 0.00100 g L -1 significantly reduced 344 the nematodes' longevity. 345

19
The sod genes encode superoxide dismutases (SODs), which comprise an antioxidant system for 348 C. elegans against oxidative stress after GO NP exposure (Ren et al., 2018). SODs which exist in 349 three isoforms of sod1, sod2, and sod-3 are a class of the antioxidant protein. The increased 350 folds in expression from the induced sod-1, sod-3, and ctl-2 genes after C. elegans had undergone 351 prolonged exposure to 0.00100, 0.100, and 1.00 g L -1 GO NP compared with the untreated control 352 are shown in Fig. 5. The activated expressions of sod-1, sod-3, and ctl-2 at the concentrations of 353 0.00100, 0.100, and 1.00 g L -1 in the GO NP-exposed C. elegans were significantly higher than 354 those in the untreated control. SOD is a key enzyme in the detoxification function of free 355

radicals. It removed free radicals generated from GO NPs in extracellular sources in 356
nematodes. Results similar to those found in the present study were also found in previous studies 357 (Wu et al., 2013;Zhao et al., 2016c), which indicates that GO NPs could induce sod-1 or sod-3 358 activation. The findings from Wu's study suggested that oxidative stress induced in the treated GO 359 NP nematodes may be related to changes of SOD activities (Wu et al., 2013). Based on these findings, 360 it can be inferred that oxidative stress is a possible mechanism causing adverse effects on 361 neurodevelopment and neurobehavioral development after prolonged GO NP exposure, as suggested 362 in previous reports (Wu et al., 2013;Zhao et al., 2016c), in combination with the results of induced 363 SOD activation and neurotoxicity in the GO-exposed nematodes in the present study ( Fig. 3 and 5). 364 Furthermore, sod-1, sod-3, and ctl-2 activation may be associated with the shortened longevity in the 365 GO-exposed worms, based on Fig. 4 and 5. In Zhou's study (Zhou et al., 2016), C. elegans ctl-2 gene 366 encoded peroxisomal catalase was linked to environmental oxidative stress after worms were exposed 367 to bisphenol A. Few studies have addressed to link between ctl-2 expression and GO exposure in C. 368 elegans. Although a positive association between ctl-2 expression and GO NP exposure was shown in 369 the present study, the mechanism is still unclear. 370 Finally, it was concluded in the present study that extremely low doses of GO NPs, compared

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20 toxicity and induce several-fold increases of sod-1, sod-3, and clt-2 gene expression. It is worth 373 noting that in the present study, the potentially toxic effects of environmental levels of GO NPs in in-374 vivo C. elegans models were evaluated to show the negative impacts on reproduction, 375 neurobehavioral development, and oxidative stress. It is thus reiterated that based on our findings, GO 376 NPs at environmental levels may cause chronically toxic effects.  . 20: 1974-1986. 427 Cole, R.D., Anderson, G.L. and Williams, P.L. (2004). The Nematode Caenorhabditis Elegans as 428 a Model of Organophosphate-Induced Mammalian Neurotoxicity. Toxicol.  De Marchi, L., Pretti, C., Gabriel, B., Marques, P.A.A.P., Freitas, R. and Neto, V. (2018). An 431 overview of graphene materials: properties, applications and toxicity on aquatic environments.  Environ. Sci. Technol. 44: 4583-4589. 515 Sanchez, V.C., Jachak, A., Hurt, R.H. and Kane, A.B. (2012). Biological interactions of 516 graphene-family nanomaterials: an interdisciplinary review. Chem.

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6 and (c) ctl-2 (Y54G11A.5); Actin-1 (T04C12.6) as the internal control. Bars shown as mean±SD.

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6 C o n c e n tr a tio n s (µ g L -1 ) S u r v iv a l r a te (% ) c.