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Alkylation, Protein leads to Cell injury/death
Key Event Relationship Overview
AOPs Referencing Relationship
Life Stage Applicability
Key Event Relationship Description
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. 
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.  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. 
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.
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. 
Evidence Collection Strategy
Evidence Supporting this KER
Uncertainties and Inconsistencies
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.  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 
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
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
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