*3.2. Epigenetic Toxicity*

Epigenetic regulatory mechanisms can be observed after exposure to NPs, including DNA methylation, histone modification, non-coding RNA (ncRNA) gene expression regulation, and dynamic chromatin organization [88,89]. As a response to internal and external stimuli, these above epigenetic regulations and complex, time-specific, and tissue-specific control of gene expression were allowed during development and differentiation [90]. DNA methylation, a covalent modification of cytosine residues in DNA, plays a supreme role in the stabilization and regulation of gene expression during development or differentiation [91,92]. Ting et al. [91] firstly proved that GQDs can inhibit the DNA methylation of transcription factor Sox2 and regulated DNA methyltransferase and demethyltransferase expressions. Global DNA hypomethylation of caprine fetal fibroblast cells, which are exposed to GO-AgNPs, might result from oxidative stress [93]. Histone modifications containing phosphorylation, methylation, and acetylation also are major components of epigenetic regulatory mechanisms [92]. The role of epigenetic regulation about toxicity of GFNs has been described in human embryonic kidney 293T cells [89]. The results showed that the GO triggered the formation of new intra-chromosomal looping (A1–A3) and enhanced and promoted cyclo-oxygenase-2 (Cox2) expression and activation. The epigenetic mechanisms of GO on transgenerational reproductive toxicity were determined using a house crickets generational experiment [94].

GO can activate microRNA (miRNA) protection regulation and inhibit the reproductive toxicity of *Caenorhabditis elegans*, which was also an epigenetic signal encoded protection mechanism [95]. Moreover, miRNAs can activate death receptor pathways by altering the expression of caspase-3 and tumor necrosis factor α receptor in GO-exposed pulmonary adenocarcinoma (GLC-82) cells [96]. Therefore, the epigenetic process induced by GFNs are complex and multi-layered. Currently, the existing studies are mainly limited to the reactions of epigenetic toxicity induced indirect genotoxicity of GFNs. How to explain the causal epigenetic mechanisms induced by GFNs remains challenging. Future experimental studies should be carefully designed for better understanding the genotoxic effects of GFNs induced epigenetic modifications that directly or indirectly cause DNA damage.

## *3.3. The DNA Replication, Repair, and Transcription Affected by GFNs*

GFNs have the ability to alter gene expression by interacting with signal transduction cascades or replication/repair/transcription mechanisms [97,98]. GO exposure activates a variety of signaling pathways, triggering the expression of many kinds of genes related to autophagy, apoptosis, and necrosis [89,99]. Cell apoptosis and the upregulation of the tumor protein p53 gene in the cell cycle induced by both nano- and microsized GO was detected [99]. In the work, both nano- and microsized GO block the cell cycle in the S phase, a critical period in the cell cycle. The GQDs (100 mg/L) can induce genotoxicity through ROS generation and inhibition of gene regulation in the cell cycle of rat alveolar macrophage cells [100]. The key genes (such as RAD51, BRCA2, ATM, and PARP1) regulate some key biological processes (e.g., nucleosome assembly, stress response, protein folding, and DNA damage) in FLG-exposed human primary endothelial cells [97]. Moreover, related study have shown that GFNs may cause genotoxicity by affecting the nucleotide excision repair and the repair system of non-homologous end connections [101].

### *3.4. Inflammation*

Inflammation, including acute and chronic inflammation, is a complex biological response to harmful stimuli such as pathogens, poisons, or dead cells [102]. GO induced

high expression of Cox2, a hallmark of inflammation and which is involved in acute and chronic diseases [103]. Inflammation is also one of the reactions of ROS induced indirect genotoxicity [104]. Chronic inflammation can induce secondary genotoxicity, which is manifested in the accumulation of reactive oxygen species, after GFNs exposed to cells [43,105]. Interestingly, there was no oxidative damage and a weak anti-inflammatory response for assessing the potential genotoxicity of GO and graphene nanoplatelets in the human intestinal barrier in vitro model simulation [106]. However, both GO and GNPs can induce DNA breaks, and GO can activate the nuclear factor kappa-B signaling pathway, which may lead to macrophage inflammation [107]. Excess inflammatory cytokines can cause DNA damage [108]. There are complex causal interactions between inflammation and ROS, and they may have independent induction mechanisms. In summary, the genotoxicity of GFNs mediated by inflammation can be attributed to the direct stimulation, secondary effect of cytokine release or ROS accumulation.

#### *3.5. Autophagy*

Autophagy, a cell survival mechanism, is described as a highly regulated intracellular catabolic pathway involving degradation of unnecessary or dysfunctional components to maintain cell homeostasis [109,110]. Autophagy controls transformation of nuclear components (e.g., nuclear lamina, chromatin, and DNA), which is important for maintaining genomic stability [111]. Inhibition of autophagy obstructs normal DNA damage repair and induces cell death in response to genotoxic stress. GFNs can induced ROS generation in mitochondria, which begin to exert autophagy to avoid oxidative damage and to reduce mutation of mitochondrial DNA [112]. GO was able to result in accumulation of autophagosomes, reduction in autophagic degradation, and lysosomal impairment [113]. Autophagy and epigenetic changes jointly regulate cell survival, and autophagy may be a downstream mechanism of epigenetic changes, one of the manifestations of secondary genotoxicity [114]. Graphene oxide quantum dot exposure induced autophagy in a ROS-dependent manner [115]. The relationship between autophagy and DNA damage is complex, while autophagy can regulate the levels of various proteins participating in the repair and detection of damaged DNA [116]. The relationship between autophagy and other toxicity mechanisms (e.g., oxidative stress, epigenetic changes, apoptosis, and inflammation) of other GFNs is still unclear [114]. Understanding GFNs-mediated autophagy is of great significance to explain the genotoxicity of GFNs.

#### **4. Factors Influencing Genotoxicity of GFNs**

As is known to all, there is a strong correlation between cytotoxicity and the physicochemical properties of NPs, such as particle size and shape, surface characteristics, and surface functionalization. Similarly, the genotoxicity of GFNs can be affected by these factors [117]. The genotoxicity of GFNs is greatly varied in the literature, which can be attributed to numerous factors including physicochemical properties (morphology, surface chemistry, size, shape, and purity), dose, test species, exposure time, and exposure assay [80,118].

#### *4.1. Surface Properties*

The oxygen-containing functional groups play a key role in the genotoxicity of GFNs [58,81–83,119]. For example, the rGO with lower oxygen content can induce stronger genotoxicity on ARPE-19 cells than these GO with higher oxygen content, suggesting that GO has a better biocompatibility owing to more saturated C–O bonds [81]. The remove of epoxy groups from the GO surface mitigates GO in vivo genotoxicity toward *Xenopus laevis* tadpoles [58]. Compared with GO, graphene, rGO, and graphite all induce higher levels of genotoxicity in glioblastoma multiforme cells, and the difference was attributed to the hydrophilic and hydrophobic surface and edge structure of GFNs [119]. GO has hydrophilic properties and smooth and regular edges, while rGO and graphene have hydrophobic properties and sharp and irregular edges, which can damage the integrity of cell membranes

greatly. The carboxyl groups in the surface of carboxyl-FLG may scavenge oxidative radical on bronchial epithelial cells to alleviate the genotoxicity of FLG [83]. Moreover, different immunological mechanisms triggered by GFNs can be attributed to the proportion of hydroxyl groups [82]. Cells produce a stronger inflammatory response after being exposed to GO than rGO by detecting transcriptomic changes, and the reason is attributed to the large number of hydroxyl groups on the surface of GO [82]. The surface functionalization also can significantly modulate the toxicity of GFNs [53,85,86,120]. For example, amino functionalized GQDs induced lower ferroptosis effects than nitrogen-doped GQDs [85]. Similarly, the DNA methylation of various tissues induced by GQDs was depend on their different surface chemical modifications [53]. Increased cytotoxicity and genotoxicity of the aminated GO were detected by following 24 h exposure on Colon 26 cells [120]. A study on the genotoxicity reduced by GO and rGO showed that the GTPs-rGO reduced by green tea polyphenols (GTPs) yielded more biocompatible and reduced sheets with lower genotoxic effects, as compared to the N2H4–rGO, which were reduced by hydrazine (N2H4) [121]. The acid-polyethylene glycol (LA-PEG) and PEG modified GO induced gentle DNA damage and decreased the genotoxicity of GO to HLF cells [86]. Surface charge also influences significantly the genotoxicity of GFNs [86,122]. The genotoxic effect of GO on cells is proportional to the amount of positive charge on the surface [86]. The surface charge density of graphene in aqueous solution can transform to chemically-converted graphene, leading to the capture of large amounts of DNA [122]. The different hydrophilic and hydrophobic properties of GO/rGO regulated by differential surface chemistry (especially the O/C ratio) determine the potential of graphene to interact with organisms [123–125]. Despite hydrophilic and hydrophobic rGO exhibiting similar toxic responses (e.g., cytotoxicity, DNA damage, and oxidative stress) to cells, their biological and molecular mechanisms are different [123]. The hydrophilic GO and hydrophobic rGO induce both kinds of DNA damage, namely single stranded and double stranded breaks, but the dose dependency was very significant and evident in GO exposure in DNA damage but not in rGO exposure [123]. Hydrophilicity, also an important factor in determining the biocompatibility and colloidal stability of GFNs, leads to different interactions with cells and bio-distribution of GFNs [124,125]. For example, simple accumulation of hydrophobic pristine graphene on the surface of monkey kidney cells without any cellular internalization led to severe metabolic toxicity, whereas hydrophilic GO was internalized by the cells and concentrated near the perinuclear region without causing any toxicity under lower concentrations [124]. Therefore, the surface properties play an important role in understanding the genotoxicity manifestations and biological and molecular mechanisms of GFNs.

#### *4.2. Size and Structure*

The genotoxicity of GFNs within organisms is size-dependent. Compared with large GFNs, small GFNs have bigger surface areas and provide more sites to interact with cells, leading to greater cellular uptake of GFNs [126]. The size effect plays a key role in the genotoxicity of GFNs. For example, small rGO (average lateral dimensions 114 nm) induce higher genotoxicity in the hMSCs than large rGO (3.8 ± 0.4 μm) at 0.1 and 1.0 μg/mL after 1 h exposure. The lateral size and extremely sharp edged structure of GFNs can result in higher permeability to the cell and nucleus, resulting in greater genotoxicity. Similarly, the size of GFNs is an important determinant of subcellular penetration [126]. Li et al. [127] suggested that the larger the lateral size of GO, the more severe is the pyroptosis induced by GO in Kupffer cells. Moreover, there is a strong correlation between the size of GO and the structural change in small-interfering RNAs [128]. The large GO merely reduces the A-helical pitch, while small GO inserted into the double strands can wreak havoc on the RNA conformation [129]. In addition, Kong et al. [74] proved that the DNA damage mechanism of GQDs was limited by the size of GQDs through molecular dynamics simulations. Briefly, the relatively large GQDs (61 benzene rings) tend to stick to the ends of the DNA molecule, causing the DNA to unfold, while the small GQDs (seven benzene rings) are easily embedded in DNA molecules, leading to DNA base mismatches. The planar structure of GFNs may also have an effect on DNA damage. The dsDNA bases have a stronger binding affinity with wrinkled GFNs and even cause more DNA damage than with planar GFNs [75]. Given these discordant results, it is necessary to clarify the size- and structure-related genotoxicity of GFNs.
