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 adapted 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. 

A substantial portion of the documentation and supporting references for this AOP was derived 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. Searches combined terms representing upstream and downstream KEs, including bulky adduct and chromosome; bulky adducts and chromosomal aberrations; bulky adducts and mutations; inadequate repair and mutation; and inadequate repair and DNA strand breaks. 

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.