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Event: 244

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. 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. The short name should be less than 80 characters in length. More help
Alkylation, Protein

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.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. More help
Cell term
eukaryotic cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.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. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). 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 signalling 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. 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 TFHA/WNT Endorsed
Renal protein alkylation leading to kidney toxicity MolecularInitiatingEvent Angela Mally (send email) Not under active development Under Development

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. 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

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help

Sex Applicability

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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. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. 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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. 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).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

Human, rat and mouse [11]

Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event

When a specific MIE can be defined (i.e., the molecular target and nature of interaction is known), in addition to describing the biological state associated with the MIE, how it can be measured, and its taxonomic, life stage, and sex applicability, it is useful to list stressors known to trigger the MIE and provide evidence supporting that initiation. This will often be a list of prototypical compounds demonstrated to interact with the target molecule in the manner detailed in the MIE description to initiate a given pathway (e.g., 2,3,7,8-TCDD as a prototypical AhR agonist; 17α-ethynyl estradiol as a prototypical ER agonist). Depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). Known stressors should be included in the MIE description, but it is not expected to include a comprehensive list. Rather initially, stressors identified will be exemplary and the stressor list will be expanded over time. For more information on MIE, please see pages 32-33 in the User Handbook.

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]

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help
  1. The European Bioinformatics Institute http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0008213 (accessed on 20 January 2016).
  2. NLM, Medical Subject Headings, National Library of Medicine, http://www.nlm.nih.gov/cgi/mesh/2011/MB_cgi?mode=&term=Alkylating+agents (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 http://livertox.nlm.nih.gov/AlkylatingAgents.htm
  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: http://cfpub.epa.gov/ncea/iris/iris_documents/documents/toxreviews/0020tr.pdf (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.