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Relationship: 269


A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Alkylation, Protein leads to Cell injury/death

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Downstream event
The responding Key Event (KE) in a Key Event Relationship (KER). More help

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name Adjacency Weight of Evidence Quantitative Understanding Point of Contact Author Status OECD Status
Protein Alkylation leading to Liver Fibrosis adjacent Moderate Brigitte Landesmann (send email) Open for citation & comment WPHA/WNT Endorsed

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 KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER.  More help
Term Scientific Term Evidence Link
human Homo sapiens High NCBI
Rattus norvegicus Rattus norvegicus High NCBI

Sex Applicability

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

Life Stage Applicability

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

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Alkylating agents are highly reactive chemicals that may produce cellular damage by covalently binding to cellular macromolecules to form adducts and thereby preventing their proper functioning. Covalent protein alkylation by reactive electrophiles was identified as a key triggering event in chemical toxicity; it disturbs the cellular redox balance - contributing also to the development of oxidative stress - through interaction with glutathione, which leads to disruption of multiple biochemical pathways in exposed cells and is associated with mitochondrial dysfunction, which in turn, can trigger the death of exposed cells via either apoptosis and/or necrosis. [1][2][3][4][5]

For example, Acrolein, the metabolite of Allyl Alcohol is a highly reactive electrophilic aldehyde and rapidly binds to cellular nucleophiles like glutathione. Thiol redox balance is critical for numerous cell functions Acrolein has been identified as both a product and initiator of lipid peroxidation. [6] The high toxic potential of Acrolein reflects its possession of two strongly electrophilic centres which ensure it readily reacts with nucleophilic groups on biological molecules including glutathione and proteins. These reactions typically proceed via Michael addition of nucleophiles to the a,b-unsaturated bond of Acrolein, generating carbonyl-retaining adducts with the ability to undergo further crosslinking. Reaction of the carbonyl group in the first instance to form Schiff base adducts is typically much less preferred. Adduction of a diverse range of targets, in addition to disruption of the cellular redox balance, appears to underlie the disruption of multiple biochemical pathways in Acrolein-exposed cells. Such events can trigger the death of exposed cells via either apoptosis and/or necrosis. [7]

It has been suggested that the alkylation of nucleophilic groups of cellular macromolecules effected by Acrolein after glutathione depletion is the event actually leading to cell injury.[8]

Another example for an alkylating agent is Carbon Tetrachloride (CCl4), for which consensus has emerged that its toxicity is a mutifactorial process involving the generation of CCl4-derived free radicals, lipid peroxidation, covalent binding to macromolecules, loss of calcium homeostasis, nucleic acid hypomethylation and inflammatory cytokines. CCl4-derived free radicals are highly reactive species that are able to alkylate proteins and nucleic acids to generate CCl4-derived adducts. [9]

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER.  For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings.  Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help
Biological Plausibility
Addresses the biological rationale for a connection between KEupstream and KEdownstream.  This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured.   More help

Cell injury caused by covalent binding is biologically plausible. The mechanistic relationship between MIE and KE 1 consistent with established biological knowledge. [10] [11][6]

Uncertainties and Inconsistencies
Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Though covalent protein alkylation by reactive electrophiles was identified as a key triggering event in chemical toxicity already over 40 years ago and despite the intense effort expended over the past few years, our understanding of the mechanism and consequences of protein modification by reactive intermediates – both oxidizing and alkylating agents - is still quite limited. Covalent protein alkylation is a feature of many hepatotoxic drugs and the overall extent of binding does not adequately distinguish toxic from non-toxic binding. Directly relating covalent binding to hepatotoxicity is likely an oversimplification of the process whereby adduct formation ultimately leads to toxicity. Understanding underlying complexities (e.g., which macromolecules are important covalent binding targets) will be essential to any understanding of the problem of metabolism-dependent hepatotoxicity and predicting toxicity from in vitro experiments. [30][31] Data from Codreanu et al. suggest that non-toxic covalent binding may largely be survivable damage to cytoskeletal components and other highly reactive protein targets, whereas toxic covalent binding produces lethal injury by targeting protein synthesis and catabolism and possibly mitochondrial electron transport. Future studies with appropriate probe molecules for toxic and non-toxic drugs could test these hypotheses and provide a better mechanistic basis for interpreting protein alkylation in drugsafety evaluation [10]

For this AOP it is not known whether protein alkylation to certain proteins is required and whether particular proteins and various binding sites influence the further downstream process. Further we do not know whether there is a threshold and if this threshold would refer to the number of alkylation of a single protein or of a threshold number of proteins.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references.  More help
Response-response Relationship
Provides sources of data that define the response-response relationships between the KEs.  More help
Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help
Known Feedforward/Feedback loops influencing this KER
Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

human:[23] rat:[29]


List of the literature that was cited for this KER description. More help
  1. Tanel, A. et al. (2007), Activation of the death receptor pathway of apoptosis by the aldehyde Acrolein, Free Radic Biol Med, vol. 42, no. 6, pp. 798-810.
  2. 2.0 2.1 Boll, M. et al. (2001), Pathogenesis of Carbon Tetrachloride-induced hepatocyte injury bioactivation of CCI4 by cytochrome P450 and effects on lipid homeostasis. Z Naturforsch C, vol. 56, no. 1-2, pp. 111-121.
  3. 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.
  4. Rudolph, T.K. and B.A. Freeman (2009), Transduction of redox signaling by electrophile-protein reactions, Sci Signal, vol. 2, no. 90, re7.
  5. Schopfer, F.J. et al. (2011), Formation and Signaling Actions of Electrophilic Lipids, Chem Rev, vol. 111, no. 10, pp. 5997–6021.
  6. 6.0 6.1 Kehrer, J.P. and S. Biswal (2000), The Molecular Effects of Acrolein, Toxicol. Sciences, vol. 57, no. 1, pp. 6-15.
  7. 7.0 7.1 Thompson, C.A. and P.C. Burcham (2008), Protein alkylation, transcriptional responses and cytochrome c release during Acrolein toxicity in A549 cells: influence of nucleophilic culture media constituents, Toxicol In Vitro, vol. 22, no. 4, pp. 844-853.
  8. Pompella, A. et al. (1991), Loss of membrane protein thiols and lipid peroxidation in allyl alcohol hepatotoxicity, Biochemical Pharmacology, vol. 41, no. 8, pp. 255-1259.
  9. 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.
  10. 10.0 10.1 10.2 10.3 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.
  11. Liebler, D.C. (2008), Protein Damage by Reactive Electrophiles: Targets and Consequences, Chem Res Toxicol, vol. 21, no. 1, pp. 117-128.
  12. Golla, U., G. Bandi and R.S.Tomar (2015), Molecular cytotoxicity mechanisms of allyl alcohol (acrolein) in budding yeast, Chem Res Toxicol. vol. 28, no. 6, pp.1246-1264.
  13. Nardini, M. et al. (2002),Acrolein-induced cytotoxicity in cultured human bronchial epithelial cells. Modulation by alpha-tocopherol and ascorbic acid, Toxicology, vol.170, no. 3, pp. 173-185.
  14. Randall, M.J., M. Hristova and A. van der Vliet (2013), Protein alkylation by the α,β-unsaturated aldehyde acrolein. A reversible mechanism of electrophile signaling? FEBS Lett, vol. 587, no. 23, pp. 3808-3814.
  15. Reddy, S. et al. (2002), Identification of glutathione modifications by cigarette smoke, Free Radic Biol Med, vol. 33, no.11, pp. 1490-1498.
  16. Moghe, A. et al. (2015), Molecular mechanisms of acrolein toxicity: relevance to human disease, Toxicol Sci, vol. 143, no. 2, pp. 242-255.
  17. Dong, L. et al. (2013), Magnolol protects against oxidative stress-mediated neural cell damage by modulating mitochondrial dysfunction and PI3K/Akt signaling, J Mol Neurosci, vol. 50, no. 3, pp. 469-481.
  18. Roy, J. et al. (2009), Acrolein induces a cellular stress response and triggers mitochondrial apoptosis in A549 cells, Chem Biol Interact, vol. 181, no. 2, pp. 154-167.
  19. Tanel, A. and D.A. Averill-Bates (2005), The aldehyde acrolein induces apoptosis via activation of the mitochondrial pathway, Biochim Biophys Acta, vol. 1743, no. 3, pp. 255-267.
  20. Kern, J.C. and J.P. Kehrer (2002), Acrolein-induced cell death: a caspase-influenced decision between apoptosis and oncosis/necrosis, Chem Biol Interact, vol.139, no.1, pp.79-95.
  21. Hristova, M. et al. (2012), The tobacco smoke component, acrolein, suppresses innate macrophage responses by direct alkylation of c-Jun N-terminal kinase, Am J Respir Cell Mol Biol, vol. 46, no. 1, pp. 23-33.
  22. 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.
  23. 23.0 23.1 23.2 Schwend, T. et al. (2008), Alkylation of adenosine deaminase and thioredoxin by acrylamide in human cell cultures, Z Naturforsch, vol. 64, no. 5-6, pp. 447-453.
  24. Cai, Y. et al. (2005), Apoptosis initiated by carbon tetrachloride in mitochondria of rat primary cultured hepatocytes, Acta Pharmacol Sin, vol. 26, no. 8, pp. 969-975.
  25. Perrissoud, D. et al. (1980), The effect of carbon tetrachloride on isolated rat hepatocytes,Virchows Archiv B, vol. 35, no 1, pp.83-91.
  26. Johnston, D.E. and C. Kroening, Mechanism of early carbon tetrachloride toxicity in cultured rat hepatocytes, Pharmacol Toxicol. vol. 83, no.6, pp. 231-239.
  27. Boll, M. et al., 2001, Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites, Z Naturforsch C, vol. 56, no. 7-8, pp. 649-659.
  28. Zwilling, R. and C. Balduini (eds) (1992), Biology of Aging, vol. 1, Springer Berlin Heidelberg.
  29. 29.0 29.1 Boot, J.H. (1996), Hepatotoxic effects of SH-reagents in human and rat hepatocyte cultures and in situ perfused rat livers, Cell Structure and Function, vol. 2, no. 4, pp. 221-229.
  30. Bauman, J.N. et al. (2008), Comparison of the bioactivation potential of the antidepressant and hepatotoxin nefazodone with aripiprazole, a structural analog and marketed drug, Drug Metab. Dispos, vol. 36, no. 6, pp. 1016–1029.
  31. 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.