**6. Conclusions, Challenges, and Perspectives**

On the basis of the existing literatures, we propose several genotoxic effects for GFNs in Figure 3. To date, there are few studies on genotoxicity mediated by direct interactions with DNA for GFNs (only GO and GQDs). That oxidative stress induced by GFNs causes DNA damage has been well established and studied. Regarding other indirect genotoxicity (e.g., epigenetic toxicity, inflammation, and autophagy), the studies largely focus on genotoxic effects induced by GFNs, and there is a lack of studies on the mechanisms underlying the observed effects. The genotoxicity of GFNs will depend on both inherent physicochemical properties (e.g., surface functionalization and coatings), exposure dose and times, and their fate in organisms or the environment. Although this review paper provides preliminary information on the genotoxicity of GFNs, the data is still very limited, especially with regard to the type of GFNs and exposure dose. The traditional techniques are limited by low observation efficiency and large errors of quantitative results, which are disadvantages in the detection of GFNs.

**Figure 3.** Direct and indirect effects of GFNs on DNA.

A number of issues remain in this area: (1) a lack of nuclear detecting and tracking techniques for GFNs to investigate the direct interactions of GFNs with DNA; (2) a challenge to reveal mechanisms underlying the indirect genotoxicity of GFNs, such as causal epigenetic mechanisms; and (3) an incomplete evaluation database regarding the type of GFNs, applied dosages, and exposure times, etc. These limitations are expected since genotoxicity research of NPs, especially GFNs, is still in their infancy when compared to other areas of toxicity (e.g., cytotoxicity, immunotoxicity, neurotoxicity, reproductive and developmental toxicity). Overall, further studies should address the questions mentioned above to clarify the genotoxic mechanisms of GFNs.

**Author Contributions:** Conceptualization, K.W. and S.O.; methodology, K.W.; software, K.W.; validation, K.W., Q.Z. and S.O.; formal analysis, K.W.; investigation, S.O.; resources, Q.Z.; data curation, K.W.; writing—original draft preparation, K.W.; writing—review and editing, S.O.; visualization, K.W.; supervision, Q.Z.; project administration, S.O.; funding acquisition, S.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the National Natural Science Foundation of China for grant number No. U1906222, the National Key Research and Development Project for grant number No. 2019YFC1804104, the Fellowship of China Postdoctoral Science Foundation for grant number No. 2020M680867, and the Ministry of Education, People's Republic of China as a 111 program for grant number No. T2017002.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank Jing Sun and Zhicheng Bi for their assistance in paper discussion. We thank all the reviewers for their constructive comments.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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