Bulky DNA adducts leading to chromosomal aberrations and mutations

This AOP network describes the linkage of bulky DNA adduct formation to irreversible genomic damage (chromosomal aberrations and mutations). Irreversible genomic damage is of interest by regulators due to its association with multiple adverse health effects such as cancer and heritable disorders.  

Mutagens are genotoxic substances that bind to the DNA sequence, altering the conformation through substitutions, deletions, adductions, or other complex methods. These mutations are possible in both the non-coding and coding regions of DNA and can lead to a silent or functional mutation. Functionality is determined by the location and type of mutation. Bulky DNA adducts are formed by the covalent bonding of a large organic compound to the DNA structure, causing large conformational shifts. Like mutagens, these chemicals' extent of effect depends on location and size of the compound.  

The molecular initiating event (MIE) of this AOP is an increase in bulky DNA adducts. These adducts form in all cell types due to exogenous (i.e., exposure to pollutants) as well as endogenous chemicals. These adducts require specific methods of repair that are complex due to the structure changes. For these reasons, it is possible that the repair mechanisms that usually perform adequately can become overwhelmed and thus inadequate (KE1). Due to the inadequate repair (KE1), as well as the conformational change from the adducts forming (MIE), mutations (AO1) can arise. DNA adducts that have failed repair can cause lesions in the DNA (KE2). With an inadequate repair (KE1) that is already struggling to repair the adducts (MIE), it can fail to repair the lesions (KE2) which can lead to chromosomal aberrations (AO2) and/or mutations (AO1). 

This AOP’s support is strong due to the depth of understanding of the mechanisms involved. Many of the KERs have robust empirical support while certain KE’s have strong evidence of essentiality. For example, inadequate repair (KE1) has been observed leading to both mutations (AO1) and chromosomal aberrations (AO2) in multiple studies. 

We predict that this AOP will gain traffic due to the increasing interest and understanding of the growing abundance of prototypical stressors found in the environment. This AOP points to critical gaps in research and understanding of this pathway. It will also help to guide research to develop quantitative associations as well as modulating factors connecting the KEs across the AOP while providing novel test methods to understand and evaluate the implications of bulky DNA adducts. 

This AOP examines the effects of increased bulky DNA adducts (MIE), on increased mutations (AO1), and chromosomal aberrations (AO2). In addition to summarizing the current state of knowledge, the AOP aims to identify important evidence gaps, support the development of future AOPs, inform the design of novel testing approaches, and contribute to regulatory decision-making. 

The evidence-gathering strategy focused on individual key events (KEs) and key event relationships (KERs). Bulky DNA adducts can form in any organism containing DNA, thus this AOP is not restricted to a specific taxonomic group, species, sex, or life stage. 

Most KEs and KERs included in this AOP were reused from AOP 296, Oxidative DNA Damage Leading to Chromosomal Aberrations and Mutations. Rather than oxidative DNA damage, the present AOP introduces bulky DNA adducts as the MIE while retaining much of the downstream biological framework. The objective is to determine whether the existing AOP structure remains valid when an alternative MIE is incorporated, thereby demonstrating the adaptability of the AOP framework to related mechanisms of genotoxicity. 

To support this objective, four new KERs are being developed. The first is the adjacent relationship between increased bulky DNA adducts (MIE) and inadequate repair (KE). Three additional non-adjacent KERs are also being evaluated: increased bulky DNA adducts leading directly to increased DNA strand breaks (KE), increased mutations (AO1), and increased chromosomal aberrations (AO2). 

This AOP was developed through the collaborative expertise of researchers at the University of Ottawa, particularly Dr. Carole Yauk, and contributors from the Partnership for the Assessment of Risks from Chemicals (PARC). The project seeks to establish the environmental and regulatory relevance of bulky DNA adduct-forming pollutants and their role in genetic damage. 

Polycyclic aromatic hydrocarbons (PAHs) serve as a representative stressor for this AOP. Following metabolic activation, PAHs can interact with DNA and form covalent bonds with nucleotides, generating bulky DNA adducts (Henkler et al., 2012; Millen et al., 2012). These adducts represent a critical initiating event in the pathway and can disrupt normal DNA structure and function. 

Several validated methods are available for detecting bulky DNA adducts. The ^32P-postlabeling assay is among the most widely used and has undergone numerous methodological refinements to improve sensitivity and application-specific performance (Kovács et al., 2010). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) has also been successfully used for the identification and quantification of bulky DNA adducts (Olsen et al., 2010). 

When bulky DNA adducts are not effectively repaired, they can contribute to altered chromosomal structures, genomic instability, and mutations (Baiken et al., 2021; Henkler et al., 2012). Increased mutation frequencies associated with bulky DNA adduct formation have been observed across multiple tissues and species, demonstrating the broad biological relevance of this mechanism (Akerman et al., 2004; Henkler et al., 2012; Long et al., 2018; Schuster et al., 2024). 

Mutations and genome instability resulting from bulky DNA adducts can be quantified using several experimental approaches. These include in vitro modeling systems (Broschard et al., 1994) and modern error-corrected sequencing technologies, which enable highly sensitive detection of rare mutations (Olsen et al., 2010; Schuster et al., 2024). 

Bulky DNA adducts can also interfere with DNA repair processes, leading to inadequate repair. Deficiencies in repair capacity increase the likelihood that DNA lesions persist and subsequently contribute to DNA strand breaks, chromosomal aberrations, and mutations (Fischer et al., 2018; Liu et al., 2012; Matullo et al., 2001; Mimmler et al., 2016; Thompson et al., 1983). 

DNA strand breaks resulting from inadequate repair can be measured using established methods such as the Comet Assay (Burlinson et al., 2012). In human populations, reduced DNA repair capacity has been associated with elevated mutation frequencies (Matullo et al., 2001), further supporting the importance of repair processes within the pathway. 

Evidence from knockout and knockdown animal models has also highlighted the role of multiple DNA repair pathways in mitigating the effects of bulky DNA adducts. Studies using repair-deficient mice exposed to chemicals known to induce bulky adducts—including PAHs, heterocyclic aromatic amines, and other genotoxic compounds—have demonstrated increased susceptibility to DNA damage and mutagenesis (Fischer et al., 2018; Liu et al., 2012; Matullo et al., 2001; Mimmler et al., 2016; Thompson et al., 1983). 

Overall, the available evidence indicates that inadequate repair is a central mechanistic link within this AOP. Failure to repair bulky DNA adducts increases the persistence of DNA damage, promotes DNA strand breaks, and contributes to the development of both mutations and chromosomal aberrations. These downstream effects collectively support the proposed pathway from bulky DNA adduct formation to adverse genetic outcomes. 

My search strategy was supplemented by documentation and supporting references Overall Assessment from previously developed AOP descriptions, as well as established key events (KEs) and key event relationships (KERs). 

The initial literature identification strategy was informed by expert input from Dr. Carole Yauk, Emmanuelle Demuynck, and contributors at PARC. In parallel, developers of the original AOP had begun investigating the newly proposed KEs and KERs and provided an Excel file containing previously screened studies classified as either relevant or not relevant to the developing AOP. Studies confirmed to be relevant were imported into Covidence for further review. 

Beginning with a key paper recommended by Dr. Yauk, Connected Papers was used to identify related publications through citation mapping. This included both articles that cited the original paper and those referenced within it. 

Because relatively few studies were identified for developing KEs and KERs, through connected papers, the search strategy was expanded using a modified approach based on Huliganga et al. (2022). Broad literature searches were conducted through the University of Ottawa’s Omni search platform using Boolean queries with the “any field contains” option. Due to time constraints and the use of a single reviewer, only the first 50 results from each search were exported in RIS format and uploaded to Covidence. Inclusion criteria were developed using a modified PECO framework adapted from Huliganga et al. (2022). 

During title and abstract screening, notes were recorded in Covidence to indicate potential relevance to specific KEs or KERs. Full-text screening was then conducted to confirm these preliminary classifications and identify any additional relationships. Studies that provided support for a KE or KER were documented and retained for evidence of evaluation. 

Both biological plausibility and empirical evidence were assessed for each developing KE and KER to identify evidence gaps. This assessment revealed a lack of temporal evidence supporting the relationship between increased bulky DNA adducts (MIE) and chromosomal aberrations (AO2). 

To verify whether supporting studies existed for this relationship, additional targeted searches were conducted. These searches focused exclusively on the KER linking bulky DNA adducts and chromosomal aberrations, including variations of both terms and the use of the “any field contains the exact phrase” search option. The complete search queries are provided below. 

Because these targeted searches returned fewer than 50 results on average, title and abstract screening were conducted directly within Omni rather than through Covidence. However, the same inclusion criteria were applied. In total, 156 articles were screened across all search stages. 

The final search day was the 16th of July using OMNI, supplied by the University of Ottawa, to find any existing support for the KER: Increasing Bulky adducts -> Increase chromosomal aberrations. In this Boolean search, “Bulky DNA adducts” and “chromosomal aberrations" was used to yield 24 articles. Each article was read, first starting with discussion and results to determine if observations were of the desired key events. If the article included one of the desired key events, it was then read for background information and methods to determine inclusion based on the initial criteria. 

A key limitation of this review is the relatively small number of articles assessed. Additionally, the literature searches were conducted on different dates, which may have resulted in the omission of relevant studies or inconsistencies in article retrieval. 

Future reviews would benefit from conducting all searches on a single date and performing a final update search immediately before publication. This approach would help capture newly published studies and improve the completeness and reproducibility of the evidence-gathering process. 

Overall, the biological plausibility of this AOP network is strong. Most of this network was originally developed by a team of experts within the Health and Environmental Sciences Institute’s Genetic Toxicology Technical Committee who have experience in research on DNA repair and genetic toxicology for decades. Most of the data, weight of evidence, and overall assessments for the original KEs and KERs were reused from the original AOP; it was past the scope of this initial project, but the resources will be updated in the future with new publications.  

Most of the new KEs of this AOP are lacking essentiality as this was out of the scope of this project but will be included in the future. For the KE inadequate repair, studies were found that overexpressed or even inhibited the repair mechanisms for removal of bulky adducts. In these studies, it was found that without inhibition there were lower levels of both mutations and aberrations and when repair was inhibited, the aberrations and mutations both increased significantly.  

Due to the limited time of this project and focusing on a single KER, not all evidence was able to be assessed for weight just yet but will be completed in the future. The evidence reused from the original AOP was mostly strong and moderate weight, lending a decent weight to the current AOP. The new KERs have yet to be developed except for one, which has overall Moderate weight, will require reassessment in the future.  

There are multiple modulating factors for this AOP. These, at the time of writing, mainly focus on polymorphisms in metabolization enzymes as well as repair insufficiencies. These have an impact on both upstream and downstream as they are affecting the concentration of adduct forming chemicals as well as the repair of the adducts preventing leading into aberrations and mutations.  

Our quantitative understanding of this AOP is limited at the time of writing. It is complex, due to the differences in tissues metabolism, damage rate, and repair. More understanding of these AOP’s quantitative relationships will be developed in the future as the rest of the AOP is developed and researched.  

Research is increasingly moving away from the use of animals and other living organisms toward in vitro and in silico models. To support this transition, it is important to first understand the basic biological mechanisms (i.e., the MIEs and KEs) to accurately develop future studies focused on relevant endpoints, while also preventing unnecessary testing for which relevant data already exist. 

This understanding is also important in the risk assessment process, even when the AOP is not biologically active at clinically relevant doses. This AOP can be used as a framework for determining the probability of agents inducing DNA adducts through different mechanism-based tests. In accordance with the Bradford–Hill criteria, this AOP can help determine whether a stressor induces bulky DNA adducts as a primary mechanism of action. 

Bulky DNA adducts are a well-established biomarker of exposure to a wide range of chemicals and can serve as a useful endpoint in risk assessment. Once it has been demonstrated that a chemical follows this AOP, it becomes possible to develop a quantitative assessment of the stressor using in vivo genotoxicity data (e.g., micronuclei formation or mutations). 

This AOP can inform various testing approaches, including more specific fit-for-purpose assays that quantitatively relate KEs to adverse genotoxic outcomes. In addition, this AOP document helps address gaps in the quantitative understanding of genomic damage induced by bulky DNA adducts. 

In conclusion, this AOP, as well as others like it, can be applied in many aspects of the regulatory assessment of chemicals. These applications include highlighting gaps and uncertainties in mechanisms of action (MOAs) and in the quantitative understanding of specific AOPs; facilitating the development of new testing strategies; identifying appropriate methods for evaluating untested chemicals and linking them to relevant AOPs; and ultimately influencing the research paradigm to improve the assessment of MOAs, apical endpoints, and the overall risk assessment framework. 

G.S Akerman, B.A Rosenzweig, O.E Domon, L.J McGarrity, L.R Blankenship, C.A Tsai, S.J Culp, J.T MacGregor, F.D Sistare, J.J Chen, S.M Morris, Gene expression profiles and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Volume 549, Issues 1–2, 2004, Pages 43-64, ISSN 1386-1964, https://doi.org/10.1016/j.mrfmmm.2003.11.013.  

Baiken Y, Kanayeva D, Taipakova S, Groisman R, Ishchenko AA, Begimbetova D, Matkarimov B and Saparbaev M (2021) Role of Base Excision Repair Pathway in the Processing of Complex DNA Damage Generated by Oxidative Stress and Anticancer Drugs. Front. Cell Dev. Biol. 8:617884. doi: 10.3389/fcell.2020.617884 

Bi, Xiaohui, et al. “DNA Polymerase κ Is Specifically Required for Recovery from the Benzo[a]Pyrene-Dihydrodiol Epoxide (BPDE)-Induced S-Phase Checkpoint.” The Journal of Biological Chemistry [United States], vol. 280, no. 23, June 2005, pp. 22343–55, https://doi.org/10.1074/jbc.M501562200. 

Fischer, J.M.F., Zubel, T., Jander, K. et al. PARP1 protects from benzo[a]pyrene diol epoxide-induced replication stress and mutagenicity. Arch Toxicol 92, 1323–1340 (2018). https://doi.org/10.1007/s00204-017-2115-6 

Georgiadis, P., et al. “Impact of Phase I or Phase II Enzyme Polymorphisms on Lymphocyte DNA Adducts in Subjects Exposed to Urban Air Pollution and Environmental Tobacco Smoke.” Toxicology Letters [Shannon], vol. 149, no. 1, April 2004, pp. 269–80, https://doi.org/10.1016/j.toxlet.2003.12.038. 

Georgiadis, Panagiotis, et al. “Interactions between CYP1A1 Polymorphisms and Exposure to Environmental Tobacco Smoke in the Modulation of Lymphocyte Bulky DNA Adducts and Chromosomal Aberrations.” Carcinogenesis (New York) [Oxford], vol. 26, no. 1, January 2005, pp. 93–101, https://doi.org/10.1093/carcin/bgh294. 

Gillet, Ludovic C. J., and Orlando D. Schärer. “Molecular Mechanisms of Mammalian Global Genome Nucleotide Excision Repair.” Chemical Reviews [United States], vol. 106, no. 2, February 2006, pp. 253–76, https://doi.org/10.1021/cr040483f.

Henkler, F., Stolpmann, K., Luch, A. (2012). Exposure to Polycyclic Aromatic Hydrocarbons: Bulky DNA Adducts and Cellular Responses. In: Luch, A. (eds) Molecular, Clinical and Environmental Toxicology. Experientia Supplementum, vol 101. Springer, Basel. https://doi.org/10.1007/978-3-7643-8340-4_5  

Huliganga E, Marchetti F, O'Brien JM, Chauhan V, Yauk CL. A Case Study on Integrating a New Key Event Into an Existing Adverse Outcome Pathway on Oxidative DNA Damage: Challenges and Approaches in a Data-Rich Area. Front Toxicol. 2022 Apr 28;4:827328. doi: 10.3389/ftox.2022.827328. PMID: 35573276; PMCID: PMC9097222.  

Kaufmann, William K. “Pathways of Human Cell Post-Replication Repair.” Carcinogenesis (New York) [Oxford], vol. 10, no. 1, January 1989, pp. 1–11, https://doi.org/10.1093/carcin/10.1.1. 

Lan Chun Tu, Sei ichi Matsui, Terry A. Beerman; Hedamycin, a DNA alkylator, induces γH2AX and chromosome aberrations: Involvement of phosphatidylinositol 3-kinase–related kinases and DNA replication fork movement. Mol Cancer Ther 1 August 2005; 4 (8): 1175–1185. https://doi.org/10.1158/1535-7163.MCT-05-0054  

Leslie Recio, Lee R. Shugart, Abraham W. Hsie, The relationship between benzo(a)pyrene diol-epoxide-DNA adducts and mutagenicity in the CHOHPGRT assay, Fundamental and Applied Toxicology, Volume 8, Issue 2, 1987, Pages 243-252, ISSN 0272-0590, https://doi.org/10.1016/0272-0590(87)90123-0.  

Lívia Anna, Reetta Holmila, Katalin Kovács, Erika Győrffy, Zoltán Győri, Judit Segesdi, János Minárovits, Ibolya Soltész, Szilárd Kostič, Attila Csekeő, Kirsti Husgafvel-Pursiainen, Bernadette Schoket, Relationship between TP53 tumour suppressor gene mutations and smoking-related bulky DNA adducts in a lung cancer study population from Hungary, Mutagenesis, Volume 24, Issue 6, November 2009, Pages 475–480, https://doi.org/10.1093/mutage/gep031  

Long, A.S., Wills, J.W., Krolak, D. et al. Benchmark dose analyses of multiple genetic toxicity endpoints permit robust, cross-tissue comparisons of MutaMouse responses to orally delivered benzo[a]pyrene. Arch Toxicol 92, 967–982 (2018). https://doi.org/10.1007/s00204-017-2099-2  

Matullo, G., Guarrera, S., Carturan, S., Peluso, M., Malaveille, C., Davico, L., Piazza, A. and Vineis, P. (2001), DNA repair gene polymorphisms, bulky DNA adducts in white blood cells and bladder cancer in a case-control study. Int. J. Cancer, 92: 562-567. https://doi.org/10.1002/ijc.1228 

Millen, A. L., Sharma, P., & Wetmore, S. D. (2012). C8-Linked Bulky Guanosine DNA Adducts: Experimental And Computational Insights Into Adduct Conformational Preferences And Resulting Mutagenicity. Future Medicinal Chemistry, 4(15), 1981–2007. https://doi.org/10.4155/fmc.12.138  

Mimmler, Maximilian, et al. “DNA Damage Response Curtails Detrimental Replication Stress and Chromosomal Instability Induced by the Dietary Carcinogen PhIP.” Nucleic Acids Research [England], vol. 44, no. 21, December 2016, pp. 10259–76, https://doi.org/10.1093/nar/gkw791.  

Mohammad Shoeb, Helen C.S. Meier, James M. Antonini, Telomeres in toxicology: Occupational health, Pharmacology & Therapeutics, Volume 220, 2021, 107742, ISSN 0163-7258, https://doi.org/10.1016/j.pharmthera.2020.107742.  

Nikolova, Teodora, et al. “The γH2AX Assay for Genotoxic and Nongenotoxic Agents: Comparison of H2AX Phosphorylation with Cell Death Response.” Toxicological Sciences [United States], vol. 140, no. 1, July 2014, pp. 103–17, https://doi.org/10.1093/toxsci/kfu066. 

Olsen AK, Andreassen Å, Singh R, Wiger R, Duale N, et al. (2010) Environmental Exposure of the Mouse Germ Line: DNA Adducts in Spermatozoa and Formation of De Novo Mutations during Spermatogenesis. PLOS ONE 5(6): e11349. https://doi.org/10.1371/journal.pone.0011349 

Paul A White, George R Douglas, David H Phillips, Volker M Arlt, Quantitative relationships between lacZ mutant frequency and DNA adduct frequency in Muta™Mouse tissues and cultured cells exposed to 3-nitrobenzanthrone, Mutagenesis, Volume 32, Issue 2, 1 March 2017, Pages 299–312, https://doi.org/10.1093/mutage/gew067  

Pavel Rossner, et al. “Genetic Toxicology and Carcinogenesis.” Toxicology for the Health and Pharmaceutical Sciences, edited by Antonio Peña-Fernández et al., 1st ed., CRC Press, 2022, pp. 81–97, https://doi.org/10.1201/9780203730584-5. 

Reardon, Joyce T., and Aziz Sancar. “Nucleotide Excision Repair.” Progress in Nucleic Acid Research and Molecular Biology, edited by Kivie Moldave, vol. 79, Elsevier Science & Technology, 2005, pp. 183–235, https://doi.org/10.1016/S0079-6603(04)79004-2.

Rossner, Pavel, et al. “Molecular Epidemiology Focused on Airborne Carcinogens.” Air Pollution and Health Effects, edited by Srikanth S. Nadadur and John W. Hollingsworth, Springer London, Limited, 2015, pp. 185–212, https://doi.org/10.1007/978-1-4471-6669-6_7. 

Schuster DM, LeBlanc DPM, Zhou G, Meier MJ, Dodge AE, White PA, Long AS, Williams A, Hobbs C, Diesing A, Smith-Roe SL, Salk JJ, Marchetti F, Yauk CL. Dose-related Mutagenic and Clastogenic Effects of Benzo[b]fluoranthene in Mouse Somatic Tissues Detected by Duplex Sequencing and the Micronucleus Assay. bioRxiv [Preprint]. 2024 Jul 26:2024.07.26.605228. doi: 10.1101/2024.07.26.605228. Update in: Environ Sci Technol. 2024 Dec 10;58(49):21450-21463. doi: 10.1021/acs.est.4c07236. PMID: 39211269; PMCID: PMC11360995.  

Thomas H. Broschard, Manfred Wiessler, Claus-Wilhelm von der Lieth, Heinz H. Schmeiser, Translesional synthesis on DNA templates containing site-specifically placed deoxyadenosine and deoxyguanosine adducts formed by the plant carcinogen aristolochic acid, Carcinogenesis, Volume 15, Issue 10, October 1994, Pages 2331–2340, https://doi.org/10.1093/carcin/15.10.2331  

L.H. Thompson, E.P. Salazar, K.W. Brookman, C.A. Hoy, Hypersensitivity to cell killing and mutation induction by chemical carcinogens in an excision repair-deficient mutant of CHO cells, Mutation Research/DNA Repair Reports, Volume 112, Issue 6, 1983, Pages 329-344, ISSN 0167-8817, https://doi.org/10.1016/0167-8817(83)90027-5.  

Tu, Lan Chun, et al. “Hedamycin, a DNA Alkylator, Induces γH2AX and Chromosome Aberrations: Involvement of Phosphatidylinositol 3-Kinase–Related Kinases and DNA Replication Fork Movement.” Molecular Cancer Therapeutics, vol. 4, no. 8, August 2005, pp. 1175–85, https://doi.org/10.1158/1535-7163.MCT-05-0054. 

Xiaoshan Liu, Jinyoung Lee, Kyunghee Ji, Shunichi Takeda, Kyungho Choi, Potentials and mechanisms of genotoxicity of six pharmaceuticals frequently detected in freshwater environment, Toxicology Letters, Volume 211, Issue 1, 2012, Pages 70-76, ISSN 0378-4274, https://doi.org/10.1016/j.toxlet.2012.03.003.  

Yuji Ishii, Shinji Takasu, Petr Grúz, Kenichi Masumura, Kumiko Ogawa, Takehiko Nohmi, Takashi Umemura, The role of DNA polymerase ζ in benzo[a]pyrene-induced mutagenesis in the mouse lung, Mutagenesis, Volume 36, Issue 2, March 2021, Pages 155–164, https://doi.org/10.1093/mutage/geab007