Upstream eventAlkylation, Protein
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Protein Alkylation leading to Liver Fibrosis||adjacent||Moderate|
|Rattus norvegicus||Rattus norvegicus||High||NCBI|
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 Supporting this KER
Even though protein alkylation is a generic process having an impact on multiple physiological processes in the cell, certain connections related to which alkylated proteins lead and/or contribute to cell injury have been described with high biological plausibility. Better understanding of the effects of alkylating agents at the molecular level is aided by recent application of new toxicogenomics technologies.  However, further efforts are certainly needed. Here we list literature-based evidence on how protein alkylation induced by allyl alcohol and carbon tetrachloride (CCl4) could be leading to cell injury (apoptosis/necrosis).
Allyl alcohol/Acrolein-induced apoptosis of human cells is associated with depletion of cellular GSH and intracellular generation of oxidants , achieved by alkylation of various proteins involved in the process. More specifically, allyl alcohol/acrolein is considered a mitochondrial toxin that leads to cell death. Whether apoptosis or necrosis ensues after acrolein exposure appears to be related to dose and cell type. In regards to activation of caspases as part of the mitochondrial death pathway it was shown that apoptosis could be both caspase-dependent: in human neuroblastoma cells  and in A549 lung cells , as well as caspase-independent: in CHO cells.  It was suggested that the activation of certain caspases may arise from a partial inhibition of their active site cysteine residue through direct alkylation by acrolein.
Furthermore, using biotin hydrazide labeling, it was shown that NF-κB RelA and p50, as well as JNK2, were revealed as direct targets for alkylation by acrolein, affecting the GSH depletion. Mass spectrometry analysis of acrolein-modified recombinant JNK2 indicated adduction to Cys(41) and Cys(177), putative important sites involved in mitogen-activated protein kinase (MAPK) kinase (MEK) binding and JNK2 phosphorylation. In complimentary work, exposure of cultured hepatocytes to acrolein led to a sustained activation of ERK1/2, JNK, and p38, which was associated with ER and mitochondrial stress and apoptosis. The cytotoxic effects of acrolein were decreased by JNK inhibitor, suggesting that kinase activation may be linked to cell death and liver injury. 
Schwend et al. (2008) tried to identify new proteins that undergo alkylation by acrylamide by treating three human cell lines (Jurkat, Caco-2 and HepG2 cells) with acrylamide and analyzing extracted proteins by MALDI-TOF for potentially alkylated candidates. They could identify two novel acrylamide target proteins that may contribute to the toxicity of acrylamide in cell cultures. Acrylamide showed dose-dependent cytotoxic effects in all three tested cultures (IC50 2-4 mM for the three cell lines). Protein alkylation could be observed already at lower, sub-cytotoxic doses (10uM). Their data confirmed that acrylamide causes cytotoxicity effects in cell cultures and this cytotoxicity is most likely mediated by protein alkylation. 
Thompson and Burcham (2008) studied the impact of culture media composition on the extent of damage occurring at protein targets within acrolein-exposed cells (A549 cells), and saw that acrolein induced concentration- dependent damage to cell proteins and increased cytochrome c release as marker of apoptotic cell death. 
Cai et al. (2005) investigated the mitochondria-initiated apoptosis pathway involved in CCl4 hepatotoxicity in vitro and observed a time-and dose-dependent decrease in cellular glutathione content, along with a concomitant increase in malondialdehyde levels following the application of CCl4. Caspase 3 activity was stimulated at all doses of CCl4, with the most significant activation at 3 mmol/L. Cytochrome c was released obviously after CCl4 treatment. A time-dependent decrease in Bcl-XL expression was observed. DNA fragmentation results revealed apoptosis and necrosis following CCl4 treatment. They concluded that oxidative damage is one of the essential mechanisms of CCl4 hepatotoxicity, which triggers apoptosis via the mitochondria-initiated pathway.
Perrissoud et al. (1980) investigated the effect of CCl4 on isolated rat hepatocytes. The ultrastructural alterations and release of lactate dehydrogenase (LDH) and glutamate-oxaloacetate transaminase (GOT), were recorded after different periods of incubation. After 5 min incubation with CC14, morphological changes observed by electron microscopy, involved the plasma membrane. The endoplasmic reticulum and mitochondria were altered later. These morphological alterations were accompanied by an early release of LDH and GOT into the incubation medium. .
Johnston and Kroening (1998) investigated mechanisms of early carbon tetrachloride toxicity in cultured rat hepatocytes and found that primary rat hepatocytes in culture were killed after a 2 hr incubation with CCl4 gas at partial pressures above a threshold between 45 and 54 mmHg. They concluded that early hepatocyte death in cell culture is independent of metabolism of CCl4, and may be related to direct effects of CCl4 on intracellular membranes.
Boll et al. (2001) modelled CCl4-induced liver damage in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CC14 metabolites to cell components and/ or peroxidative damage as the cause of injury. They observed that covalent binding of 1 4 C-labelled metabolites was detected in hepatocytes immediately after exposure to CC14. Electrophoresis of microsomal proteins from [14C]-CCl4-treated hepatocytes revealed that, aside of the start and the front of the gel, radioactive label was found primarily between 70 and 80 kDa.
In a study performed on isolated hepatocytes it was demonstrated that direct alkylation of critical sulfhydryl groups in proteins leads to a sustained increase in free Ca++ concentrations which, via Ca++ - dependent protease activates the enzyme xantine oxidase. This activation generates a substantial amount of superoxide anion free radical and other ROS that oxidize other protein sulfhydryl groups. Thiol depletion in the cytoplasm is primarily linked to the alkylation by the reactive metabolite acrolein.
Boot (1996) described toxicological data of organic mercury compounds (alkylating agents) in rat hepatocytes, primary human hepatocytes, and in situ perfused total rat livers. Significant effects like induction of glutathione depletion, inhibition of cellular glucose and amino acid uptake with blocked albumin synthesis were observed in almost all tested physiological parameters. 
Codreanu et al. (2014) intended to profile the accumulation of proteome damage in human cells (RKO and THP-1 cells) treated with lipid electrophile probes. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation- susceptible proteins mapping to networks involved in cytoskeletal regulation. 
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.
Quantitative Understanding of the Linkage
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.
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).
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
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