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Event: 1879

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Bulky DNA adducts, increase

Short name

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Biological Context

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Level of Biological Organization
Molecular

Cell term

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Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). 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 signaling 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. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Process	Object	Action
	deoxyribonucleic acid	increased

Key Event Overview

AOPs Including This Key Event

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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 Do not cite	Under Development

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.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	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

An indication of the the relevant life stage(s) for this KE. More help

Life stage	Evidence
All life stages

Sex Applicability

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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. 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

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.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). Do not provide detailed protocols. More help

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. A 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)

https://aopwiki.org/events/373

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). 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.

References

List of the literature that was cited for this KE description. 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 transactions, 46(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 America, 104(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 Sciences, 78(9), 5445-5449.

Gupta, R. C., Reddy, M. V., & Randerath, K. (1982). 32 P-postlabeling analysis of non-radioactive aromatic carcinogen—DNA adducts. Carcinogenesis, 3(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 reviews, 39(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 research, 48(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 research, 4(4), 763-776.