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Event: 244

Key Event Title

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

Alkylation, Protein

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. More help
Alkylation, Protein
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Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. More help
Level of Biological Organization

Cell term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help
Cell term
eukaryotic cell

Organ term

The location/biological environment in which the event takes place.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.  Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

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; 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
protein alkylation 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
Protein Alkylation to Liver Fibrosis MolecularInitiatingEvent Brigitte Landesmann (send email) Open for citation & comment WPHA/WNT Endorsed
Renal protein alkylation leading to kidney toxicity MolecularInitiatingEvent Angela Mally (send email) Not under active development 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 Evidence Link
human Homo sapiens High NCBI
Rattus norvegicus Rattus norvegicus High NCBI
mouse Mus musculus High NCBI

Life Stages

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

Sex Applicability

An indication of the the relevant sex for this KE. More help

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

Alkylation is the transfer of an alkyl group from one molecule to another. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion or a carbene (or their equivalents). Protein alkylation is the addition of an alkyl group to a protein amino acid. An alkyl group is any group derived from an alkane by removal of one hydrogen atom. Alkylating agents are highly reactive chemicals that introduce alkyl groups into biologically active molecules and thereby prevent their proper functioning. Alkylating agents are classified according to their nucleophilic or electrophilic character. Nucleophilic alkylating agents deliver the equivalent of an alkyl anion (carbanion). These compounds typically can add to an electron-deficient carbon atom such as at a carbonyl group. Electrophilic alkylating agents deliver the equivalent of an alkyl cation. Alkyl halides can also react directly with amines to form C-N bonds; the same holds true for other nucleophiles such as alcohols, carboxylic acids, thiols, etc. Alkylation with only one carbon is termed methylation. [1] [2]

Covalent protein alkylation by reactive electrophiles was identified as a key triggering event in chemical toxicity over 40 years ago and these reactions remain a major cause of chemical-induced toxicity. Interestingly, some chemical molecules produce significant protein covalent binding without causing toxicity, which suggests that only a critical subset of protein alkylation events contributes to injury. The study by Codreanu et al. (2014) describes an inventory of electrophile- mediated protein damage in intact cells and suggests that non-toxic covalent binding may largely be survivable damage to cytoskeletal components, whereas toxic covalent binding produces lethal injury by targeting protein synthesis and catabolism and possibly mitochondrial electron transport. [3] [4] [5] [6] [7]

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

HPLC-ESI-MS/MS analysis

High Performance Liquid Chromatography – electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) is the most popular MS technique. It combines the separation ability of HPLC along with the sensitivity and specificity of detection from MS. One of the advantages of HPLC-MS is that it allows samples to be rapidly desalted online, so no sample preparation is required unlike samples for GC-MS. Electrospray ionisation can produce singly or multiply charged ions. Typically high molecular weight compounds have multiple charges i.e. peptides and proteins. This technique is particularly suited to analysing polar molecules of mass <2000 Dalton and requires no prior derivatisation in most applications. [8] [3] [9]

MALDI-TOF/MS (Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry)

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. MALDI methodology is a three-step process. First, the sample is mixed with a suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized by being protonated or deprotonated in the hot plume of ablated gases, and can then be accelerated into whichever mass spectrometer is used to analyse them. [10]

Domain of Applicability

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

Human, rat and mouse [11]


List of the literature that was cited for this KE description. More help
  1. The European Bioinformatics Institute (accessed on 20 January 2016).
  2. NLM, Medical Subject Headings, National Library of Medicine, (accessed on 20 January 2016).
  3. 3.0 3.1 3.2 Codreanu, S.G. et al. (2014), Alkylation damage by lipid electrophiles targets functional protein systems, Molecular & Cellular Proteomics, vol. 13, no. 3, pp.849–859.
  4. Grattagliano, I. et al. (2009), Biochemical mechanisms in drug-induced liver injury: certainties and doubts, World J Gastroenterol, vol. 15, no. 39, pp. 4865-4876.
  5. Livertox
  6. 6.0 6.1 Kehrer, J.P. and S. Biswal (2000), The Molecular Effects of Acrolein, Toxicol. Sciences,vol.57,pp.6-15.
  7. Schopfer, F.J., C. Cipollina and B.A. Freeman (2011), Formation and Signaling Actions of Electrophilic Lipids, Chem Rev, vol. 111, no. 10,pp.5997–6021.
  8. Zhang F et al. (2005), Differential adduction of proteins vs. deoxynucleosides by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro, Mass Spectrom, vol 19, no. 4, pp. 438–448.
  9. Gundry, R.L. et al. (2009), Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow, Curr Protoc Mol Biol, chapter 10, section VI, unit 10.25, pp. 1-23.
  10. Kislinger, T. et al. (2005), Analysis of protein glycation products by MALDI-TOF/MS, Ann N Y Acad Sci, vol. 1043, pp. 249-259.
  11. 11.0 11.1 EPA Toxicological review of Carbon Tetrachloride (CAS No. 56-23-5). March 2010 EPA/635/R-08/005F available at: (accessed 24.10.2015)
  12. Auerbach, S.S. et al. (2008), A comparative 90 day toxicity study of allyl acetate, allyl alcohol and acrolein, Toxicology, Vol. 253, No.1-3, pp.79–88.
  13. Huang, L. et al. (2008), Genes related to apoptosis predict necrosis of the liver as a phenotype observed in rats exposed to a compendium of hepatotoxicants, BMC Genomics, vol. 9: 288.
  14. Mohammad, M.K. et al. (2012), Acrolein cytotoxicity in hepatocytes involves endoplasmic reticulum stress, mitochondrial dysfunction and oxidative stress, Toxicol Appl Pharmacol, vol. 265, no. 1, pp. 73-82.
  15. Yamada T et al., (2013), A category approach to predicting the repeated-dose hepatotoxicity of allyl esters, Regulatory Toxicology and Pharmacology, vol. 65, no. 2, pp. 189–195.
  16. Basu, S. (2003), Carbon tetrachloride-induced lipid peroxidation: eicosanoid formation and their regulation by antioxidant nutrients, Toxicology,vol.189, no.1-2, pp. 113-127.
  17. Calabrese, E.J., L.A. Baldwin and H.M. Mehendale (1993), G2 subpopulation in rat liver induced into mitosis by low-level exposure to carbon tetrachloride: an adaptive response, Toxicol Appl Pharmacol, vol. 121. no. 1, pp. 1-7.
  18. Clawson, G.A. (1989), Mechanisms of carbon tetrachloride hepatotoxicity, Pathol Immunopathol Res, vol. 8, no. 2, pp.104-112.
  19. Knockaert, L. et al. (2012), Carbon tetrachloride-mediated lipid peroxidation induces early mitochondrial alterations in mouse liver, Lab Invest, vol. 92, no. 3, pp. 396-410.
  20. Lee Kwang-Jong et al. (2004), Induction of molecular chaperones in carbon tetrachloride-treated rat liver: implications in protection against liver damage, Cell Stress Chaperones, vol. 9, no. 1, pp. 58-68.
  21. Li, Xiaowei et al. (2014), NMR-based metabonomic and quantitative real-time PCR in the profiling of metabolic changes in carbon tetrachloride-induced rat liver injury, J Pharm Biomed Anal; vol. 89, pp.42-49.
  22. Manibusan, M.K., M. Odin and D.A. Eastmond (2007), Postulated carbon tetrachloride mode of action: a review, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev, vol. 25, no. 3,pp. 185-209.
  23. Masuda, Y. (2006), [Learning toxicology from carbon tetrachloride-induced hepatotoxicity],Yakugaku Zasshi, vol. 126, no. 10, pp. 885-899.
  24. Nagano, K. et al. (2007), Inhalation carcinogenicity and chronic toxicity of carbon tetrachloride in rats and mice, Inhal Toxicol, vol 19, no. 13, pp. 1089-1103.
  25. Recknagel, R.O. (1976), Carbon tetrachloride hepatotoxicity, Pharmacol Rev, vol. 19, no. 2,pp.145-208.
  26. Weber, L.W., M. Boll and A. Stampfl (2003), Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model, Crit Rev Toxicol, vol. 33, no. 2, pp. 105-136.
  27. Bauman, J.N. et al. (2009), Can in vitro metabolism-dependent covalent binding data distinguish hepatotoxic from nonhepatotoxic drugs? An analysis using human hepatocytes and liver S-9 fraction, Chem Res Toxicol, vol. 22, no. 2, pp. 332-340.
  28. Chen, Q. et al. (1990), The mechanism of cysteine conjugate cytotoxicity in renal epithelial cells. Covalent binding leads to thiol depletion and lipid peroxidation, J Biol Chem, vol. 265, no. 35, pp. 21603-21611.
  29. Dennehy, M.K. et al. (2006), Cytosolic and nuclear protein targets of thiol-reactive electrophiles, Chem Res Toxicol, vol. 19, no. 1, pp. 20-29.
  30. Shin, N.Y. et al. (2007), Protein targets of reactive electrophiles in human liver microsomes, Chem Res Toxicol, vol. 20, no. 6, pp. 859-867.