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Relationship: 269
Title
Alkylation, Protein leads to Cell injury/death
Upstream event
Downstream event
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 | Brendan Ferreri-Hanberry (send email) | Open for citation & comment | WPHA/WNT Endorsed |
Taxonomic Applicability
Sex Applicability
Life Stage Applicability
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]
| ID | Experimental Design | Species | Upstream Observation | Downstream Observation | Citation (first author, year) | Notes |
|---|
| Title | First Author | Biological Plausibility |
Dose Concordance |
Temporal Concordance |
Incidence Concordance |
|---|
Biological Plausibility
Dose Concordance Evidence
Temporal Concordance Evidence
Incidence Concordance Evidence
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. [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.
Quantitative data are hardly available.
Schwend et al found that Acrylamide concentrations causing serious cytotoxicity were 2 – 4 mM. Acrylamide toxicity in vivo and in vitro is most likely the result of protein alkylation.Protein alkylation could be observed already at lower, sub-cytotoxic doses (10uM). The effects were dose-dependent and these IC 50 values were found for the three treated cell types: Jurkat cells: 2mM, HepG2 cells: 2mM, Caco-2 cells: 4mM. Cells were grown in 96-well plates and treated with acrylamide for 48 h. Cell viability was measured by the MTT assay (0.05 mg/mL MTT). IC50 values were calculated from dose-response curves 48 h after acrylamide treatment.[23]
Codreanu et al. performed adduct profiling experiments with alkynyl analogs of the prototypical lipid electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) in human colorectal carcinoma (RKO) cells and human monocytic leukemia (THP-1) cells. Treatment with aHNE and aONE produced widespread protein alkylation in both cell types. IC50 concentrations for HNE and ONE and their alkynyl analogs in both cell types were 20 uM. Protein alkylation could be observed already at nontoxic concentrations (5 and 10uM).[10]