**1. Introduction**

Graphene, a two-dimensional crystal repeatedly peeled from graphite, is a single layer of carbon atoms with a sp2-hybridized structure (Figure 1a) [1,2]. Graphene and its derivatives, including graphene oxide (GO), reduced graphene oxide (rGO), and graphene quantum dots (GQDs), exhibit various excellent physical, electrochemical, and optical advantages [3–5]. GO is an amphiphilic sheet-like graphenic carbon and contains fewer oxygen functional groups (Figure 1b) [6,7]. rGO is prepared by oxidative exfoliation of graphite and has lower C/O ratios than GO (Figure 1c) [8]. GQDs are similar to graphene but have unique zero-dimensional structures due to their nanoscale sized lateral dimensions (Figure 1d) [9].

Currently, GFNs, as promising nanomaterials, have attracted increasing attention in the scientific community and are in commercial production for many applications, such as energy storage [10–17], medicine [18–25], environmental protection [26–31], and industrial manufacturing [32–34]. For example, the market for graphene-based products is forecast to reach \$675 million by 2020 [35]. With rapid developments in application and production of GFNs, their potential for release into the environment and the environmental risks of GFNs have become emerging issues [36–38]. Consequently, many studies have shown that adverse effects can be induced by GFNs in vivo and in vitro, such as organ (e.g., lung, liver, and spleen) toxicity, cytotoxicity, immunotoxicity, neurotoxicity, and reproductive and developmental toxicity [3,39]. Moreover, the toxicity mechanisms of GFNs to organisms, including physical destruction, oxidative stress, inflammatory response, apoptosis,

**Citation:** Wu, K.; Zhou, Q.; Ouyang, S. Direct and Indirect Genotoxicity of Graphene Family Nanomaterials on DNA—A Review. *Nanomaterials* **2021**, *11*, 2889. https://doi.org/10.3390/ nano11112889

Academic Editor: Marta Marmiroli

Received: 21 September 2021 Accepted: 21 October 2021 Published: 28 October 2021

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autophagy, and necrosis, are summarized in Table 1. However, the genotoxicity of GFNs on DNA (e.g., DNA damage) remains largely unknown.

**Figure 1.** Structural models of single-layer graphene (**a**), graphene oxide (**b**), reduced graphene oxide (**c**), and graphene quantum dots (**d**).

Genotoxicity is broadly defined as 'damage to the genome' and also a distinct and important type of toxicity, as specific genotoxic events are considered hallmarks of cancer [40]. Generally, the genotoxicity can be sub-classified into direct genotoxicity and indirect genotoxicity in cells or the nucleus [41–43]. Nanoparticles (NPs) can be uptaken by the nucleus and induce DNA damage, leading to direct genotoxicity on organisms [42]. While many studies have shown that most NPs cannot enter the nucleus, they still indirectly affect genotoxicity by oxidative stress, epigenetic changes, inflammation, and autophagy [42]. Moreover, genotoxicity plays a key role in assessing the safety of NPs on human health and the environment [44–47]. Although there has been many researches about the genotoxicity of NPs in recent years, it is mainly focused on traditional artificial nanomaterials, such as TiO2, carbon nanotubes, and silver and gold NPs [48–50]. However, the existing literature on genotoxicity of GFNs remains limited and conflicting. A few studies showed that GFNs had no adverse effects on genotoxicity [51]. In contrast, many researchers have reported that the small size and sharp edges of GFNs (e.g., GO and GQDs) can induce genotoxicity on aquatic organisms (e.g., fish and algae) [52–54]. However, the direct and indirect genotoxicity mechanisms of GFNs remain unclear, despite genotoxic phenomena being widely reported.

The purpose of this article is to critically review the existing literatures on the genotoxicity of GFNs. This review will focus mainly on the genotoxicity mechanisms of GFNs in order to (1) expand our understanding of possible mechanisms underlying the promotion of DNA damage by GFNs; (2) highlight the direct and indirect genotoxicity of different subsets of GFNs; and (3) explore the factors that influence the genotoxicity of GFNs. This review will provide new insights into the genotoxicity and environmental risks of engineered nanoparticles (ENPs).

