Key Event Title
|Level of Biological Organization|
Key Event Components
|DNA alkylation||deoxyribonucleic acid||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Alkylation of DNA leading to heritable mutations||MolecularInitiatingEvent|
|DNA alkylation -> cancer 1||MolecularInitiatingEvent|
|DNA alkylation -> cancer 2||MolecularInitiatingEvent|
|Syrian golden hamster||Mesocricetus auratus||High||NCBI|
|Homo sapiens||Homo sapiens||High||NCBI|
Key Event Description
The event involves DNA alkylation to form a variety of different DNA adducts (i.e., alkylated nucleotides). Alkylation occurs at various sites in DNA and can include alkylation of adenine- Nl, - N3, - N7, guanine- N3, - O6, - N7, thymine-O2, - N3, - O4, cytosine- O2, -N3, and the phosphate (diester) group (reviewed in detail in Beranek 1990). In addition, alkylation can involve modification with different sizes of alkylation groups (e.g., methyl, ethyl, propyl). It should be noted that many of these adducts are not stable or are readily repaired (discussed in more detail below). A small proportion of adducts are stable and remain bound to DNA for long periods of time.
How It Is Measured or Detected
There is no OECD guideline for measurement of alkylated DNA, although technologies for their detection are established. Reviews of modern methods to measure DNA adducts include Himmelstein et al,. 2009 and Philips et al., 2000.
High performance liquid chromatography (HPLC) methods can be used to measure whether an agent is capable of alkylating DNA in somatic cells. Alkyl adducts in somatic cells can be measured using immunological methods (described in Nehls et al. 1984), as well as HPLC (methods in de Groot et al. 1994) or a combination of 32P post-labeling, HPLC and immunologic detection (Kang et al. 1992). We note that mass spectrometry provides structural specificity and confirmation of the structure of DNA adducts.
DNA alkylation can also be measured using a modified comet assay. This method involves the digestion of alkylated DNA bases with 3–methyladenine DNA glycosylase (Collins et al., 2001; Hasplova et al., 2012) followed by the standard comet assay to detect where alkyl adducts occur. The advantage of this method is that the alkaline version of the comet assay, as a core method, has an in vivo OECD guideline.
Finally, structure-activity relationships (SARs) have been developed to predict the possibility that a chemical will alkylate DNA (e.g., Vogel and Ashby, 1994; Benigni, 2005; Dai et al., 1989; Lewis and Griffith, 1987).
Measurement of alkylation in male germ cells:
In rodent testes, studies have detected adducts in situ by immuhistocytological staining. For example, fixed testes are incubated with O6-EtGua -specific mouse monoclonal antibody and subsequently with a labeled anti-mouse IgG F antibody. Nuclear DNA is counterstained with DAPI 4,6-diamidino- 2-phenylindole. Fluorescence signals from immunostained O6-EtGua residues in DNA are visualized by fluorescence microscopy and quantitated using an image analysis system. Methods are described in (Seiler et al. 1997). An immunoslot blot assay for detection of O6-EtGua has been described previously in (Mientjes et al. 1996).
Alternatively, rodents have also been exposed to radio-labeled alkylating agents. Examples from the literature include [2-3H] ENU, [1-3H]di-ethyl sulfate, or [1-3H]ethyl-methane sulfonate. Following treatment with the labeled chemical, testis and other tissues of interest are removed. Germ cells are released from tubuli by pushing out the contents with forceps. Using this procedure all germ-cell stages are liberated from the tubuli, with the possible exception of part of the population of stem-cell spermatogonia that remain attached to the walls of the tubuli. DNA is then extracted from germ cells, empty testis tubuli and other tissues of interest. DNA adduct formation is determined after neutral and acid hydrolysis of DNA followed by separation of the various ethylation products using HPLC (described in van Zeeland et al. 1990).
Domain of Applicability
Alkylated DNA has been measured in somatic cells in a variety of species, from prokaryotic organisms, to rodents in vivo, to human cells in culture. Theoretically, DNA alkylation can occur in any cell type in any organism.
Evidence for Perturbation by Stressor
Overview for Molecular Initiating Event
Alkylating agents are prototypical DNA-reactive compounds and have been extensively studied for decades (reviewed in Beranek 1990). The chemicals can be direct-acting electrophiles, or can be converted from non-reactive substances to reactive metabolites via metabolism. A prototypical alkylating agent is N-ethyl-N-nitrosourea (chemical formula C3H7N3O2) (ENU). ENU is rapidly absorbed following oral exposure and intraperitoneal injections and distributed widely across the tissues. ENU is unstable and readily reacts with somatic and germ cell DNA in mice, rats, flies and hamsters, to alkylate DNA. Very generally, mono-functional (referring to the transfer of a single alkyl group) alkylating agents include: 1. Alkyl sulfates: e.g., diethyl (DES) and dimethyl sulfate (DMS); 2. Alkyl alkanesulfonates: e.g., methyl (MMS) and ethyl methanesulfonate (EMS); 3. Nitrosamides: e.g., methyl (MNU) and ethyl nitrosourea (ENU), methyl- (MNNG) and ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and the indirect-acting (i.e., requiring metabolic activation) dimethyl (DMN) and diethyl nitrosamines (DEN).
ENU is the most widely studied and understood alkylating agent and as such has been instrumental in contributing to the knowledgebase in this field. Immunohistochemistry studies clearly indicate the presence of alkylated DNA following exposure to ENU in both somatic cells and spermatogonia (Kamino et al. 1995; Seiler et al. 1997; van Zeeland et al. 1990).
Benigni, R. (2005), "Structure-activity relationship studies of chemical mutagens and carcinogens: mechanistic investigations and prediction and approaches", Chem. Rev., 105: 1767-1800.
Beranek, D.T. (1990), "Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents", Mutation Res., 231: 11-30.
Collins, A.R., M. Dusinská and A. Horská (2001), "A Detection of alkylation damage in human lymphocyte DNA with the comet assay". Acta Biochim Pol., 48: 611-4.
Dai, Q.H. and R.G. Zhong (1989), "Quantitative pattern recognition for structure-carcinogenic activity relationship of N-nitroso compounds based upon Di-region theory", Sci China B., 32:776-790.
de Groot, A.J., J.G. Jansen, C.F. van Valkenburg and A.A. van Zeeland (1994), "Molecular dosimetry of 7-alkyl- and O6-alkylguanine in DNA by electrochemical detection", Mutat Res., 307: 61-6.
Hašplová, K., A. Hudecová, Z. Magdolénová, M. Bjøras, E. Gálová, E. Miadoková and M. Dušinská (2012), "DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD)", Toxicol Lett., 208: 76-81.
Himmelstein, M.W., P.J. Boogaard, J. Cadet, P.B. Farmer, J.J. Kim, E.A. Martin, R. Persaud and D.E. Shuker (2009), "Creating context for the use of DNA adduct data in cancer risk assessment: II. Overview of methods of identification and quantitation of DNA damage", Crit. Rev. Toxicol., 39: 679-94.
Kamino, K., F. Seiler, M. Emura, J. Thomale, M.F. Rajewsky and U. Mohr (1995), "Formation of O6-ethylguanine in spermatogonial DNA of adult Syrian golden hamster by intraperitoneal injection of diethylnitrosamine", Exp. Toxicol. Pathol., 47: 443-445.
Kang, H.I., C. Konishi, G. Eberle, M.F. Rajewsky, T. Kuroki and N.H. Huh (1992), "Highly sensitive, specific detection of O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of high-performance liquid chromatography prefractionation, 32P postlabeling, and immunoprecipitation", Cancer Res., 52: 5307-5312.
Lewis, D.F. and V.S. Griffiths (1987), "Molecular electrostatic potential energies and methylation of DNA bases: a molecular orbital-generated quantitative structure-activity relationship", Xenobiotica, 17: 769-776.
Mientjes, E.J., K. Hochleitner, A. Luiten-Schuite, J.H. van Delft, J. Thomale, F. Berends, M.F. Rajewsky, P.H. Lohman and R.A. Baan (1996), "Formation and persistence of O6-ethylguanine in genomic and transgene DNA in liver and brain of lambda(lacZ) transgenic mice treated with N-ethyl-N-nitrosourea", Carcinogenesis, 17: 2449-2454.
Nehls, P., M.F. Rajewsky, E. Spiess, D. Werner (1984), "Highly sensitive sites for guanine-O6 ethylation in rat brain DNA exposed to N-ethyl-N-nitrosourea in vivo", EMBO J., 3:327-332.
Phillips, D.H., P.B. Farmer, F.A. Beland, R.G. Nath, M.C. Poirier, M.V. Reddy and K.W. Turteltaub (2000), "Methods of DNA adduct determination and their application to testing compounds for genotoxicity", Environ. Mol. Mutagen., 35: 222-233.
Scherer, E., A.A. Jenner and L. den Engelse (1987), "Immunocytochemical studies on the formation and repair of O6-alkylguanine in rat tissues", IARC Sci. Publ., 84: 55-58.
Sega, G.A., C.R. Rohrer, H.R. Harvey and A.E. Jetton (1986), "Chemical dosimetry of ethyl nitrosourea in the mouse testis", Mutat. Res., 159: 65-74.
Seiler, F., K. Kamino, M. Emura, U. Mohr and J. Thomale (1997), "Formation and persistence of the miscoding DNA alkylation product O6-ethylguanine in male germ cells of the hamster", Mutat. Res., 385: 205-211.
van Zeeland, A.A., A. de Groot and A. Neuhäuser-Klaus (1990), "DNA adduct formation in mouse testis by ethylating agents: a comparison with germ-cell mutagenesis", Mutat. Res. 231: 55-62.
Vogel, E.W., Ashby, J. (1994), "Structure-activity relationships: experimental approaches." In: Methods to asses DNA Damage and repair: Interspecies comparisons. Edited by R.T. Tardiff, P.H.M. Lohman and G.N. Wogan, SCOPE, Wiley and Sons LTD.