Introduction 

Graphene family materials (GFM) are presently regarded as one of the most fascinating nanomaterials with numerous applications. Graphene is the fundamental structural component of a variety of carbon allotropes. Graphite (GR) is composed of graphene sheets arranged in sequential stacks through carbon-carbon bonds with sp²-hybridization. The arrangement of carbon atoms in graphene forms a honeycomb lattice, with van der Waals interactions binding adjacent planes within each layer. Consequently, the exfoliation of GR yields isolated graphene sheets, which can be accomplished by chemical and mechanical methods ¹.

Unlike graphene, graphene oxide (GO) has functional groups, including carboxylic acid, epoxide, and hydroxyl, attached to a carbon sheet ². The reduction can partially remove these functional groups ³. Thermal, chemical, or UV treatment of GO produces reduced graphene oxide (rGO) with fewer O2 groups and enhanced electrical conductivity ⁴. After reduction, rGO resembles an isolated graphene sheet; the existence of O groups in its structure only makes the difference ^{5, 6}.

Graphene has become a robust material for the twenty-first century owing to its extraordinary characteristics, such as notable mechanical strength, impressive flexibility, highly efficient electrical/thermal conductivity, and chemical stability⁷. As drug carrier materials and scaffolds, graphene and its byproducts have been extensively evaluated in various applications, particularly biomedicine. In addition, it is employed in gene delivery, biosensors, tissue engineering, and neurosurgery⁸.

Rutin (3, 3′, 4′, 5, 7- pentahydroxyflavone-3-rhamnoglucoside) is a bioflavonoid present in various natural sources, like fruits and vegetables, especially buckwheat, tea, apple and passion flower⁹. It has numerous biological functions and pharmaceutical applications, including antioxidant, antiproliferative, anti-inflammatory, antiviral, and anticarcinogenic properties. While inhibiting platelet aggregation, it reduces hyperlipidemia. In addition, its low toxicity makes it an excellent candidate for potential clinical applications ^10-12.

Owing to its exceptional structural and biological characteristics, zebrafish (Danio rerio) has been employed as an in vivo model to investigate the environmental health and safety (EHS) implications of engineered nanomaterials and nano-related products ¹³. Thereafter, many remarkable research using embryos, larvae, adults, and transgenic zebrafish have been carried out on accumulation, morphology, behaviour, neurological disorders, molecular mechanisms, and other potential targets of GPN ^14-19.

In recent years, a few studies have explored carbon nanoparticles’ environmental implications and investigated GO and rGO’s toxicity across various cell types (bacterial, mammalian, and plant) and animal models (mice and zebrafish). Moreover, these studies have collected data on the impact of GFNs in both soil and aquatic ecosystems ^20-23. The specific mechanism by which graphene derivatives interact with cells remains unclear; however, for GO, its toxicity is impacted by factors like the amount and type of O²-containing functional groups and the size, charge, and aggregation of the sheets². Zhu et al.²⁴ found that the aggregation of Buckminster-fullerene (C60) decreased the survival and hatching rates of zebrafish. Additionally, Zhang et al.²⁵ and Yan et al.²⁶ examined the effects of graphene and rGO on human health. Carbon nanoparticles have an impact on human health, plants, and animals. However, the specific toxicity of rGO in the water ecosystem remains unknown.

The objective of this research was to identify the optimum dose of rutin for mitigating the harm resulted by exposure to rGO NPs in zebrafish, a well-established vertebrate model for toxicity evaluation, by assessing behavioural responses, biochemical parameters, and morphological alterations.

Methodology 

Synthesis and Characterization of rGO NPs 

GO was formed using Hummer’s technique ²⁷. Initially, 1g of natural graphite powder and 1g of NaNO3 were combined with a specific quantity of H2SO4 at 15°C, resulting in a suspension. Subsequently, 6g of KMnO4, a potent oxidizing agent, was gradually added to the suspension, and the mixture was continuously stirred for 2 hours in an ice bath while maintaining the temperature below 20°C. The mixture was stirred until it exhibited a brownish tint. After KMnO4 had completely dissolved, the temperature was maintained at 35°C for an additional 30 minutes. The suspension was then diluted with deionized water, and a specific amount of 30% H₂O₂ was introduced with continuous stirring, resulting in a bright yellow-coloured suspension. To eradicate any remaining unreacted salt, the mixture underwent treatment with 10% HCl and was rinsed multiple times with distilled water. After centrifugation, a gel-like solution was obtained and subsequently dried for 24 hours at 70°C in a hot air oven, yielding GO powder. rGO was produced as a brownish-black powder through thermal reduction at 200°C for three hours. The produced reduced graphene oxide was subjected to characterization using UV-Visible absorption spectroscopy, X-ray diffraction (XRD), Fourier Transform Infra-Red (FTIR) spectroscopy and Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray (EDX) analysis.

Fish and the experimental conditions 

Adult zebrafish were obtained from a commercial breeding centre in Chennai, Tamil Nadu, India. One week prior to the trial, fish were acclimated to the aquatic facility’s dechlorinated tap water. The photoperiod (12L:12D) and the water temperature (26.0±1.0°C) were maintained at steady levels. Daily readings of the water’s pH and dissolved oxygen concentration were taken. Two times a day, fish were given commercial feed.

Experimental design 

Fish were randomly classified into 8 groups. Each group comprised 6 animals in 3 L of dechlorinated tap water (2 fish/L). Each group was treated as below:

Group 1: Control, Group 2: rGO (10 mg/L), Group 3: Rutin (50 mg/L), Group 4: rGO (10

mg/L) + Rutin (50 mg/L), Group 5: Rutin (100 mg/L), Group 6: rGO (10 mg/L) + Rutin (100 mg/L),

Group 7: Rutin (200 mg/L), Group 8: rGO (10 mg/L) + Rutin (200 mg/L).

The H₂O, rGO NPs, and rutin administered to both the control and treated groups were rehabilitated daily. Each group was subjected to the same treatment mentioned above for 14 days.

Behavioural analysis 

The T-maze is a multi-species operative task used to evaluate memory and investigate the learning and memory processes of zebrafish. The Novel Tank Test (NTT) examined the anxiety- like behaviour and changes in the exploratory behaviour of zebrafish. The Light/Dark Preference Test (LDT) analyzed the anxiety and exploratory behaviour of zebrafish in the presence of a motivational conflict between light and dark sleeves. The T-maze, NTT, and LDT experiments were carried out following the procedures outlined in the protocol provided by Devaraj et al.²⁸.

Sample Collection 

After 14 days, the fish were submerged in ice-cold water and sacrificed. The liver was carefully dissected and rinsed twice with PBS. Subsequently, the tissue was stored at -80 °C for future analysis. Prior to homogenization, the liver was washed with an ice-cold saline solution. Homogenization was performed using a chilled mortar and pestle with 1 mL of homogenization buffer at pH 7.4 (composed of 50 mM Tris-HCl buffer with 1 mM EDTA and 0.25 mM sucrose). The resulting homogenate was subsequently centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected for further investigation.

Analyses of Biochemical Parameters 

The supernatant obtained from the tissue homogenate was utilized to assess various biochemical parameters. Lipid peroxidation (LPO), an indicator of oxidative stress, was estimated based on the protocol defined by Devasagayam and Tarachand²⁹. Superoxide dismutase (SOD) activity was determined using the protocol established by Marklund and Marklund³⁰, while catalase (CAT) activity was estimated following the protocol developed by Aebi³¹. Glutathione peroxidase (GPx) activity was estimated using the procedure defined by Rotruck et al.³². Reduced glutathione (GSH) levels were analyzed as stated by the protocol outlined by Ellman³³. Glutathione reductase (GR) activity was assessed based on the procedure developed by Calberg and Mannervik³⁴. Glutathione S-transferase (GST) activity was estimated following the technique established by Habig et al.³⁵. Lastly, the concentration of vitamin C was determined using the procedure described by Roe and Kuether³⁶.

Histological examination 

The liver of the adult fish was removed, thoroughly cleaned in 0.9% saline, then preserved in 10% neutral buffered formalin, embedded in low melting point paraffin (56°C), and sectioned (5 µm). Sections underwent deparaffinization, graded ethanol dehydration for several hours, H&E staining, and microscopy examination (Nikon Eclipse 50i).

Statistical Analysis 

The data are stated as mean ± standard error of means (SEM) and encompass the entire dataset. To compare the outcomes among the various groups, a one-way analysis of variance (ANOVA) was conducted, followed by a Duncan’s Multiple Range Test (DMRT). SPSS version

Software package was employed for the statistical A significance level of p < 0.05 was employed to assess the statistical significance.

Results 

Characterization of rGO NPs UV-Visible spectroscopy

The optical characteristics of nanoparticles were evaluated using the UV-Vis absorption spectrum. The absorption spectrum was captured, between 200 and 800 nm. The maximum absorption peak of GO appears at 230 nm and rGO was discovered at 278 nm as shown in Fig. 1 (a & b)

Figure 1: UV-Visible spectrum of reduced graphene oxide  Click here to view Figure

X-Ray diffraction (XRD)

The average crystallite size of rGO nanoparticles was analyzed with the XRD technique. The XRD pattern of produced rGO nanoparticles is depicted in Fig. 2. The main strong peak of rGO was observed at 26.4° which corresponds to the plane (002). It matched the peak described by Pratima et al. [43] and so the targeted nanoparticle size was confirmed. The Scherrer’s formula in the following equation, D = (0.94λ)/(βcosθ) was used to determine the particle size based on the width of XRD peaks. The calculated crystal size measured at 12.4 nm.

Figure 2: XRD pattern of reduced graphene oxide  Click here to view Figure

Fourier transform infrared spectroscopy (FTIR)

The targeted nanoparticles existing functional groups are identified using Fourier transform infrared spectroscopy. Fig. 3 depicts the rGO FTIR spectra. The peak at 3796 cm^-1 is attributed to O-H stretching vibration, while a peak in the region 3686 cm^-1 may be due to O-H stretching. The peak at 2923 cm^-1 is attributed to C-H stretching, and the peak at 1752 cm^-1 corresponds to C=O stretching. The peak at 1592 cm^-1 corresponds to C=C stretching. The peak at 1454 cm^-1 is attributed to C-H bending, and the peak at 1156 cm^-1 corresponds to C-N stretching. Finally, the peak at 857 cm^-1 is attributed to C-H bending vibration, which predicts the targeted nanoparticle. During the thermal reduction process, the elimination of O2 resulted in a reduction in the spectrum size and a substantial decrease in oxygen-containing functional groups.

Figure 3: FTIR spectra of reduced graphene oxide  Click here to view Figure

Scanning Electron Microscopy (SEM) with Energy Dispersive X-ray (EDX)

Scanning Electron Microscopy (SEM) is a powerful method for generating insightful data for describing nanostructures. The SEM was utilized to assess the morphology of the synthesized rGO NPs, which provided significant magnification of the material’s surface. Fig. 4 (a & b) describes the micrographs of rGO NPs with diverse magnifications, which demonstrate individual thin sheet-like structures, randomly aggregated with distinct edges through self-assembly techniques demonstrating the successful reduction of GO into rGO.

The EDX analysis indicates that the synthesized NPs contain elemental mass % C (86.14 ± 0.21) and O (13.86 ± 0.31), endorsing its higher purity without any impurities. Further, the EDX analysis confirms that the synthesized NPs are rGO. Fig. 5 represents the EDX analysis and the details are shown in Table 1.

Figure 4: (a – b) Micrographs of rGO NPs with different magnifications Click here to view Figure

Table 1: Energy Dispersive X-ray measurements of reduced graphene oxide. 

Figure 5: EDX of reduced graphene oxide Click here to view Figure

Behavioural analysis of Danio rerio 

The behavioural tests, including T-maze, NTT and LDT significantly analyzed the adult zebrafish’s learning, memory, anxiety and exploratory behaviours. The fish in our study subjected to rGO showed exceptionally dull behaviour compared to the control, rutin alone and rGO + rutin groups. Rutin significantly reduced the behavioural alterations caused by the rGO in the rGO + rutin groups. Thus, behavioural improvement could be observed in the rGO + rutin-treated groups compared to the rGO alone treated zebrafish groups.

T-maze for learning and memory 

The total time spent on the green and red arm of the T-maze and the number of entries to the green and red arm of the T-maze by the control, rGO, rutin, rGO + rutin treated groups have been graphically represented in Fig. 6 (a & b).

Figure 6: T-maze (a) Graphs depicting the time spent in the red and green arms of the T-maze by the control, rGO, rutin, rGO + rutin supplemented groups.  Click here to view Figure

Novel Tank Test (NTT) for checking anxiety-induced behavioural changes 

The total time spent in the top and bottom zones of the tank and the number of entries to the top and bottom zones by the control, rGO, rutin, rGO + rutin treated groups are graphically represented in Fig. 7 (a & b).

Figure 7: NTT (a) Graphs depicting the time spent in the top and bottom zone of the tank by the control, rGO, rutin, rGO + rutin supplemented groups.  Click here to view Figure

Light and Dark Test (LDT) for assessing anxiety-like behavioural changes 

The total time spent in the light and dark compartments and the total number of entries to the light and dark compartments by the control, rGO, rutin, rGO + rutin treated groups are graphically represented in Fig. 8 (a & b).

Figure 8: LDT (a) Graphs depicting the time spent in the light and the dark zone by the control, rGO, rutin, rGO + rutin supplemented groups.  Click here to view Figure

Oxidative stress markers 

The adult zebrafish gill tissue was examined to assess the impact of rGO NPs and rutin on oxidative stress markers. The groups that received only rGO (10mg/L) and those subjected to the combination of rGO with the lowest dose of rutin (50mg/L) exhibited a significant decrease in antioxidants, including SOD (Fig. 9a), CAT (Fig. 9b), GPx (Fig. 9c), GSH (Fig. 9d), GR (Fig. 9e), GST (Fig. 9f), and Vit C (Fig. 9g), compared to the control and rutin-only treated groups (50mg/L, 100mg/L, and 200mg/L). A steady increase in the oxidant level LPO (Fig. 9h) was detected in the rGO NPs treated zebrafish compared to the control and rutin alone groups. In addition, the antioxidant levels in the rGO + rutin treated groups (different doses as mentioned above) were elevated as compared to rGO alone treated fish in a dose-dependent manner. However, the markers exhibited significant changes in response to varying doses with the decrease in the antioxidant levels. This suggests that the treated groups encountered elevated oxidative stress.

Figure 9: The oxidative stress markers such as SOD (a), CAT (b), GPx (c), GSH (d), GR (e), GST (f), Vit C (g) and LPO (h) in the gill tissue of control, rGO, rGO + rutin supplemented groups. Click here to view Figure

Histology 

Fig.10 (a-h) depicts the morphological analysis of adult zebrafish liver tissue from both the control and treated groups. The liver samples of the control group and rutin (50 mg/L, 100 mg/L and 200 mg/L) exposed zebrafish showed normal morphological structure. Histological abnormalities were found in the liver tissues treated with rGO (10 mg/L), with clear evidence of sinusoidal and venous congestion, cytoplasmic vacuolation of hepatocytes, necrosis and increased hepatic plate gap. However, zebrafish treated with rGO and rutin supplementation (distinct doses mentioned above) showed markedly reduced changes, in the irregular shape of hepatocytes and cytoplasmic vacuolation of hepatocytes. Less harm was demonstrated in the groups treated with rGO along with rutin.

Figure 10: Morphology of the liver in adult zebrafish after 14 days of rGO nanoparticles and rutin exposure. Click here to view Figure

Discussion 

Carbon nanomaterials have garnered significant attention in numerous studies owing to their remarkable mobility as charge carriers, high thermal conductivity, and advantageous electronic and mechanical properties. These materials find applications in various fields, including electrochemical devices, catalysis, energy storage, cell imaging, photothermal therapy, biosensors, and drug delivery^37,38. The two most widely used and produced nanoparticles are rGO and GO. Due to structural modifications, rGO performs better than GO in activity, biocompatibility, dispersion, and binding sites^39-42.

As a model organism, zebrafish (Danio rerio) are frequently utilized in scientific research because of their genetic similarity to humans, rapid reproduction, and transparent embryos that make it simple to see internal organs. They are accommodative in researching the toxic effects of various substances, such as pharmaceutical compounds and environmental pollutants.

Most of the research has been concentrated on the impacts of graphene-based materials on zebrafish’s embryonic and larval stages, so there are few toxicology studies involving adult zebrafish and rGO. According to Pratima et al.,⁴³, rGO toxicity significantly impaired behavioural responses and disturbed antioxidant levels. In addition, zebrafish exposed to a sub-lethal dose of rGO (10 mg/L) experienced oxidative stress, metabolic abnormalities, and immune responses.

This study examined the potential ameliorative effects of rutin against the sublethal dose (10 mg/L) of rGO-induced toxicity in adult zebrafish. To assess the protective impact of rutin, zebrafish were treated with various concentrations of rutin (50 mg/L, 100 mg/L, and 200 mg/L) over 14 days, while rGO was administered during the same timeframe. The fish underwent various behavioural, biochemical, and histological examinations following the study period.

Exposure to rGO has been associated with behavioural and physiological changes in fish. Fish exposed to graphene-based materials exhibited changes in swimming behaviour, feeding patterns, and reproductive parameters. These effects may disrupt normal ecological interactions and reduce the population’s fitness ⁴⁴. By examining LDT, NTT, and T-maze tests, this research reveals that rutin supplementation effectively countered the behavioural modifications induced by rGO administration in zebrafish. For instance, in the LDT, the zebrafish exhibited increased movement towards the brightly lit zone and spent more time in the light zone compared to the group that received only rGO. NTT demonstrated that the co-supplementation of rutin after exposure to rGO significantly altered the bottom-dwelling behaviour of zebrafish compared to the other groups. T maze revealed that rutin treatment also improves the zebrafish’s impaired learning and memory caused by rGO exposure.

Fish have been shown to exhibit signs of oxidative stress when exposed to RGO. Reactive oxygen species (ROS) are produced in excess compared to an organism’s detoxification capacity, which results in oxidative stress. Excessive ROS can impact the organism’s overall health because they can cause cellular damage and inflammation⁴⁵.

In response to ROS, zebrafish activate an antioxidant defence mechanism similar to mammals⁴⁶. Antioxidants act as the foremost line of defence against oxidative stress, effectively scavenging free radicals to safeguard cells from potential damage^47,48. When the antioxidant defence system fails to neutralize excessive ROS, it results in the inactivation of enzymes. One potential cause of this inactivation is the overproduction of ROS. To counteract this, Superoxide Dismutase (SOD) plays a vital role by converting oxygen-free radicals into hydrogen peroxide (H₂O₂). Subsequently, Catalase (CAT) and Glutathione Peroxidase (GPx) work together to break down H₂O₂ into water (H₂O) and oxygen (O₂) molecules. This enzymatic process is crucial for maintaining the intracellular redox balance⁴⁹. However, along with the observed inhibition of SOD and CAT activities, the activity of GPx was also reduced⁵⁰. Therefore, reducing the activities of these essential primary antioxidants could lead to the accumulation of H₂O₂ and its degradable products⁵¹.

Sublethal exposure to rGO resulted in a dysfunction of the essential primary antioxidant defence system and a decrease in SOD, CAT, and GPx activity in the gill tissues of zebrafish. This may be due to an excess of superoxide ion production. However, the antioxidant levels of the groups supplemented with the combination of rGO and rutin showed some improvement. This could be due to rutin’s protective effect against rGO.

The glutathione system, encompassing the non-enzymatic antioxidant defence, is the secondary line of protection against oxidative damage. GSH and its associated enzymes, GR and GST, play vital roles within this system. GSH, the predominant non-protein thiol found in cells, is a scavenger of free radicals and a substrate for GPx and GST. The regeneration of GSH, crucial for combating oxidative stress, heavily relies on the activity of GR ⁵¹. GR transforms oxidized glutathione (GSSG) to its reduced form (GSH). In its reduced state, glutathione regulates ROS within the cell. Conversely, GST catalyzes the conjugation of GSH with various xenobiotic substrates to facilitate detoxification⁵².

Additionally, GSH helps maintain other antioxidants like vitamins E and C in their active, reduced state within the cellular environment. Its depletion serves as a significant biomarker of oxidative stress in animals and humans⁵³. In a previous study by Lushchak⁵⁴, zebrafish exposed to CuO nanoparticles displayed a notable reduction in GSH levels. Another non- enzymatic antioxidant, vitamin C, acts as an oxidative stress inhibitor through various mechanisms. It scavenges reactive oxygen species by trapping radicals in the aqueous phase and halting peroxidation, thereby safeguarding cell membranes against oxidative damage⁵⁵. Zhang et al.⁵⁶ reported significantly lower levels of vitamin C in fish exposed to CuO-NPs compared to the control group.

However, in our research, the gill tissue of adult zebrafish treated solely with rGO and a combination of rGO and rutin (50mg/L) showed reduced GSH, GR, GST and vitamin C activities. At the same time, rGO and rutin supplementation at higher doses (100 mg/L and 200 mg/L) showed better activities than rGO alone groups.

Lipid peroxidation (LPO) is a process where polyunsaturated fatty acids (PUFAs) undergo oxidative degradation. It plays a vital role in maintaining membrane function, structural integrity, and the deactivation of certain membrane-bound enzymes⁵⁷. Excessive ROS can induce LPO, damaging tissue. However, the presence of antioxidant and detoxification systems can help mitigate LPO by eliminating excess ROS [49]. Malondialdehyde (MDA) is commonly utilized as a biomarker to assess the level of LPO and indicates oxidative damage or stress⁵⁸.

Our study revealed that the rGO alone groups exhibited higher levels of LPO than the control group. In contrast, the rGO and rutin-treated groups demonstrated lower levels of LPO than the rGO-treated group alone. These findings suggest that rutin treatment may help to mitigate LPO. Moreover, Wang et al.⁵⁹ propose that SOD is the first-line protective enzyme against ROS and LPO. Therefore, a decline in SOD activity may lead to an increase in LPO.

Histological abnormalities including sinusoidal and venous congestion, cytoplasmic vacuolation of hepatocytes, increased hepatic plate gap and necrosis were observed in the liver tissues of rGO alone (10 mg/L) treated group. Nevertheless, zebrafish treated with rGO along with rutin supplementation (50 mg/L, 100 mg/L and 200 mg/L) showed markedly reduced changes like irregular shape of hepatocytes and cytoplasmic vacuolation of hepatocytes. The histopathological changes observed in our study were consistent with the findings stated by Rajini et al. ⁶⁰, Liu et al. ⁶¹, and Sezgi Arman et al. ⁶² in their respective investigations on zebrafish liver.

Conclusion 

According to the results of this research, exposure to a sublethal dose of rGO (10 mg/L) led to oxidative stress in zebrafish, resulting in reduced antioxidant levels. In addition, it led to behavioural changes, liver tissue morphological alterations and elevated LPO levels in the adult zebrafish. However, the alterations were markedly decreased in the groups treated with rutin and rGO in combination. After exposure to rGO, rutin exhibits a therapeutic ability to scavenge ROS and hydroxy radicals effectively. The desirable quantity of rutin supplementation for alleviating the detrimental effects of rGO exposure falls within the range of 100 and 200 mg/L. Further study is essential to entirely comprehend the potential toxicity of rGO in adult zebrafish and evaluate its long-term effects on individual and population levels. It is also essential to consider the potential environmental impacts of rGO release and its accumulation in aquatic ecosystems. This study would help to investigate the safety and potential risks associated with graphene-based materials and ensure their responsible usage in various applications.

Acknowledgement 

The authors would like to acknowledge the Department of Biochemistry and Biotechnology, Annamalai University for providing the laboratory facilities and all other required consumables and equipment.

Conflict of Interest 

The authors declare no conflict of interest to disclose.

Funding Sources

There are no funding sources

Author Contributions

The authors confirm contribution to the paper as follows: study conception and design: Briska
Jifrina Premnath; data collection: Manoj Kumar Srinivasan; analysis and interpretation of results:
Briska Jifrina Premnath; draft manuscript preparation: Nalini Namasivayam. All authors
reviewed the results and approved the final version of the manuscript.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Ethical Statement

Since the work was done before the CCSEA decision on fish ethics. There are no ethical issues
involved to carry out the research.

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