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Event Title

Protein, Alkylation
Short name: Protein, Alkylation

Key Event Overview

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AOPs Including This Key Event

AOP Name Event Type Essentiality
Protein Alkylation leading to Liver Fibrosis MIE Strong

Chemical Initiators

The following are chemical initiators that operate directly through this Event:

  1. Allyl Alcohol
  2. Carbon tetrachloride
  3. Retinol
  4. Dimethyl nitrosamine
  5. Thioacetamide

Taxonomic Applicability

Name Scientific Name Evidence Links
human Homo sapiens Strong NCBI
Rattus norvegicus Rattus norvegicus Strong NCBI
mouse Mus musculus Strong NCBI

Level of Biological Organization

Biological Organization

How this Key Event works

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

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]

Evidence Supporting Taxonomic Applicability

Human, rat and mouse [11]

Evidence for Chemical Initiation of this Molecular Initiating Event

Two prototypical chemicals acting via protein alkylation are Allyl Alcohol [12][13][6][14][15] and Carbon Tetrachloride (CCl4)[11][16] [17] [18] [19][20][21][22] . [23] [24] .[25] [26]

Covalent protein alkylation is a feature of many cytotoxic drugs but the overall extent of binding does not adequately distinguish toxic from non-toxic binding. [27] Interestingly, some chemicals significantly alkylate proteins without causing toxicity, which suggests that only alkylation of a specific protein subset critical subset contributes to injury. Indeed, Codreanu presented an inventory of proteins affected by electrophile-mediated alkylation in intact cells and suggested that non-toxic covalent binding largely affects cytoskeletal protein components, whereas toxic covalent binding induces lethal injury by targeting factors involved in protein synthesis and catabolism and possibly mitochondrial electron transport. [3] In vitro covalent binding studies to macromolecules have been used to elucidate the biochemical mechanisms of chemical-induced toxicity. Experimental work with kidney epithelial cells by Chen et al suggested that following alkylation of cellular macromolecules as initial cytotoxic event both sulfhydryl depletion and lipid peroxidation are components of the cytotoxic mechanism [28] Dennehy et al have analyzed the protein targets in nuclear and cytoplasmic proteomes from human embryonic kidney cells (HEK293) treated in vitro with two biotin-tagged, thiol-reactive electrophiles and mapped the adducts. Certain protein families appeared particularly susceptible to alkylation. [29] Shin et al have identified protein targets of two biotin-tagged model electrophiles in human liver microsomes through LC-MS-MS and showed that different target selectivities of the two electrophile probes correlated with different biological outcomes and that alkylation reactions of specific targets could be quantified. [30]


  1. The European Bioinformatics Institute http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0008213 (accessed on 20 January 2016).
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  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 http://livertox.nlm.nih.gov/AlkylatingAgents.htm
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  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: http://cfpub.epa.gov/ncea/iris/iris_documents/documents/toxreviews/0020tr.pdf (accessed 24.10.2015)
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  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.