Genome stability is critical for the survival of a cell and, by extension, the organism. Cellular DNA is constantly under attack from a variety of lesions caused by either exogenous agents such as genotoxic chemicals or endogenous factors such as reactive oxygen species produced during normal cellular metabolism (Chatterjee and Walker, 2017). Different types of damage can result in DNA single-strand and double-strand breaks, base modification, cross-links, etc. As a result of the damage, cells have developed DNA repair mechanisms that can prevent the accumulation of DNA injuries. To ensure error-free replication in the adult somatic cell cycle, DNA damage is detected and repaired before or during genome replication (Hu et al., 2015).

Fish live in a variety of marine and freshwater ecosystems and are exposed to DNA-damaging factors at all stages of development. Externally fertilized fish embryos develop in water bodies, making them susceptible to DNA damage caused by xenobiotic agents like genotoxic chemicals, metals, pesticides, synthetic hormones, etc., and environmental factors like radiation, heat, etc. Vertebrates developed several pathways of xenobiotic resistance, such as ABC transporters or UDP-glucouronyl transferases (Ugts) (Li and Wu, 2007). Ugts are a family of phase-II drug metabolizing enzymes playing role in detoxification of xenobiotics (Huang and Wu, 2010; Wang et al., 2014). Nevertheless, numerous human-made chemicals find their way inside the cells and can induce significant damage to DNA. The early developmental stages are particularly susceptible to xenobiotic influence since a development-dependent expression of ugt transcripts was recorded (Christen and Fent, 2014). Furthermore, genotoxicants can have long-term effects as demonstrated for Gasterosteus aculeatus (Santos et al., 2013) and induce epigenetic changes with transgenerational effects in fish (reviewed by Terrazas-Salgado et al., 2022). DNA replication and cell proliferation are rapid during early embryonic development, and the cell cycle progresses without G1 and G2 before the mid-blastula transition (Kermi et al., 2017). Thus, to prevent mutations in actively proliferating cells during early embryo development, DNA repair is critical (Levine, 2004; Nordman and Orr-Weaver, 2012).

The majority of studies on DNA repair pathways have been conducted in zebrafish (Danio rerio) (reviewed by Pei and Strauss, 2013; Canedo and Rocha, 2021). Several literatures, however, described DNA repair in other fish species, including Xiphophorus (reviewed by David et al., 2004), Kryptolebias marmoratus (Rhee et al., 2011), and medaka, Oryzias latipes (Armstrong et al., 2002; Barjhoux et al., 2014). Base excision repair (BER), nucleotide excision repair (NER), photoenzymatic repair, direct reversal (DR), mismatch repair (MMR), homologous recombination (HR), and non-homologous end joining (NHEJ) are all proven DNA repair pathways in fish (Pei and Strauss, 2013). However, the proteins involved in DNA repair pathways appear to have more functions than just correcting DNA errors. They also take part in crucial DNA metabolism pathways during development (Shin et al., 2021). The fact that DNA damage response (DDR) proteins exhibit complex stage- and tissue-specific patterns of expression and activity (Sun et al., 2019) may indicate a role in development and organogenesis regulation (reviewed by Friedberg and Meira, 2000; Figure 1).

Furthermore, we speculate that some exogenous damaging agents can impair DNA repair protein function. Any change in the expression or localization of these proteins as a result of genotoxic stress can impact their developmental role and steer malformation during development (Figure 1). The goal of this review is to summarize the existing literature on the roles of DNA repair genes and proteins in fish embryo development and to investigate the relationship between phenotype formation and disruption of developmental pathways under genotoxic stress.

The DDR is a signal transduction pathway that detects damage and triggers a coordinated cellular response to protect the cell. The response to DNA damage can differ depending on the developmental stage. As a result, determining the timing of gene expression in embryos is critical for understanding the dynamics of gene regulation (Chechik and Koller, 2009).

We have summarized different stages of embryo development with respect to DDR pathways (Figure 2). Most of the studies available in literature have been performed on zebrafish model; however, it is important to keep in mind that timing of development vary significantly between different fish species. Furthermore, information is scarce about DDR gene expression at different stages of development in other fish. Therefore, in the current review we present data about embryo development and development of DNA repair mechanisms mostly for zebrafish (Figure 2). Future studies should complete the gap in knowledge about DDR in embryos of non-model fish species.

Fish embryo development begins with a series of synchronous and rapid cell cycles (cleavage stage) (Kane and Kimmel, 1993). Unlike adults, embryos at the cleavage stage cycle through S and M phases without G1 and G2 (Siefert et al., 2015). Most zygotic genes are silent, and the embryo relies on maternally deposited mRNAs for development (Zhang et al., 2014). The maternally loaded DNA repair transcripts include: xrcc1, unga, lig3 for BER; xpc, ccnh, xpd and xpb for NER; msh6 and msh2 for MMR; rad51b, rad51c, rad54, brca2 and mre11a for HR; xrcc4, xrcc5, xrcc6 and lig4 for NHEJ (Honjo and Ichinohe, 2019; Silva et al., 2012; Wu et al., 2014; Figure 2).

Though some DNA repair activity is present in zygotes and early-stage embryos, their ability to recognize DNA damage and to respond is reduced. Thus, checkpoint regulations differ between embryonic and adult somatic cells (Munisha and Schimenti, 2021). DNA damage checkpoints are mechanisms that link DNA repair to cell cycle events. Checkpoints stop the cell cycle and give the damaged DNA time to get repaired, or they initiate apoptosis if repair is not possible (Hartwell and Kastan, 1994; Ford and Kastan, 2020). However, in fish embryos at cleavage stage, cell divisions are unaffected by inhibitors of DNA replication or DNA damaging agents (Ikegami et al., 1997). This fact indicates impaired checkpoint activity at early stages. Following cleavage, the mid-blastula transition (MBT) occurs, which is characterized by cell cycle lengthening, loss of cell synchrony, and activation of checkpoints (Kane and Kimmel, 1993; Ikegami et al., 1999). MBT in fish corresponds with zygotic transcription activation (Tadros and Lipshitz, 2009). The S-phase lengthens at MBT which corresponds to the need for a DNA replication checkpoint in zebrafish embryos (Siefert et al., 2015).

Additionally, machinery for detecting damaged DNA is inefficient until 4hpf in zebrafish (Honjo and Ichinohe, 2019). The 2hpf embryonic cells were found to be incapable of forming phosphorylated H2AX foci (γH2AX). The formation of γH2AX is a conserved mechanism of early recognition and response to DNA double-strand breaks (Podhorecka et al., 2010). As a result, available data indicate that the mechanisms such as DNA damage recognition, checkpoint activation, and apoptosis are compromised at an early stage of fish embryo development. This could be an adaptation strategy to ensure embryonic cell divisions take place even in adverse conditions (Warkentin, 1995; Kermi et al., 2019). The delay in activation of checkpoint after MBT can provide a time frame for the induction of cytogenetic lesions by DNA-damaging agents in the developing embryo (Ikegami et al., 1997).

After cleavage stage, blastulation starts from 128-cell stage until the time of gastrulation (Kimmel et al., 1995; Figure 2). After MBT, yolk syncytial layer forms and epiboly starts. Further, at gastrulation stage, morphogenetic cell movements result in primary germ layers and embryonic axis. The epiblast and hypoblast are formed, which will give rise to ectoderm, mesoderm, and endoderm. At segmentation stage, somites develop and organogenesis starts. The tail bud becomes prominent, and first body movements appear. At the next stage, pharyngula, the body axis straightens from its early curvature around the yolk sac, and circulation, pigmentation, and fins begin to develop. Finally, hatching occurs when morphogenesis of primary organs is complete, cartilage developed in head and pectoral fin (Kimmel et al., 1995).

The expression of DNA repair proteins is temporally regulated during organogenesis and can be localized to specific organs and tissues (Vinson and Hales, 2002). We will summarize briefly the stage-specific activity of the major DNA repair pathways in developing embryos (Figure 2; Table 1).

Apart from their function in DNA repair pathways some of the above-mentioned genes and proteins play important roles in fish embryo organogenesis. In this part we will briefly summarize what is known about dual functioning of DNA repair factors. Most of the studies available in literature evaluated the role of DNA repair in organogenesis through knockout and knockdown of corresponding genes. This allows researchers to evaluate a resulting embryo phenotype, indicating which developmental pathway requires a specific DNA repair protein. Another approach focuses on localization of gene or protein expression inside the embryo. We have summarized available information on several DNA repair genes concerning their localization and knock-down specific phenotypes in Table 1.

Genotoxicants are substances found in the environment that directly bind to DNA or affect the DNA repair enzymes causing damage to DNA. Cells detect and respond to genotoxic stress by altering gene transcription (Gasch et al., 2017), protein abundance (Nouspikel, 2009; Soufi et al., 2009), protein post-translational modification (Peng et al., 2003; Ptacek et al., 2005), or protein intracellular localization (Tkach et al., 2012). Pollutants can also cause DNA damage by interfering with DNA repair mechanisms. We hypothesize that any disruption in the expression of DNA repair genes in response to genotoxicants during embryonic development can perturb their developmental role, causing organogenesis to be disrupted. Table 2 summarizes the effect of different xenobiotics on developing fish embryos, DDR gene expression, and induced phenotypes. In this chapter we review studies on toxicant exposures associated with changes in DDR gene expression, and phenotype formation.

Phthalate acid esters are widely used plasticizers frequently detected in water that have been reported to cause oxidative DNA damage (Berge et al., 2013). Exposure of four hpf zebrafish embryo for 96 h to phthalate esters, including dibutyl phthalate (DBP), mono-(2-ethylhexyl) phthalate (MEHP), and di-(2-ethylhexyl) phthalate (DEHP), resulted in altered expression of BER pathway (Lu et al., 2021). The mRNA levels of ogg1, lig3, unga, pcna, pold, fen1, and lig1 were increased after DBP exposure. Exposure to DEHP led to upregulated mRNA levels of ogg1, parp1, pcna, fen1, and lig1, and downregulated pold expression. The mRNA levels of ogg1, nthl1, apex1, parp1, lig3, pcna, and polb increased after 10 and 25 µM MEHP exposure while decreasing at 50 µM (Lu et al., 2021). Interestingly, the changes in gene expression were associated with changes in behavior, malformation, and hatching rates in all treatments.

Graphene oxide (GO) is a carbon nanomaterial with numerous applications in biology and medicine (Ni et al., 2013). GO exposure has been linked to oxidative DNA damage (Seabra et al., 2014). After exposing blastula-staged zebrafish embryos to GO nanoparticles for 24 h, the genes apex1, ogg1, polb, and creb1 were upregulated (Lu et al., 2017). The authors found no significant differences in embryo morphology, survival or hatching rate. Treatment with the same nanoparticles at 72 hpf, on the other hand, was linked to larval malformations, and neurotoxicity (Ren et al., 2016).

When exposed to sediment-spiked methylpyrene from one dpf to nine dpf, medaka (O. latipes) showed an increase in ogg1 expression (Barjhoux et al., 2014). Methylpyrene is a polycyclic aromatic hydrocarbon (PAH), a potential carcinogen, released into the environment by incomplete combustion of organic matter, fossil fuels, and other sources. In this study, there was no significant increase in the level of DNA strand breaks after treatment, which could be due to an efficient DNA damage repair mechanism (Barjhoux et al., 2014). Non-etheless, ogg1 upregulation was linked to cardiovascular injuries and, to a lesser extent, skeletal deformities in medaka embryos. This is consistent with Yan et al. (2013), who proposed that ogg1 plays a role in cardiomyocyte formation.

Pesticides are commonly used in agriculture, but they have been linked to oxidative stress in non-target organisms (Yang et al., 2020). Thus, fenvalerate, a pyrethroid insecticide that causes oxidative DNA damage, has been shown to reduce ogg1 gene expression in 24–96 hpf zebrafish embryos (Gu et al., 2010). Similarly, Shi et al. (2011) discovered that when four hpf zebrafish embryos were exposed to cypermethrin (an insecticide used in agriculture; that causes oxidative DNA damage) until 96 hpf, the expression of ogg1 was downregulated and the expression of tp53 was upregulated. Downregulation of ogg1 expression was associated with skeletal malformations and increased apoptosis in the brain region in both studies, which is similar to the ogg1 knockdown phenotype observed by Gu et al. (2013) (Table 2). Overall, these studies showed that when fish embryos were exposed to xenobiotics which changed gene expression in BER pathway, these exposures were associated with skeletal malformations, neurotoxicity and cardiovascular injuries.

Several studies reported alterations in DDR gene expression in response to heavy metals and xenobiotics. Ling et al. (2017) detected downregulation of xpc, and upregulation of UV-ddb2 and ogg1 genes when one hpf zebrafish embryos were exposed to Cd²⁺ and paraquat for 9 h. The authors suggested that the observed upregulation of NER gene, ddb2, is associated with oxidative stress. Cadmium-induced oxidative stress also ensued downregulation in the expression of MMR genes, msh2 and msh6, in one hpf zebrafish embryos when subjected to sub-lethal concentrations (3–5 µM) of Cd²⁺ (Hsu et al., 2013). The inhibition of Msh6 protein binding activity was recorded after exposure of one hpf zebrafish embryos to Cd²⁺ (Ho et al., 2015). Exposure to Cd²⁺ during zebrafish embryo development was not associated with significant mortality or teratogenicity at larval stage but led to changes in behavior compared to control (Shankar et al., 2021). Furthermore, Monaco et al. (2017) reported an increased rate of apoptotic events in the brain of embryos after 24 h of treatment with Cd²⁺ (Monaco et al., 2017). This is in agreement with the localization of ogg1, ddb1, and msh2 expression (Table 1). However, further studies are needed to show the possible long-term consequences of cadmium-induced neurodegeneration in fish.

Hg²⁺ has been shown to cause DNA strand breaks through oxidative stress and to impede DNA repair machinery (Rooney, 2007; Barcelos et al., 2011). Hg²⁺ had different effects on Msh6 protein synthesis and msh2, msh6 mRNA expression (Ho et al., 2015). Msh6 protein synthesis was inhibited without causing msh6 mRNA expression to be downregulated. It was proposed that Hg²⁺ could target a DNA repair protein function rather than transcription. Chang et al. (2017) then demonstrated that Hg²⁺ inhibited NER-associated damage incision activity in zebrafish embryos. Interestingly, Hg²⁺ exposure did not inhibit NER factor gene expression but did affect endonuclease activity, such as that of Xpg.

17α-ethinylestradiol (EE2) is a semi-synthetic hormone used in oral contraceptives and is known to promote mutation (Notch et al., 2007; Notch and Mayer, 2013). Co-exposure of 17α-ethinylestradiol (EE2) and its metabolite, estrone (E1), elicited an increased NER gene transcription, namely, xpa at 48 hpf and 72 hpf zebrafish embryos (Notch and Mayer, 2013). However, the ability of EE2 to cause DNA damage has not been described.

Benzo [a]pyrene (BaP) is a carcinogen and a model PAH produced by incomplete combustion of organic material (Soltani et al., 2019). DSBs and increased expression of xpc were observed in sturgeon (Acipenser ruthenus) embryos exposed to BaP from two cell stage to eight dpf (Gazo et al., 2021). Changes in xpc expression were associated with higher mortality when compared to controls. In contrast to zebrafish (Šrut et al., 2015), sterlet embryos effectively repaired DNA damage and did not show malformations by eight dpf (Gazo et al., 2021). This finding could point to the species-specificity of DDR mechanisms.

Camptothecin (CPT) is an inhibitor of DNA topoisomerase I widely used as an antitumor drug. CPT had an effect on the transcriptome profile of sturgeon embryos, according to Gazo et al. (2022). CPT treatment increased the expression of xpc, rad50, xpa, xrcc1, msh2, rpa1, ercc5, pold3, ercc2, fen1, blm, rad51ap1, nbn, and eme1. Upregulation of these genes was linked to a higher rate of malformations and decreased embryo viability. Embryos exposed to CPT during neurulation, on the other hand, showed only a slight increase in the expression of genes involved in DNA repair. Because viability and hatching rates in this group were comparable to those in the control group, these findings highlight stage-dependent sensitivity to CPT exposure in sturgeon embryos.

Fenthion is an organophosphate pesticide that causes oxidative stress and DNA damage, resulting in a decrease in the expression of the HR genes rad51 and rad18 in zebrafish embryos exposed for 24 h (Wahyuni et al., 2021). Further, 48 h of exposure inhibited the expression of rad51, rad18, xrcc2, and NHEJ genes (xrcc6/Ku70). In addition, exposure to terbufos (another organophosphate pesticide) for 48 h inhibited the expression of rad51, rad18, xrcc2, xrcc6/Ku70 (Wahyuni et al., 2021). In the same study, combined fenthion and terbufos exposure for 48 h increased the expression of rad51 and xrcc2. The authors hypothesized that single pesticide exposure disrupted DSB repair pathways, whereas terbufos and fenthion combined showed less genotoxicity. Future studies should investigate whether xenobiotic-induced changes in HR gene expression at embryo stage could lead to impaired meiosis, fertility, or sex differentiation at later stages of development.

So far, numerous studies considered DNA damage as an endpoint of xenobiotic toxicity (reviewed by Kienzler et al., 2013; Canedo and Rocha, 2021). However, several toxicants can decrease the expression of key genes involved in DDR, subsequently causing the cell defenseless to DNA damage (Notch and Mayer, 2013). This necessitates future studies on the vulnerability of DNA repair pathways to stress. In this review, we discussed the various roles of DNA repair genes and proteins in fish embryogenesis. We also summarized studies in which a change in DNA repair genes was linked to malformation during development. However, few studies have looked into the relationship between the type of DNA damage, changes in DDR gene expression, and embryo phenotype (Gu et al., 2010; Shi et al., 2011; Lu et al., 2017). Thus, more research is needed to understand the relationship between DDR and phenotype formation, specifically, how changes in expression level, protein post-translational modifications, and protein translocation under genotoxic stress may affect embryo development.

The rapidly developing embryos of externally fertilizing fish are exposed to environment or hatchery conditions where they deal with numerous DNA damaging factors. Inhibition of apoptosis and checkpoints during early embryo development indicates sensitivity to genotoxic damage. Cells proliferating with damaged DNA can acquire mutations, slowing the growth of the individual fish and, in the long run, the entire population. Thus, efficient DNA repair machinery is critical for avoiding damage tolerance.

Lethality can result from defects in the expression of DNA repair genes. Proteins involved in DDR are also vulnerable to damage. As a result, it is critical to identify the complex pathways by which DDR proteins participate in organogenesis. Overall, available literature indicates that alterations in BER pathway gene expression could affect cardiovascular system, CNS, brain development, and lead to changes in behavior (Wang et al., 2006; Gu et al., 2010; Gu et al., 2013; Yu et al., 2013; Barjhoux et al., 2014; Pei et al., 2019; Lu et al., 2021). Similarly, Cd²⁺-induced stress led to alterations in NER pathway and downregulation of MMR genes, which could be related to observed neurodegeneration (Hsu et al., 2013; Ling et al., 2017; Monaco et al., 2017; Shankar et al., 2021). However, more studies are required to understand changes in expression, structural modification, translocation, and inhibition undergone by repair proteins in response to genotoxicants. These future studies are necessary to understand the link between genotoxicant exposure and resulting phenotype formation.

Furthermore, the efficacy of DNA repair mechanisms appears to be stage dependent. As a result, it is necessary to describe the DDR pathways involved at various stages of fish embryo development to determine which stages are most vulnerable to stress and require immediate attention. Aside from zebrafish, there is a striking lack of research on DDR in other fish species; however, damage sensitivity and DDR may be species-specific. As a result, we believe that more research is needed to elucidate DDR in non-model species important for aquaculture, as well as more research to understand the level of DNA damage tolerance and repair in non-model fish species at the embryonic stage. This could lead to the discovery of new DDR genes and proteins, particularly during the embryonic stage.