- freely available
Int. J. Mol. Sci. 2017, 18(7), 1515; https://doi.org/10.3390/ijms18071515
- DNA damage signalling (DDS): this pathway is induced in response to DNA damage caused by various agents including environmental, ENM and endogenous. DDS pathways are programmed to induce several cellular responses including checkpoint activity, triggering of apoptotic pathways and DNA repair .
- Direct reversal repair (DRR): reverses/eliminates the DNA damage caused by chemical reversal or modification by restoring the original nucleotide. It is also known as direct DNA damage reversal.
- Base-excision repair (BER): this repair mechanism is initiated by the excision of modified bases from DNA by DNA glycosylases. The length of the DNA that needs to undergo re-synthesis can be variable; thus, the pathway can be subdivided into short-path or long-path BER. Although various pathways are involved in this repair process, one of the most widely studied mechanisms that triggers the BER pathway is oxidatively induced damage. Since oxidative stress is one of the most common mechanisms of ENM-induced DNA damage, oxidatively induced DNA lesions are predominantly repaired by the BER pathway (see Table 1). The key enzymes involved in the BER process are DNA glycosylases, which remove damaged bases by cleavage of the N-glycosylic bonds (between the bases and deoxyribose moieties) of the nucleotide residues. The DNA glycosylase action is followed by an incision step, DNA synthesis, an excision step, and DNA ligation. Various metal oxide based ENMs, quantum dots and carbon nanomaterials have been implicated in activating the BER pathway (Table 1).
- Nucleotide excision repair (NER): is involved in removing bulky DNA adducts. The damage from the active strand of transcribed DNA and DNA damage elsewhere in the genome is removed in this pathway by transcription-coupled repair and global genome repair, respectively. Silver and cadmium based ENMs have been shown to interfere with the NER pathway (Table 1).
- Mismatch repair (MMR): this pathway is involved in post-replicational DNA repair that removes errors including mismatched nucleotides, insertions, deletions, etc.
- Homologous recombination repair (HRR): this pathway involves repair of DSBs using the homologous DNA strand as a template for re-synthesis.
- Non-homologous end joining repair (NHEJ): helps to ligate the DNA ends resulting from DSBs.
- Translesion synthesis (TLS): this pathway employs specialized polymerases that use damaged DNA as templates, to finish replication across lesions. Although the mechanism is error-prone, and cell survival may be associated with an increased risk of mutagenesis/carcinogenesis, it helps to prevent a stalled replication fork.
2. Activation/Up-Regulation of DNA Damage Signalling Pathways
3. Up-Regulation of DNA Repair Genes
4. Inactivation/Downregulation of DNA Repair Pathway Genes
5. Conclusions & Future Considerations
- Characterisation of induced DNA damage lesions: a given ENM may have a primary mechanism for the induction of DNA damage, which triggers the initiation of a specific repair pathway. For example, metal and metal oxide based ENMs tend to cause oxidatively induced DNA damage, which is mainly repaired via the BER pathway. Therefore, characterising the type of DNA damage is critically important in future studies, as it will enable predictive models to be developed that can be used to predict which types of ENMs might affect specific DNA repair pathways.
- Role of ions: inorganic NPs could via corrosion and dissolution release metal ions such as Cd2+, Fe3+, Zn2+, and Ag+ and hence influence the upregulation/downregulation (measured as excision activity) of various pathways. Additionally, metal ions released from ENMs have been shown to interact/bind with protein domains and amino acids of DNA repair proteins (e.g., zinc finger structures contained in the DNA repair protein, XPA) resulting in distorted protein structure and inefficient DNA repair activity . Therefore, a thorough physicochemical characterisation of ENMs is imperative, to discriminate between the actual causative factor (ENM vs. metal ions), as the impact of ENMs on DNA repair pathway may be strongly associated with the presence of metal(s) either in their composition, or as undesirable impurities.
- Dose-dependent DNA damage response: presently, the doses of ENMs administered in in vitro studies/test species to generate dose-response analysis may not mimic a potential human exposure level. This is because concentration-dependent activation of genes/pathways as well as transition in gene changes can be highly dose dependent. Therefore, dose ranges that are relevant to true exposure levels of ENMs need to be included when studying DNA damage responses pertaining to repair pathways. However, ENM exposure assessment currently presents a technical challenge and more work is needed to evaluate emissions of ENMs into the environment . For example, it will be necessary to perform more thorough background measurements at workplaces to determine accurate occupational exposure levels, to develop appropriate metrics for ENM exposure assessments and to validate personal air samplers.
- Method/technique: various techniques and methods with different endpoints are utilized for evaluating DNA damage repair and/or DNA damage responses, e.g., Western blots for translational changes/modifications and/or phosphorylation events; RT-PCR for transcriptional alterations; excision or incision assays for DNA repair enzyme activity; mass spectrometry methods for measuring adduct or lesion formation and multiplexed excision/synthesis assays for DNA repair enzyme inhibition activity . Each method has its own sensitivity, specificity and endpoints, which makes it challenging to compare results across different studies. Hence, to enable an appropriate intra-laboratory/interlaboratory comparison of DNA damage repair responses, statistically appropriate analysis on normalised data must be performed in order to identify reproducible upregulation or downregulation of ENM-induced DNA repair responses.
- Tissue specific detection /expression: different tissues and cell types (including primary cells, normal/cancer cell lines) exhibit varying DNA repair responses, which may correlate with the degree of DNA damage and susceptibility following exposure to some ENMs. Hence, it is imperative to measure the levels and activity of DNA repair genes and proteins, respectively, in all relevant cells, tissues or organs of interest as their expression and responses are largely “site-specific”.
- Effect of acute vs. chronic exposure of ENMs: the type of exposure may affect the severity of the DNA damage and the resultant activation of specific DNA repair pathway(s). The human population may be exposed to natural, environmental or ENMs in a cumulative manner . On the other hand, occupational, lifestyle or behaviour-related exposure to various nano-entities may induce acute responses [8,43]. Therefore, it is important to understand how various kinds of exposure scenarios dictate not only DNA damage, but also trigger specific repair pathways.
- Effect of potential ENM artefacts on the interpretation of DNA damage repair or DNA damage response: as described in previous reports, the solution state physico-chemical properties of ENMs are not like the solution state physicochemical properties of chemicals . Depending upon the category of ENMs under investigation, ENMs are prone to disparate rates of dissolution, aggregation/agglomeration phenomena, nutrient depletion and other behaviours that can potentially result in false-positive and/or false-negative responses in DNA damage repair and DNA damage response assays. These types of artefactual effects have been observed in many types of nanotoxicity  and nanoecotoxicity , but can be avoided by including appropriate experimental controls in the assays and having a thorough understanding of the assay variability parameters.
Conflicts of Interest
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|Study||Analysis Technique Applied||Cell/Tissue Used||NP||DNA Repair Pathway and Its Corresponding Component Involved|
|Homologous Recombination Repair (HRR)||Non-Homologous End Joining (NHEJ)||DNA Damage Signalling (DDS)||Base Excision Repair (BER)||Nucleotide Excision Repair (NER)||Mismatch Repair (MMR)|
|AshaRani et al., 2012 ||mRNA and array hybridisation RT-PCR||Human lung fibroblast, IMR 90||AgNPs||↓ |
|Kovvuru et al., 2014 ||DNA repair RT2 Profiler PCR array||Liver||AgNPs||↓ |
|Asare et al., 2015 ||PCR||Lung tissue||AgNPs||↑ |
|Satapathy et al., 2014 ||In Vivo Base Excision Repair (BER) Assay||Oral squamous cell carcinoma||QAgNPs||↓ |
|Van Berlo et al., 2010 ||mRNA expression||Lung tissue||Carbon||↑ |
|Tang et al., 2013 ||RT-PCR||Daphnia pulex||CdSO4 or CdTeQDs||↑ |
|Tang et al., 2015 ||RT-PCR||Daphnia pulex||CdTe/ZnS||↑ |
|Ahamed et al., 2010 ||Western blotting||Human pulmonary epithelial cells (A549)||CuO||↑ |
|Khatri et al., 2013 ||RT-PCR||THP-1, Primary human nasal, Small airway epithelial||ENMs emitted from photocopiers||↑ |
|Prasad et al., 2013 ||Western blot (phosphorylation)||Human dermal fibroblasts||TiO2||↑ |
Activation of ATM/Chk2 DNA damage signalling pathway
|El-said et al., 2014 ||RT-PCR||HepG2||TiO2||↑ |
|Hanot-Roy et al., 2014 ||Western blot (phosphorylation)||Alveolar macrophages (THP-1), Epithelial cells (A549), Human Pulmonary Endothelial Cells (HPMEC-ST1.6R cells)||TiO2||↑ |
|Pati et al., 2016 ||Western blot||Macrophages||Zinc oxide nanoparticles (ZnO-NPs)||↓ |
|ATM (ataxia-telangiectasia mutated)||Cell cycle checkpoint kinase protein, which belongs to the PI3/PI4- kinase family. Serves as a DNA damage sensor and regulator of a wide variety of downstream proteins, including, 1) Tumour suppressor protein p53 and 2) Serine/threonine protein kinase that activates checkpoint signalling upon double strand breaks (DSBs), apoptosis, and genotoxic stresses.|
|ATR Rad3-related kinase||PI3 kinase-related kinase family member (like ATM), which phosphorylates multiple substrates on serine/ threonine residues (that are followed by a glutamine) in response to DNA damage or replication blocks. Causes cell cycle delay, in part, by phosphorylating checkpoint kinase (CHK)1, CHK2, and p53.|
|CHK1 and CHK2 (Checkpoint kinase 1 and 2)||Downstream protein kinases of ATM/ATR, which play an important role in DNA damage checkpoint control.|
|APEX1 (Apurinic/apyrimidinic endonuclease 1)||Multifunctional DNA repair enzyme, apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) responsible for abasic site cleavage activity. |
Plays a critical role in the DNA base excision repair (BER) pathway and in the redox regulation of transcriptional factors.
Activated/ induced by oxidative DNA damage.
Localisation signals, post-translational modifications and dynamic regulation determines the localisation of APE protein in the nucleus with subcellular localization in the mitochondria, endoplasmic reticulum and cytoplasm.
|APEX2 (Apurinic/apyrimidinic endonuclease 2)||AP endonuclease 2 is characterized by a weak AP endonuclease activity, 3′-phosphodiesterase activity and 3′- to 5′-exonuclease activity. |
Involved in removal of mismatched 3′-nucleotides from DNA and ATR-Chk1 checkpoint signalling in response to oxidative stress.
|(POLB) DNA polymerase β||Contributes to DNA synthesis and deoxyribose-phosphate removal.|
|(FEN1) Flap endonuclease 1||Possesses 5′–3′ exonuclease activity and cleaves 5′ overhanging “flap structures” in DNA replication and repair.|
|LIG1 (Ligase 1)||Seals SSB ends.|
|MBD4 (methyl-CpG binding domain protein 4)||Belongs to a family of nuclear proteins that possess a methyl-CpG binding domain (MBD). These proteins bind specifically to methylated DNA, possess DNA N-glycosylase activity and can remove uracil or 5-fluorouracil in G:U mismatches.|
|MUTYH (mutY DNA glycosylase)||Serves as DNA glycosylase (excises adenine mispaired with 8-oxoguanine). |
Maintains chromosome stability by inducing ATR-mediated checkpoint activation, cell cycle arrest and apoptosis.
|NEIL1, NEIL3 (Nei-like 1; Nei-like 3)||Generate apurinic/apyrimidinic (AP) sites and/or SSBs with blocked ends.|
|NTHL1||Serve as oxidized base-specific DNA glycosylases that remove oxidized and/or mismatched DNA bases.|
|OGG1 (8-oxoguanine DNA glycosylase)||Excises and repairs oxidatively damaged guanine bases in DNA, which occur as a result of exposure to ROS.|
|PCNA (Proliferating cell nuclear antigen)||Co-factor for DNA polymerase and essential for DNA synthesis and repair.|
|PARP1 (Poly ADP ribose polymerase)||PARP1—serves as sensor of SSBs.|
|XRCC1 (X-ray repair cross-complementing protein 1)||XRCC1—serves as a scaffold for recruiting and activating BER proteins.|
|RPA1 (replication protein A1)||Largest subunit of the replication protein A (RPA), the heterotrimeric single-stranded DNA-binding protein involved in replication, repair, recombination and DNA damage check point activation.|
|XPC (xeroderma pigmentosum group C protein)||Recognizes bulky DNA adducts. Pairs up with RAD23 and helps in the assembly of the other core proteins involved in NER pathway progression.|
|XPA (xeroderma pigmentosum group A protein)||Attaches to damaged DNA, interacts along with other proteins in the NER pathway to unwind, excise and replace the damaged DNA.|
|BRCA1/ BRCA2 (breast cancer type 1 and type 2 susceptibility proteins)||BRCA1 and BRCA2 are coded by human tumour suppressor genes that are involved in DNA damage repair, cell cycle progression, transcription, ubiquitination and apoptosis. |
Aberrant proteins coded by mutated genes are found in hereditary breast and ovarian cancers; activation of various kinases in response to DNA-damage have been shown to phosphorylate sites on BRC1 and BRC2 in a cell cycle-dependent manner.
|RAD51||Involved in the homologous recombination and repair of double strand DNA breaks.|
|Ku||Ku, a heterodimer of two related proteins, Ku70 and Ku80, is involved in DSB repair and V(D)J recombination.|
|LIG4 (Ligase 4)||LIG4 is the DNA ligase required for, and specific to, c-NHEJ. It catalyzes the same ATP-dependent transfer of phosphate bonds that results in strand ligation in all eukaryotic DNA repair. |
LIG4 is the only ligase with the mechanistic flexibility to ligate one strand independently of another or to ligate incompatible DSB ends as well as gaps of several nucleotides.
|XRCC4 (X-ray repair cross-complementing protein 1)||XRCC4 is a non-enzymatic protein that is required for the conformational stability and functioning levels of LIG4. |
XRCC4 interacts with LIG4 facilitated by carboxy-terminal repeats at the LIG4 carboxyl terminus, resulting in a coiled-coil like conformation.
Most of the enzymatic domain of LIG binds to and interacts with XRCC4, except for the small region implicated in DNA binding.
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