Graphene Quantum Dots Alter Proliferation and Meiosis of Germ Cells

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Graphene Quantum Dots Alter Proliferation and Meiosis of Germ Cells only in Genetic Females of Japanese Medaka during Early Embryonic Development Sreelakshmi Krishnakumar, Vinod Paul, Ajish Ariyath, Puthiyoth Dayanandan Anoop, Sajini Sreekumar, Deepthy Menon, and Bindhu Paul-Prasanth ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00606 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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On exposure to graphene quantum dots, XX embryos revealed a drastic decline in germ cell number and meiosis along with significantly high levels of reactive oxygen species. 239x100mm (150 x 150 DPI)

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Graphene Quantum Dots Alter Proliferation and Meiosis of Germ Cells only in Genetic Females of Japanese Medaka during Early Embryonic Development Sreelakshmi Krishnakumar, Vinod Paul, Ajish Ariyath, Puthiyoth Dayanandan Anoop, Sajini Sreekumar, Deepthy Menon*, Bindhu Paul-Prasanth* Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham, Kochi -682041, Kerala, India

*Corresponding authors 1)

Dr. Bindhu Paul Assistant Professor Amrita Centre for Nanosciences and Molecular Medicine Amrita Institute of Medical Sciences Kochi – 682041, Kerala, India

E-mail: [email protected] 2)

Dr. Deepthy Menon Professor Amrita Centre for Nanosciences and Molecular Medicine Amrita Institute of Medical Sciences Kochi – 682041, Kerala, India E-mail: [email protected]

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Abstract Graphene quantum dots (GQDs) have emerged as promising bio-labelling agents owing to their stable innate fluorescence, photostability, and biocompatibility as opposed to semiconductor quantum dots. While several studies reported GQDs to be cytocompatible, their potential for reproductive toxicity, particularly to germ cells that can cause transgenerational toxicity, remain unexplored. Here we report the intrinsic toxicity of 2 – 3 nm sized GQDs synthesized from glucose by a novel bottom-up green chemistry technique on germ cell proliferation and meiosis during early embryonic development. In vitro cell viability studies with a normal ovarian cell line, Chinese Hamster Ovarian cells (CHO), and a primary cell type, Human Umbilical Vein Endothelial Cells (HUVEC), portrayed good cytocompatibility even up to a high concentration of 800 g/ml. When embryos of Japanese medaka were exposed to GQDs, no developmental toxicity was observed up to 250 µg/ml, beyond which hatchability and survival were affected adversely. In contrast, toxicity to germ cells in developing gonads was apparent in genetically female (XX) embryos exposed to much lower doses (50, 75 and 100 µg/ml), at which in vitro cytotoxicity and embryo hatchability and survival were unaffected. A drastic decline in germ cell number and meiosis was observed at these doses in XX embryos implying anomalies in sexual differentiation of the gametes. Conversely, germ cells of genetically male embryos exposed to GQDs were unaltered. Significantly high levels of reactive oxygen species (ROS) were detected in the XX larvae exposed to GQDs, however, there was no DNA damage, suggesting ROS to be responsible for the adverse effects observed. Keywords: Graphene quantum dots, Japanese medaka, Developmental toxicity, Germ cells, Meiosis



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1. Introduction Graphene quantum dots (GQDs) are small fragments of graphene typically of sizes below 10 nm with excitons confined in all three spatial dimensions. This result in non-zero band gap that invariably renders them with innate photoluminescence (PL) property that is easily tunable and highly photostable.1-3 Unlike the parent graphene, the presence of structural defects inherited through synthesis procedures render GQDs with sp3 and sp2 carbon atoms, making them easily amenable to functionalization. Along with this property, small size, good steric stability, and presence of oxygen containing surface functional groups make GQDs well dispersed in various types of solvents. The ability to penetrate biological membranes has rendered GQDs potentially useful in applications like drug delivery, biosensing, and bioimaging4 as evidenced by development of folic acid conjugated GQDs loaded with doxorubicin for drug delivery,5 nitrogen-doped GQDs for detection of chromium (Cr) in aqueous solutions,6 label-free GQDs for colorimetric analysis of H2O27 and polyethyleneimine (PEI) coated GQDs with red, blue, and yellow luminescence under UV irradiation for multicolour cellular imaging.8 GQD synthesis has been achieved by both top-down and bottom-up approaches. Topdown synthesis involves exfoliation and break down of massive graphene-based materials using techniques that are extremely cumbersome, laborious and without proper control of size and morphology of the end product.9-13 Conversely, bottom-up synthesis that most often follows a solution chemistry technique involving monomeric nucleation and assembling of aromatic organic molecules allows better control of physical properties of GQDs as evidenced by previous reports.14-16 The vast majority of in vitro cytotoxicity studies have reported good biocompatibility of GQDs in a variety of cell lines.17-19 However, the developmental toxicity potential of GQDs demonstrated in zebrafish embryos showed developmental abnormalities like reduced heart rate, physical malformation, hypoactivity,


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and mortality in response to graphene oxide (GO) derived GQDs of 2-5 nm size distribution at doses above 25 µg/ml.20 These embryos were exposed through the rearing medium from the day of fertilization to 120 hours post fertilisation (hpf). Conversely, when zebrafish embryos were exposed to plant leaf-derived GQDs (5 nm) from 0 hpf to 84 hpf, adverse effects such as delayed hatching, stunted growth and pericardial edema were observed only at very high treatment doses like 2 mg/ml and above.21 Thus, GQDs were in general shown to be biocompatible at lower doses both in vitro and in vivo. However, there have been no studies till date that explored the intrinsic toxicity potential of GQDs on germ cell differentiation and meiosis during early embryonic development. Such studies are essential as several engineered nanoparticles (ENPs) were shown to affect reproduction through their toxic effects on gametes,22 which are responsible for transmission of genetic material across generations and thus, aid in the propagation of various species. Emerging data have revealed the alarming sensitivity of early stages of germ cell development to various exogenous factors.23-24 Germ cell proliferation and meiosis are vital for the differentiation and genesis of gametes, the sperms and the ova. Hence, damages incurred to germ cells during early development could result in developmental and functional anomalies of reproductive organs that could get manifested only in adulthood. Thus, in this study, we synthesized GQDs from a natural organic precursor, glucose, and investigated the in vitro cytocompatibility and embryonic as well as germ cell toxicity potential of these particles using Japanese medaka (Oryzias latipes) embryos and larvae. Medaka has been serving as an excellent model for such studies being a gonochoristic animal with sexually differentiated gonads at the time of birth itself. Furthermore, the germ cell development in medaka embryos and larvae were reported to be highly vulnerable to external influences, making it an ideal organism to investigate the effects of ENPs on germ cell differentiation and development.



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2. Results and Discussion 2.1. Characterization of GQDs The GQDs reported in this study were prepared using a green chemistry route from glucose as the sole precursor to yield a stable GQD colloid after dialysis. The PL spectrum of the GQD dispersion was excitation wavelength-dependent as shown in the figure (Figure 1A). Evidently, the emission wavelength (λem) red shifted from 444 nm to 570 nm with increase in excitation wavelength (λex). Concurrently, the intensity of the emission spectrum peaked at 457 nm for an excitation wavelength of 340 nm and thereafter decreased. This excitation wavelength dependence of the PL spectra is observed in most of the luminescent carbon nanoparticles and is attributed to the changes in solvation dynamics and realignment of particle dipole and solvent dipole to attain equilibrium referred to as the ‘Giant Red-Edge Effect’.25-26 The broad fluorescence spectrum can be attributed to the multiple electron energy transitions occurring between the bonding and anti-bonding molecular orbitals, resulting in simultaneous overlapping fluorescence peaks. Figures 1B and 1C show the GQD dispersion in deionised water upon illumination under ambient light and UV at 365 nm, respectively. TEM analysis of GQDs showed monodispersed particles with uniform size distribution ranging between 2 - 3 nm (Figure 1D). High resolution imaging revealed the presence of crystalline lattices (Figure 1E) with an in-plane lattice spacing of 0.21 nm corresponding to the (100) planes of graphite, suggesting that the prepared GQDs possessed graphene-like crystallinity.27 AFM analysis also showed uniformly dispersed particles with a disc-like morphology. The cross-sectional analysis yielded a thickness of 0.98 nm suggesting the presence of only few layers (1-3) of graphene28 (Figure 1F). The absorption spectrum (Figure 1G) exhibited two strong UV absorption peaks, which could be attributed to the π electron transitions that occur in GQDs. The absorption peak at 227 nm was due to the * transition of C=C and at 283 nm by the n* transition of the C=O bond.29-30 5 ACS Paragon Plus Environment

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Figure 1. (A) Photoluminescence emission spectra of GQDs when excitation wavelength is varied from 270 nm to 480 nm. (B-C) GQD dispersion in water imaged under UV at 365 nm and ambient light, respectively. (D-E) TEM and HR-TEM images of GQDs, respectively. (F) AFM analysis showing the height profile of the particles. (G) Absorbance spectrum.

XPS analysis was carried out to analyze the surface states of the GQDs. The wide spectrum showed C1s and O1s peaks at 285 eV and 532 eV (Figure 2A). The deconvoluted C1s spectrum (Figure 2B) showed sp2-carbons (283.95 eV), sp3-carbons (284.65 eV), C-O (285.95 eV), and C=O (287.85 eV and 288.45 eV).31-32 Further, FT-IR analysis showed a 6 ACS Paragon Plus Environment


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spectrum with band at 1641 cm−1 attributed to the C=C stretching in GQD core, 3257 cm−1 to the O-H stretching and the peak at 1027 cm−1 to the presence of C-O functional group.29-30 The existence of C-H in the GQDs was confirmed by the presence of bands at 1346 cm−1 and 2922 cm−1 (Figure 2C). The Raman spectrum of the GQDs was taken to confirm the graphitic nature of the particles. The broad shoulder peak at 1370 cm-1 corresponds to the D band and the peak at 1583 cm-1 is the G band (Figure 2D), indicating the disordered structure of sp2 cluster in the crystalline lattice and the in-plane stretching vibration mode E2g of single crystal graphite, respectively.33

13

C NMR spectroscopic analysis of the GQDs in D2O as

solvent was carried out. Several peaks from 60-100 ppm suggested the presence of sp3 carbon in different molecular environments.34 Absence of peaks beyond 100 ppm that correspond to sp2 carbon could be due to the masking of GQDs by surface passivated glucose molecules (supplementary figure S1). The presence of oxygen containing functional groups on the surface of the particles rendered the as-prepared GQDs water soluble and stable at ambient conditions. Thus, the caramelisation of glucose under appropriate synthesis conditions effectively produced monodispersed GQD particles with characteristic structural and optical properties, making it a fluorescent nanomaterial suitable for diverse applications.


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.

Figure 2. Physico-chemical characterization of GQDs, (A) XPS wide spectrum (B) C1s deconvoluted spectrum (C) FTIR spectrum and (D) Raman spectrum.

2.2. In vitro exposure 2.2.1. Cell viability analysis A primary cell source from humans, human umbilical vein endothelial cells (HUVECs) and a normal cell line, Chinese Hamster Ovarian cells (CHO) were chosen for the examination of the cytocompatibility of glucose-derived GQDs. As shown in Figure 3, neither of the cell types showed significant decrease in cell viability upon exposure to GQDs for 24 or 48 hours (h). This data demonstrated that glucose-derived GQDs were not harmful to either the primary cells or the cell line, up to a very high concentration tested (800 g/ml). Previous studies have shown that functionalised graphene have lower toxicity than pristine graphene particles of similar sizes.35-36 GQDs of lateral dimensions around 3 nm synthesized from pyrene as precursor were shown to decrease cell viability of MSCs to below 60% upon exposures above 100 g/ml.37 However, at lower doses they enhanced osteogenic



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differentiation in a dose and time dependent manner along with increased calcium mineralisation, which was favourable for tissue engineering applications. In contrast, graphite-derived GQDs of average diameters 5-10 nm showed 70% cell viability even at 200 g/ml when exposed to stem cells for 72 h.11 Another study compared the cytotoxicity of crude GQDs (C-GQDs) of average diameter 6 nm before and after PEGylation (P-GQDs), which is known to mitigate the toxicity of ENPs, and reported that P-GQDs were cytocompatible (75%) even at a very high concentration of 4 mg/ml, while cytocompatibility of C-GQDs were very poor (25%) at the same dose.38 However, the CGQDs were not toxic up to a concentration of 400 g/ml. Similarly, the glucose-derived GQDs synthesized here using the bottom-up approach could also be considered safe as the cell viability of normal primary cells and a cell line was found to be unaffected even when exposed to a very high particle concentration of 800 g/ml.

Figure 3. In vitro cell viability upon exposure to GQDs. (A-B) Percentage viability of CHO cells exposed to GQDs for 24 h and 48 h respectively. (C-D) Percentage viability of HUVEC cells exposed to GQDs for 24 h and 48 h respectively.


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2.2.2. In vitro oxidative stress analysis Toxicity studies in vitro and in vivo with carbon nanomaterials have evidenced increased generation of ROS resulting in anomalies like inactivation of several proteins, mitochondrial dysfunction, lipid peroxidation, etc.39 A comparative study of in vitro toxicity in HeLa cells exposed to multi-walled carbon nanotubes and graphene oxide for 24 h showed a significant increase (about 23 fold for carbon nanotubes and 17 fold for graphene oxide) in ROS levels at the highest concentration used i.e. 80 µg/ml.40 However in this study, CHO cells exposed to GQDs at different concentrations upto 1000 µg/ml for 24 h did not show any increase in ROS levels (Figure 4) implying that GQDs did not induce oxidative stress in cells.



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Figure 4. In vitro ROS analysis in CHO cells. (A-C) Representative bright field images of control, treatment (GQD at 1000 µg/ml) and H2O2 (200 µM) exposure respectively. (D-F) Corresponding fluorescent images of control, treatment and H2O2 exposure respectively. (H) Quantification of fluorescent intensity in the different groups showing no significant increase in any of the treatment groups. Statistical significance; (***) indicate p≤0.0005.

2.3. In vivo toxicity analysis 2.3.1. Survival and hatchability There have been instances, when materials with good cytocompatibility were shown to exhibit toxicity in vivo.41-42 Moreover, as afore-mentioned GO-derived GQDs were shown to affect the development of zebrafish embryos adversely, indicating the developmental toxicity potential of these particles. Quite often, embryonic development is the period most vulnerable to exogenous factors that can damage various organ systems irreparably. Therefore, the toxicity potential of glucose-derived GQDs on embryonic development was analysed in medaka. Similar to zebrafish, medaka is a freshwater teleost widely used as a model organism for toxicological and biomedical research. Fertilization and embryonic development are external in this species, making it easier to conduct such exposure studies. Moreover, the development of medaka embryos can clearly be seen under the microscope because of the optical transparency of the egg shell. In the present study, the embryos were exposed to glucose-derived GQDs for 18 days post fertilisation (dpf) at a wide range of concentrations starting from 0.01 to 500 µg/ml. The embryos were observed every day for recording any developmental abnormalities. As shown in figures 5A and 5B, GQDs did not affect the survival and hatchability of medaka embryos up to the dose of 250 µg/ml. Eye pigmentation in the treated embryos (75 µg/ml and above) was observed to be slower as compared to the controls (Figure 5C). However, after an initial slow down, the pigmentation process was regained by the day of hatching in these embryos. Embryos exposed to 2 mg/ml of GQDs for 48 h showed particle uptake through the egg chorion (Figure 5D) when illuminated under the 11 ACS Paragon Plus Environment

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GFP filter (excitation filter at 425/60 nm). However, in vivo biodistribution studies could not be carried out at the same dose due to mortality of the embryos beyond 5 dpf. The lower concentrations did not give intense fluorescence signals to determine the uptake and distribution of GQDs in medaka embryos at this excitation range. Though survivability and hatchability were severely affected at and above 250 µg/ml, no adverse structural malformations or physiological discrepancies were detected in the treatment range as in the case of zebrafish embryos exposed to GO-derived GQDs of similar size range.20 The zebrafish embryonic development was severely affected at a concentration of 50 µg/ml which was 10 times lower than the highest concentration (500 µg/ml) used in this study. Nevertheless, delayed hatching and slow eye pigmentation in the exposed larvae of this study indeed suggested potential influence of GQD exposure at the molecular level. However, the hatched larvae were all normal and able to swim up.



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Figure 5. In vivo toxicity analysis in medaka embryos. (A) Percentage survival on 18 dpf. (B) Percentage hatchability on 12 dpf. (C) Poor eye pigmentation in the treated embryo at 7 dpf. (D) Embryo exposed to GQDs at 2 mg/ml show fluorescence under the GFP filter. Statistical significance; (*) indicate p≤0.05, (**) indicate p≤0.005 and (***) indicate p≤0.0005.

2.3.2. Effects on germ cells (GCs) Primordial germ cells (PGCs) are the precursor cells of gametes, namely sperms in males and ova in females. In vertebrates, specification of these cells happen during the early stages of embryonic development and subsequent to this process, the PGCs were found to migrate towards the presumptive gonadal anlage deploying mechanisms that were speciesspecific. Unlike other organ systems, gonadal development is quite a lengthy process in vertebrates including humans and gets completed only at puberty, which takes several months to years depending on the species. During the early stages of gonadal development, most of the germ cells are undifferentiated and any adverse effect at this stage could hamper its differentiation process, ultimately resulting in various health hazards including cancers of the reproductive organs and infertility in both men and women and other species.23-24 Similar to mammals, medaka is a gonochorist, from which the male and female gonads could be clearly distinguished at the time of birth by looking at the morphological features of the developing gonads.43 Additionally, using Qurt strain of medaka was advantageous for this study as sexual genotype of the embryos could be identified with the help of sex-linked phenotypic markers (leucophores) from 3 dpf. The XY embryos have leucophores on their head and trunk regions, while XX embryos lack these pigments completely.44 XX larvae differentiating as females usually have more number of germ cells than XY larvae that were presumptive males. More importantly, germ cells in XX larvae of medaka enter into meiosis, the reduction division, from the day of hatching, while germ cells in XY larvae enter into meiotic arrest, which is released only when the XY larvae become 50 dph.45 This sexually dimorphic 13 ACS Paragon Plus Environment

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proliferation and meiosis of germ cells during early development was found to be critical for the differentiation and morphogenesis of gonads in accordance with the sexual genotype of the embryo concerned. For example, XY larvae of medaka exposed to estrogenic substances during early development showed gonads with high GC count and meiosis; and the gonads in these fish developed as ovaries even after the exposure was terminated.46 On the other hand, XX larvae exposed to androgenic substances were shown to possess low GC count with no meiosis and these fish later had testes instead of ovaries.47 Furthermore, genetically female medaka deficient in germ cells due to morpholino-mediated knockdown of cxcr4 gene was reported to develop as phenotypic males with streak gonads in adulthood.48 Hence, GC count along with presence or absence of meiosis in developing XY and XX larvae has been used as a parameter to assess the sex-reversing effects of hazardous substances and chemicals. Therefore next, it was analysed whether GQDs could affect the sexual development in XY and XX embryos of medaka. The GC count and morphology of the developing gonad on the day of hatching (0 dph) in the treated males were comparable to that of the control males (Figure 6B and 6D). However, genetic females exposed to doses of 50 µg/ml and above, could be divided into 2 groups, larvae with high GC count and meiosis as in the case of control larvae and the other group with low GC count and no meiosis (Figure 6A and 6C). Intriguingly, the number of XX larvae with low GC count and no meiosis was more at 75 µg/ml (60%) dose exposure than 50 µg/ml (33%) and 100 µg/ml (33%) as shown in Table 1. Though this data was atypical as the dose-response was non-monotonic in nature, there have been reports on similar dose-response effects of endocrine disrupting chemicals earlier.49-50 Since 60% of the larvae treated with 75 µg/ml were adversely affected, this group was subjected to long-term exposure (0 dpf to 10 dph) to examine whether this effect of GQDs on proliferation of GCs and meiosis got further worsened. The long-term treatment also showed 14 ACS Paragon Plus Environment


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30% of larvae with significantly low GC count and no meiosis, while the remainder of the larvae showed high GC count and meiosis, which was comparable to that of control XX larvae at 10 dph (Figure 6E). Notably, the number of XX larvae with low GC count and no meiosis was lesser in the long-term treatment group in comparison to the group exposed only up to hatching. To find whether this adverse effect on GCs were specific to GQDs, XX embryos were exposed to glucose at concentrations 50 µg/ml, 75 µg/ml, and 100 µg/ml. Unlike in the GQD exposure, there was no reduction in the germ cell count or lack of meiosis in the glucose exposed larvae. Rather, a significant increase in germ cell count was evident in the 75 µg/ml exposure group (supplementary figure S2). This data has unambiguously demonstrated that early stages of GC differentiation was vulnerable to glucose-derived GQDs, warranting more studies of this kind in the case of similar particles which were reported to be safe.

Figure 6. Germ cell analysis in medaka embryos. (A-B) The total germ cell count at 0 dph in females and males respectively. (C-D) H and E stained sections of 0 dph embryos of females and males respectively. Scale bar 50 µm. The gonadal region is enclosed in yellow dotted lines and the arrow points to a meiotic germ cell in a control embryo. (E) Germ cell count in females at 10 dph. Statistical significance; (***) indicate p≤0.0005.


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Table 1. Percentage of XX embryos with meiotic germ cells at 0 dph

2.3.3. Oxidative stress analysis ROS was found to play a key role in the physiology of reproduction influencing oocyte maturation, ovulation, implantation, and pregnancy. Imbalance in ROS levels in vivo has resulted in reproductive pathologies like infertility, pre-eclampsia, birth defects, and abortions.51 To analyse the influence of oxidative stress in the embryonic germ cell toxicity observed in this study, XX embryos exposed to GQDs at a dose of 75 µg/ml were subjected to H2DCFDA staining on the day of hatching (Figure 7). A significant increase in the fluorescence intensity, around 2 fold on an average, was observed in the treated XX larvae as compared to the control XX larvae (Figure 7G). To investigate whether the increase in signal was contributed by the intrinsic photoluminescence of the GQDs in the exposed embryos, a control experiment without H2DCFDA staining was done on the GQD-exposed and unexposed larvae (at 0 dph) under the GFP filter. The result showed that there was no significant increase in signal intensity in the exposed larvae when compared to the unexposed larvae in the absence of the stain (supplementary figure S3) suggesting that the increase in signal intensity observed in the H2DCFDA staining was indeed due to high ROS levels in the exposed larvae. Since there was no appreciable ROS signal from the gonadal region, the 16 ACS Paragon Plus Environment


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observed germ cell toxicity could be an indirect influence of increased oxidative stress in the exposed embryos.

Figure 7. ROS analysis in medaka embryos. (A), (C) and (E) show representative bright field images of female larvae from control, positive control (H2O2 at 2mM) and treatment group, respectively. (B), (D) and (F) show corresponding fluorescent images of female larvae from control, positive control and treatment group respectively under the GFP filter. Scale bar 400 µm. (G) Quantification of fluorescence intensity in control and treated female larvae at 0dph (n=7) showing significant increase in the treatment group. Statistical significance; (**) indicate p≤0.005.

2.3.4. DNA damage analysis Alkaline comet assay is a single cell gel electrophoresis technique widely used for sensitive detection of single and double strand DNA breaks in cells and tissue homogenates to measure the extent of genotoxicity of different materials.52-54 Several in vitro and in vivo studies with semiconductor quantum dots have shown increased genotoxic potential of these 17 ACS Paragon Plus Environment

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particles compared to their bulk counterparts.55 Carbon nanotubes and graphite nanofibers have shown dose-dependent and time-dependent increase in DNA damage in human bronchial epithelial BEAS 2B cells.56 A study on colon cancer cell line showed significant genotoxic effects on exposure to GO nanosheets for 24 h even at the lowest dose studied i.e. 10 µg/ml.57 However, comet analysis did not show any genotoxicity in zebrafish exposed to GO (20 µg/ml) for72 h.58 Since genotoxicity can arise from either a direct contact of an intoxicant with the genetic material or even through indirect influences such as increased generation of ROS species,59 we have analyzed the possibility for DNA damage due to GQD exposure in female medaka embryos. The embryos exposed to GQDs at 75 µg/ml from 0 dpf to 0 dph were sacrificed and subjected to alkaline comet analysis. The average percentage tail DNA was comparable between the treatment group and the control group (Figure 8A, 8B & 8D) suggesting that the glucose-derived GQDs did not induce DNA damage. On the contrary, DNA damage was apparent in the positive control group (Figure 8C & 8D).



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Figure 8. Comet analysis in female larvae. (A-C) Representative images of nucleoids from control, GQD-75 µg/ml and H2O2 (200 mM) exposed larvae. (D) Quantification of the average percentage tail DNA from individual larva (n=5 in each group) showing no significant difference between the control and treatment group. Statistical significance; (***) indicate p≤0.0005.

3.

Conclusion The study synthesized GQDs from glucose as the precursor using a simple, scalable,

green chemistry route. In vitro toxicity analyses showed that the glucose-derived GQDs possessed excellent biocompatibility towards HUVECs and CHO cell line and no ROS was found to be generated. Though pronounced toxic effects on embryonic development were seen only upon exposures to very high concentrations of GQDs in vivo, lower doses with no apparent toxicity was found to adversely affect germ cell proliferation and meiosis in the exposed XX larvae along with increase in oxidative stress. These results have unravelled novel insights on the potential toxicity of GQDs on germ cell development and differentiation and necessitate investigations on long term effects of these particles in view of human and environmental safety owing to their high demand for a wide variety of applications. 4. Experimental section 4.1. Materials D-Glucose was purchased from Sigma-Aldrich (purity ≥99.5%, U.S.A.). Snake skin dialysis tubing of 3.5 kDa was obtained from Thermo Scientific (U.S.A.). PbNO3 (≥99.0%), FeCl3 (≥99.99%), and ZnSO4 (≥99.0%) was provided by Sigma-Aldrich. Cell culture consumables such as minimum essential medium (MEM), Iscove’s modified Dulbecco’s medium (IMDM), heat inactivated fetal bovine serum (FBS), trypsin-EDTA (0.25%), epidermal growth factor (EGF) and penicillin-streptomycin were purchased from Invitrogen (U.S.A.) and alamar blue from Invitrogen (UK). Cell culture multi-well plates were purchased from BD Falcon (U.S.A.). 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) 19 ACS Paragon Plus Environment

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stain was provided by Invitrogen (U.S.A) and fluoromount aqueous mounting medium was obtained from Sigma Life Sciences (U.S.A). Haematoxylin and eosin stains were purchased from Sigma-Aldrich (UK) and paraffin wax and xylene were obtained from Merck Specialities Private Limited (India). Agarose and low melting agarose were purchased from Lonza (U.S.A) and Sigma-Aldrich (U.S.A.), respectively. Triton X-100 was purchased from Promega Corporation (U.S.A.) and DMSO from HiMedia Laboratories Pvt Ltd (India). NaCl, Tris base, EDTA (disodium salt dihydrate), and Ethidium Bromide (EtBr) were purchased from Sigma-Aldrich (U.S.A). 4.2. Synthesis of GQDs D-glucose (20 g) was stirred in 20 ml of MilliQ ultrapure water (Millipore, Billerica, MA) at 120 ºC for 2 ½ h using an oil bath set up for uniform heating. After the reaction, the obtained golden brown solution was transferred to a dialysis membrane (pore size 3.5 kDa) and kept for 24 h dialysis, with occasional change of the water in which the membrane was suspended. The dialyzed solution was then freeze-dried for 24 h to obtain dry GQD powder sample. 4.3. Characterization A dilute suspension of the particles in deionised water was used for further characterizations and experiments. UV-Vis spectra were obtained using Shimadzu UV-3600 spectrophotometer (Japan). Photoluminiscence (PL) spectra were recorded using fluoromax-4 spectrofluorometer (Horiba Scientific, France). Fourier transform infrared spectrum (FT-IR) of the GQDs was studied using the Shimadzu IR affinity spectrometer (Japan). High resolution transmission electron microscopy (HR-TEM) images of the particles were taken using the Tecnai G2 TF20 S-TWIN electron microscope at an operating voltage of 200 kV. The QDs were further characterised by X-ray photoemission spectrum (XPS) analysis



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(Kratos Analytical, UK) and atomic force microscopy (AFM) analysis (JEOL SPM5200, Japan) using the non-contact mode. Raman analysis was conducted using the 532 nm laser source (WITEC 300R, Germany). 4.4. In vitro exposure 4.4.1. Cell viability analysis Cytotoxicity on exposure to GQDs was assessed on two different cell types by analysing cell viability using the alamar blue assay. The CHO cells (purchased from National Centre for Cell Science, Pune) were maintained and passaged in MEM media supplemented with 10% FBS and 50 U/ml Pen-strep at 37 ºC in 5% CO2. The HUVEC cells, obtained as primary cells, were maintained in complete IMDM media supplemented with 20% FBS and 15 mg/L EGF at 37 ºC in 5% CO2. The cells were passaged twice a week upon reaching 8590% confluence. The cell viability analysis was carried out by seeding cells into 96 wells cell culture plates, in triplicates for each concentration. Prior to the cell viability assay, a standard plot was generated using various seeding densities of cells for 24 h and 48 h. The seeding density used for the study were 10000 cells per well for the 24 h study and 8000 cells per well for the 48 h study. Cells were allowed to attach for 4 h (CHO cells) and 24 h (HUVEC cells) before incubating with GQDs suspended in the media at concentrations ranging from 25 g/ml to 800 g/ml. After exposure, the media was replaced with 10% alamar blue in basal media for 4 h and the absorbance was measured at 570 nm and 600 nm using the microplate reader (BioTek Instruments, U.S.A.) and calculated as per the directions of the manufacturer. Cell viability was finally estimated as an average of 3 replicative experiments using 3 different cell culture plates of 96 wells. 4.4.2. Oxidative stress analysis Analysis of oxidative stress due to release of reactive oxygen species (ROS) on 24 h exposure to GQDs was carried out in CHO cells following H2DCFDA staining (20 µM in


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basal medium) for 45 minutes. The fluorescence intensity was measured at excitation wavelength 485 nm and emission wavelength at 527 nm in the microplate reader. Cells exposed to H2O2 (200 µM in complete medium) for 1 h, prior to staining, was used as the positive control.60 Each group was analysed in triplicates and the net fluorescence intensity was estimated as below, Net fluorescence intensity = Average fluorescence intensity from the stained wells – average fluorescence intensity from the unstained wells (to account for the innate fluorescence of the GQD particles + fluorescence from the cell free medium) 4.5. In vivo exposure 4.5.1. Developmental toxicity analysis The Qurt strain of medaka was reared under laboratory conditions (26–28 ºC and 60% humidity) with 14 h light and 10 h dark cycles. Experiments were conducted in accordance with the institute animal ethics committee guidelines after obtaining specific approval. Spawned eggs were collected immediately within an hour of mating and the fertilised eggs were separated. Ten eggs were exposed to each concentration of GQD suspensions ranging from 0.01 µg/ml to 500 µg/ml (0.01, 0.1, 1, 10, 25, 50, 75, 100, 250, and 500 µg/ml) through daily bath exposure for 18 days post fertilization (dpf). Survival, hatchability, and developmental defects were analysed during the period of study in three replicative experiments using totally 30 eggs per each dose.

4.5.2. Germ cell analysis The embryos were exposed to GQDs from 0 dpf to the day of hatching (0 dph). The hatched larvae were immediately fixed in freshly prepared Bouin’s reagent (picric acid: formalin: acetic acid in 15: 5:1 ratio) for histological analysis of the developing gonad. The fixed larvae embedded in paraffin were sectioned at 5 µm thickness using a manual



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microtome to which a section transfer system (Thermo Scientific Micron HM 340E, Germany) was attached. Further, the sections were stained with haematoxylin and eosin in accordance with the standard staining protocol. The total number of germ cells in each larva was counted under the binocular microscope (Leica DM500, Leica Microsystems, Germany). 4.5.3. Oxidative stress analysis Female embryos, exposed to GQDs at 75 µg/ml from 0 dpf to 0 dph, were subjected to staining with 2’,7’-dichlorodihydrofluoresceindiacetate (H2DCFDA) for the detection of ROS following the manufacturer’s protocol with slight modifications. The embryos were then imaged under the stereomicroscope (Leica MZ10F, Taiwan) using the GFP filter (excitation filter at 425/60 nm and barrier filter at 480 nm). The fluorescence intensity of the stain which was directly proportional to ROS levels generated in the larvae was measured using ImageJ software. Hatched larvae exposed to 2 mM H2O2 for 1h were used as the positive control.61

4.5.4. DNA damage analysis by comet assay To assess the possibility of any DNA damage due to GQD exposure in female embryos, alkaline comet assay was carried out in the hatched larvae following a previous protocol62 with minor modifications. In brief, each individual larva was macerated using a pellet homogeniser (Pellet Pestle Motor, Kontes) in 1X PBS and low melting agarose was then added to this homogenate. The total mixture was then spread over a microscopic slide precoated with normal agarose and was allowed to solidify over ice. The slide was then dipped into the lysis buffer at 4 ºC for 2 h and then placed inside the electrophoresis chamber filled with an alkaline solution (pH ˃ 13) for 30 minutes for DNA unwinding and expression of damage at alkali labile regions. After the incubation, electrophoresis was done at 24 V (current level 300 mA) for 15 minutes at 4 ºC. At the end of the run, the slides were washed in the neutralising buffer thrice and then stained with ethidium bromide for 10 minutes. The


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stained slides were then imaged under a 40X objective of a fluorescent microscope (Olympus BX51 F, Japan) at excitation 510-550 nm. Larvae exposed to 200 mM H2O2 for 10 minutes were used as the positive control.63 The comets were scored using the ImageJ software with OpenComet plugin. Five larvae per group were examined and the average percentage tail DNA of 50 nucleoids per larva was compared for statistical analysis. 4.6. Statistical Analysis Statistical data shown in the figures were analyzed by Student’s t-test and one-way ANOVA wherein the significance was determined using Tukey’s multiple comparison posthoc test using the GraphPad Prism (version 5.03). Supporting Information 13

C NMR spectrum of the GQDs, germ cell analysis in medaka embryos after glucose

exposure at different concentrations and quantification of fluorescence intensity in control and treated female larvae without H2DCFDA staining. Author information Corresponding author e-mail address: [email protected] ORCID 0000-0002-6034-7028 Conflicts of interest The authors declare that there are no conflicting interests associated with this work. Acknowledgments This work was financially supported by grant-in-aid for research (SR/NM/NS-1070/2012(G)) from the Nanomission, Department of Science and Technology, Government of India. We acknowledge National Bio-resource Project (NBRP), Japan, for providing the medaka strain.



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Graphic for manuscript


Graphene Quantum Dots Alter Proliferation and Meiosis of Germ Cells

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