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Key Event Title
Bulky DNA adducts, increase
Key Event Components
Key Event Overview
AOPs Including This Key Event
|All life stages|
Key Event Description
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
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)
Domain of Applicability
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
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).
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.
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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.