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Event: 1879

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Bulky DNA adducts, increase

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Bulky DNA adducts, increase

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
deoxyribonucleic acid increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Bulky DNA adducts leading to mutations MolecularInitiatingEvent Carole Yauk (send email) Under development: Not open for comment. Do not cite

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
human and other cells in culture human and other cells in culture NCBI
human Homo sapiens NCBI
mouse Mus musculus NCBI
rat Rattus norvegicus NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
All life stages

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Term Evidence
Unspecific

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

Bulky DNA adducts are formed when activated genotoxic aromatic compounds interact with the nitrogenous bases of DNA. This occurs at various sites. The most common reactive sites for these adduct is C8, N7, N3 and N2 positions of guanine, the N7, N6, N3, and N1 positions of adenine, the N3, N4, and O2 positions of cytosine, and the N3, O2, and O4 positions of thymine (As reviewed by Hwa Yun et al., 2020). The position of the adduct depends on the chemical structure of the activated aromatic compound. Some adducts are not stable, but some can persist. For example, the most harmful adducts formed by benzo(a)pyrene are from radicals that bind to the N7 and C8 of purines (IARC., 2012). Aristolochic Acid forms adducts at N6 of adenine and Aflatoxin B1 forms adducts at the N7 of Guanine (Arlt et al., 2002). This KE describes an increase in Bulky adducts. These adducts can cause depurination, transversions which in turn cause DNA damage and chromosome aberrations.

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

Quantification of Bulky DNA Adducts

  • 32P Post labelling is used for the detection of DNA adducts (for PAHs and also Aristolochic Acid) (Gupta et al., 1982; Klaene et al., 2013; Phillips and Arlt., 2014)
  • The DNA is isolated using the standard methods and digested into 3-deoxynucleoside monophosphates. 32P-orthophosphate from [gamma-32P] ATP is used to radiolabel the adducts in a reaction catalyzed by T4 polynucleotide kinase.
  • The radiolabelled nucleotides are separated and detected by thin-layer chromatography. They are quantified by scintillation counting. This is usually used to detect bulky adducts.
  • Nuclease P1 can be used for enrichment with PAH adducts. Using 1-Butanol to extract the adducted molecules before labelling is another optimization method and it works well with aromatic amines.
  • CometChip assay (modified by adding DNA synthesis inhibitors (Ngo et al.,2020)
  • This variation of the assay uses DNA synthesis inhibitors to convert bulky lesions into detectable SSBs.
  • HepaCometChip uses Hydroxyurea (HU) and 1-β-d-arabinofuranosyl cytosine (AraC) to detect SSBs formed from bulky adducts in the presence of the high metabolism of HepaRG™ cells.
  • HU inhibits the enzyme ribonucleotide reductase. This enzyme mediates the synthesis of deoxyribonucleotides (dNTPs). When it is inhibited dNTPs are depleted which inhibits NER.
  • AraC’s structure allows it to be incorporated into DNA and interrupts DNA elongation.
  • HU and AraC delay the removal of NER and SSB intermediates. The prolonged presence of NER intermediates are indicators of bulky lesions and can be observed as comet detectable SSBs.
  • The number of bulky lesions is then measured by detecting the % of DNA found in the tail of the comet compared to untreated samples. Percentage DNA in the comet tail is proportional to the level of strand breaks.

 

Other methods for adduct detection   A variety of other methods are available to measure bulky DNA adducts including Isotope dilution mass spectrometry (MS) liquid chromatography mass spectrometry (LC–MS), gas chromatography mass spectrometry (GC–MS), capillary electrophoresis mass spectrometry (CE–MS). (Long et al., 2018; Fischer et al., 2018; Chang et al., 2017; Woo et al., 2011)

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

Bulky adducts can occur in virtually any cell type or organism, as long as the organism/cell type has the xenobiotic metabolism enzymes necessary to activate pro-mutagens when required. Bulky adducts have been detected both in vitro (various cell lines) and in vivo in mammalian cells (human, mouse, rat), and can occur in males and females at any life stage.

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

A variety of stressors can induce bulky adducts. The most well studied are Benzo(a)pyrene, Aflatoxin B1 and Aristolochic acid. These compounds have been demonstrated to induce bulky adducts in various in vivo and in vitro methods (). Many studies use 32-P post labelling and other methods to observe the presence of bulky adducts when different doses of a carcinogenic compound are administered. A dose-dependent increase of bulky adducts measured by 32P post-labelling and comet assay was observed by Long et al., 2018 and Lemieux et al., 2011 from Benzo(a)pyrene doses as low as 0.20 mg/kg BW/day in mammalian cells from various tissues in vivo. AFB1-induced adducts were observed by Woo et al. (year) by isotope dilution mass spectrometry (MS) in mice. A dose-dependent increase in Aristolochic Acid induced adducts have been demonstrated by 32-P post labelling in in rodent models (Mei et al., 2006 and McDaniel et al., 2012). The authors noted an increase in adducts from a dose as low as 0.1 mg/kg bodyweight (Li et al., 2020).

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help

Arlt VM, Stiborova M, Schmeiser HH. Mutagenesis. 2002; 17:265–277.

Barnes, J. L., Zubair, M., John, K., Poirier, M. C., & Martin, F. L. (2018). Carcinogens and DNA damage. Biochemical Society transactions46(5), 1213–1224. https://doi.org/10.1042/BST20180519

Grollman, A. P., Shibutani, S., Moriya, M., Miller, F., Wu, L., Moll, U., Suzuki, N., Fernandes, A., Rosenquist, T., Medverec, Z., Jakovina, K., Brdar, B., Slade, N., Turesky, R. J., Goodenough, A. K., Rieger, R., Vukelić, M., & Jelaković, B. (2007). Aristolochic acid and the etiology of endemic (Balkan) nephropathy. Proceedings of the National Academy of Sciences of the United States of America104(29), 12129–12134. https://doi.org/10.1073/pnas.0701248104

Groopman, J. D., Croy, R. G., & Wogan, G. N. (1981). In vitro reactions of aflatoxin B1-adducted DNA. Proceedings of the National Academy of Sciences78(9), 5445-5449.

Gupta, R. C., Reddy, M. V., & Randerath, K. (1982). 32 P-postlabeling analysis of non-radioactive aromatic carcinogen—DNA adducts. Carcinogenesis3(9), 1081-1092.

Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011 Mar 4;144(5):646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230.

Hwa Yun, B., Guo, J., Bellamri, M., & Turesky, R. J. (2020). DNA adducts: Formation, biological effects, and new biospecimens for mass spectrometric measurements in humans. Mass spectrometry reviews39(1-2), 55-82.

IARC Some non-heterocyclic polycyclic aromatic hydrocarbons and some related exposures. IARC Monogr Eval Carcinog Risks Hum. 2010;92:1–853. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4781319/

IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Chemical Agents and Related Occupations. Lyon (FR): International Agency for Research on Cancer; 2012. (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, No. 100F.) BENZO[a]PYRENE. Available from: https://www.ncbi.nlm.nih.gov/books/NBK304415/

Jessica L. Barnes, Maria Zubair, Kaarthik John, Miriam C. Poirier, Francis L. Martin; Carcinogens and DNA damage. Biochem Soc Trans 19 October 2018; 46 (5): 1213–1224. doi: https://doi.org/10.1042/BST20180519

Li, X. L., Guo, X. Q., Wang, H. R., Chen, T., & Mei, N. (2020). Aristolochic Acid-Induced Genotoxicity and Toxicogenomic Changes in Rodents. World journal of traditional Chinese medicine, 6(1), 12–25. https://doi.org/10.4103/wjtcm.wjtcm_33_19

McDaniel, L. P., Elander, E. R., Guo, X., Chen, T., Arlt, V. M., & Mei, N. (2012). Mutagenicity and DNA adduct formation by aristolochic acid in the spleen of Big Blue® rats. Environmental and molecular mutagenesis, 53(5), 358-368.

Ngo, L. P., Owiti, N. A., Swartz, C., Winters, J., Su, Y., Ge, J., Xiong, A., Han, J., Recio, L., Samson, L. D., & Engelward, B. P. (2020). Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates. Nucleic acids research48(3), e13. https://doi.org/10.1093/nar/gkz1077

Phillips, D. H., & Arlt, V. M. (2014). 32 P-Postlabeling Analysis of DNA Adducts. In Molecular Toxicology Protocols (pp. 127-138). Humana Press, Totowa, NJ.

Yun, B. H., Sidorenko, V. S., Rosenquist, T. A., Dickman, K. G., Grollman, A. P., & Turesky, R. J. (2015). New approaches for biomonitoring exposure to the human carcinogen aristolochic acid. Toxicology research4(4), 763-776.