*3.1. Oxidative Stress*

The internalization NPs by organism can induce intracellular reactive oxygen species (ROS) generation and antioxidant defense. ROS generation can lead to typical oxidative DNA damage (e.g., single- and double-stranded DNA breaks, DNA cross-links, and base modifications) [78–80]. Indirect genotoxicity of GFNs mediated by oxidative stress has been explored in vivo and in vitro. For instance, ROS generation and ROS-dependent DNA damage and genotoxicity were observed in human retinal pigment epithelium (ARPE-19) cells after 24 h exposure to GO and rGO [81]. Similarly, GO and rGO can also trigger genotoxicity of female C57BL/6J mice by induction of oxidative stress [82]. Exposed to few-layer graphene (FLG), the indirect DNA damage in THP-1 macrophages and humantransformed type-I alveolar epithelial cells was also driven by oxidative stress [43]. The specific induced mechanisms of indirect DNA damage are identified by baseline levels of micronuclei induction. Moreover, the indirect genotoxicity induced by FLG also correlates with an increase of inflammatory mediator (IL-8), decreased antioxidant (rGSH), and a depletion in mitochondrial ATP production [83]. Zhao et al. reported that GO can induce oxidative stress and genotoxicity in earthworms and the excessive accumulation of ROS, leading to lipid peroxidation, lysosomal membrane damage, and DNA damage [84]. Organisms possess a well-developed inhibition of antioxidant defense, including ROSscavenging enzymes (e.g., superoxide dismutase (SOD), peroxidase, and catalase) and regulatory mechanisms to protect organisms from the negative effects of ROS [46,84]. The ROS generation benefitted from inhibition of fatty acid, carbohydrate, and amino acid metabolism [85]. ROS induced by GO seemed to be the main mechanism leading to human

lung fibroblast (HLF) cells of genotoxicity [86]. Natural nanocolloids (Ncs) can mediate the phytotoxicity of GO such that GO–Ncs induced stronger ROS production and DNA damage compared with GO alone [87]. The mitochondrial oxidative stress induced by GQDs in microglia can cause ferroptosis.
