MechoApedia is a tool dedicated to learning the scientific basis of the mechanisms of toxic action, termed "MechoA", classified within the MechoA+ and MechoA Premium schemes.

MechoAs are specific molecular initiating events (MIE), the first step in Adverse Outcome Pathways (AOP), responsible for toxicity to biological organisms of all kinds and therefore these classifications are relevant to both ecotoxicologists and human health specialists (Ankley et al., 2010; F. J. Bauer et al., 2018a, 2018b).

This literature review is adapted from Bauer’s thesis on MechoAs (F. Bauer, 2017; Firman et al., 2022; Sapounidou et al., 2021) from MechoA+ publication (Levet et al., 2026) and further internal work carried out by KREATiS (F. Richard, F.J. Bauer, G. Levet).

The scheme below presents the MechoA classes and subclasses that can be predicted by the model:

Figure 0.1 classes and subclasses of the MechoA scheme

Click on MechoA classes and their subclasses on the left panel to get specific information on how chemical substances interact with biological matrices to lead to toxicological effects according to that MechoA.

MechoA Premium scheme

MechoA Premium scheme is based on MechoA+. For further information on how the model was built and its use, refer to the publication of Levet et al. (2026) about MechoA+ model.

MechoA Premium is the extended version of MechoA+. Additional alerts were added; 160 alerts in version 1.0 compared to 152 alerts in the MechoA+. Thus, the MIE coverage has been extended. Additionally, a few alerts of MechoA+ were refined in MechoA Premium.

Format of MechoA predictions

MechoA predictions are given as follows. “MechoA xxY.Z: Sentence describing the MIE with an indication of the taxa concerned.”  where “xx” is the taxa code relating to the taxonomic applicability of the MIE, “Y” the class and “Z” the sub-class. In addition, a ‘C’ can appear between the prefix “xx” and the class number “Y.Z”, to designate that the toxicological outcomes of the mechanism of action can be observed in chronic timeframe only (e.g. carcinogenicity). 

In the case if taxa applicability is “for all species”, i.e., all species may be impacted by such MIE, “xx” is absent. For example, in the case of the alert about acrylates, the model would predict “MechoA 3.2: Michaël addition with sulfhydryl groups, generating protein and DNA adducts for all species.”.

“&” was needed to separate predictions when several MIEs are predicted for a substance. “/” indicate a degree of uncertainty of the results. This would be the case when the MIE leading to observed toxicological outcomes is not completely understood. For instance, this is the predicted MechoA for propham derivatives: “MechoA 6.2 / pl6.7: disruption of calcium transport in all species and inhibition of tubulin polymerisation into microtubules, which may be a direct inhibition or simply be due to the modified calcium concentration, stopping mitosis in plants.” 

Table 1: Separation characters

After the MechoA code, a text clarifies and provides details on the predicted mechanism and applicable species. This text also often describes further key events typically occurring from the predicted MIE.

Taxonomy explanation

The scheme predicts a variety of MIEs to which a taxonomical applicability is associated, i.e. defining the biological species potentially affected by the given MIE.

The taxonomical applicability varies between alerts, depending on the presence of the biological target and the ability of a species to metabolise the represented chemical structures, etc.

The taxa codes are standardised into a two-letter code.  

Table 2: MechoA+ taxa codes attribution

* The term “pl” for “plants” in this scheme refers to all photosynthesizing organisms, thus including but not limited to: viridiplantae, cyanobacteria, glaucophytes, cryptophytes, haptophytes, dinoflagellates, chromerids, ochrophytes, chlorarachniophytes, euglenids, some cnidarians, some sponges, some aquatic gastropods, some marine flatworms. 

Two methodologies were used to extrapolate the range of taxa in which a MIE is susceptible to occur, depending upon whether the alert is considered “non-specific” or “specific”.

• Non-specific MechoAs

Non-specific MIEs apply across most species, with exceptions whereby a metabolic activation can occur. These include mechanisms like narcosis which, as the “baseline toxicity”, affects all species by disrupting cell membranes. Mechanisms involving reactivity (e.g., covalent binding to DNA/proteins), and indirect enzyme disruption (e.g. oxidative phosphorylation uncoupling) also effect all species. Non-specific mechanisms impact commonly shared biological systems, and as such MechoA classes 1, 3, and 5 are considered non-specific. For these non-specific MIEs, taxonomic applicability was set to “all species”, unless (eco)toxicological data show specific metabolism in certain species, leading to significant metabolites with different MechoAs.

• Specific MechoAs

Molecules which are known to target one or several biological entities before or after metabolism enter the category of “specific” mechanisms. Inevitably, the taxonomic applicability of such alerts is more restrictive compared to alerts for non-specific mechanisms, since the MIE is related to the presence of a specific target (e.g. receptor, enzyme, etc.) in the organism.

To achieve this, a rapid cross-species search methodology with EggNOG was developed, refining the taxonomic applicability of such alerts. This approach allowed to exclude taxa that cannot be impacted by a specific MIE (such as a chemical acting on GABAergic chloride channels in animals but not in plants due to the absence of the target) while allowing to include more taxa than those which have (eco)toxicological data, if these taxa appear to have the same target. This method distinguishes MechoA+ from previous schemes using a more “traditional” method to either extrapolate like MechoA or not extrapolate the taxa for SF scheme. Though limitations exist, such as potential false positives/negatives and the exclusion of orthologs, the methodology represents a significant step forward to better characterise the taxonomic applicability domain of each alert, thus of MechoA+.

G.T. Ankley, R.S. Bennett, R.J. Erickson, D.J. Hoff, M.W. Hornung, R.D. Johnson, D.R. Mount, J.W. Nichols, C.L. Russom, P.K. Schmieder, J.A. Serrrano, J.E. Tietge, D.L. Villeneuve, Adverse outcome pathways: a conceptual framework to support ecotoxicology research and risk assessment, Environ Toxicol Chem 29 (2010) 730–741. https://doi.org/10.1002/etc.34.

F. Bauer, Une meilleure caractérisation des mécanismes d’action toxique à partir de la structure moléculaire., Université de Haute-Alsace, 2017.

F.J. Bauer, P.C. Thomas, S.Y. Fouchard, S.J.M. Neunlist, A new classification algorithm based on mechanisms of action, Computational Toxicology 5 (2018a) 8–15. https://doi.org/10.1016/j.comtox.2017.11.001.

F. Bauer, P. Thomas, S. Fouchard, N. Serge, High-accuracy prediction of Mechanisms of Action using structural alerts, Computational Toxicology 7 (2018b). https://doi.org/10.1016/j.comtox.2018.06.004.

J.W. Firman, D.J. Ebbrell, F.J. Bauer, M. Sapounidou, G. Hodges, B. Campos, J. Roberts, S. Gutsell, P.C. Thomas, M. Bonnell, M.T.D. Cronin, Construction of an In Silico Structural Profiling Tool Facilitating Mechanistically Grounded Classification of Aquatic Toxicants, Environ. Sci. Technol. 56 (2022) 17805–17814. https://doi.org/10.1021/acs.est.2c03736.

G. Levet, F.J. Bauer, P.C. Thomas, M.T.D. Cronin, J. Roberts, S. Gutsell, B. Campos, G. Hodges, J. Firman, MechoA+: A Chemical Structure Profiler Raising the Bar for the Prediction of Mechanisms of Toxic Action for Chemical Safety Assessment, Environmental Science and Technology (2026). https://doi.org/10.1021/acs.est.5c18657.

M. Sapounidou, D.J. Ebbrell, M.A. Bonnell, B. Campos, J.W. Firman, S. Gutsell, G. Hodges, J. Roberts, M.T.D. Cronin, Development of an Enhanced Mechanistically Driven Mode of Action Classification Scheme for Adverse Effects on Environmental Species, Environ. Sci. Technol. 55 (2021) 1897–1907. https://doi.org/10.1021/acs.est.0c06551.

Biological process description under ‘normal’ circumstances

Cells membranes are mostly composed of lipid (phospholipids), cholesterol and proteins (e.g. glycoprotein, transmembrane proteins). They play huge variety of roles among which they constitute an essential layer to maintain the integrity of the cells of every living organism, controlling the exchanges between the extracellular medium and the cytosol as well as the cell fluidity and rigidity (Campbell, 1993; Goodman et al., 2008; Richard, 2022).

MIE description

Narcosis is a non-specific, non-reactive and reversible process that any organic molecule can exert on cellular organisms (Verhaar et al., 1992). The principle is based on a simple accumulation of substances in cell membranes thus disturbing their functions up to the loss of physical integrity. Since cell membranes are hydrophobic components, the main property driving the intensity of the adverse outcome due to these toxic mechanisms of action is the hydrophobicity of molecules.

There is a recognised strong relationship between toxicity and octanol-water partition coefficient (K_OW). This property gives an indication of the relative fraction of the test substance in the aqueous compartment (i.e. extracellular medium or cytosol) and the fraction accumulated within hydrophobic compartments like membranes (target lipid) but also lipidic tissue (storage lipid) (Campbell, 1993; Richard, 2022). This has led to the hypothesis that narcosis is related to destabilisation of cell membranes integrity. However, the effect may be due to the impairment of cell membrane proteins (e.g. ion channel or G proteins) because their function depends on their environment and their spatial conformation (Franks & Lieb, 1998; Sikkema et al., 1995).

The narcotic effect varies according to the different affinities of the substance with the membrane and three categories are considered in MechoA Premium scheme.

The first and second categories are the so-called ‘non-polar narcotics’ (e.g. simple alkanes or alcohols) and ‘polar narcotics’ (e.g. phenols, anilines, some surfactants) described in their sub-sections MechoA 1.1 and MechoA 1.2, respectively. Widely recognised and use historically by ecotoxicologists, the separation between these two categories is not exclusively due to the polarity (a chemistry concept) of molecules. For instance, a polar compound such as an alkyl alcohol is classified as a ‘non-polar narcotic’, while it actually is a polar molecule. It should be noted that these are historical wordings describing two (probably) different biological process (described in their sub-section) and not a chemically based concept.

As an example, the illustration of the narcotic impact was shown in a study (de Sousa et al., 2012) using carvacrol or 1,8-cineole on bacteria cells (following figure). After exposition, some cavities are visible in cell membranes causing the cytosol to leak out of the cell. 1,8-cineole is an aliphatic ether considered a non-polar narcotic (MechoA 1.1), while carvacrol is classified as a polar narcotic (MechoA 1.2) as a simple alkylphenol.

Figure 1.0.1: Bacteria P. fluorescens observed with Transmission Electron Microscopy (left column) and with Scanning Electron Microscopy (right column); (a-b) control, (c-d) after exposure to carvacrol, (e-f) after exposure to 1,8-cineole (de Sousa et al., 2012).

The third category is mostly related to molecules that are cationic at any pH, such as alkylammonium, and thus was named ‘narcosis of permanently cationic molecules’ (MechoA 1.3 in the scheme).

Taxonomy application

A parallel between human toxicology and ecotoxicology could also be made. In vivo studies suggest that rats exposed to narcotics experienced dizziness or drowsiness if the xenobiotic is able to cross the blood brain barrier, and this symptom is named ‘narcosis’.

Further, the term "narcosis" here is the same as that used to describe rapid and reversible anaesthesia. Anaesthetic potential of molecules has been demonstrated to be correlated to their hydrophobicity according to the Meyer-Overton relationship (Lugli et al., 2009). That would show the mechanism of action leading to anaesthesia is the accumulation of molecules within the neural cell membranes.

However, several studies on the anaesthesia mechanisms have suggested the efficiency of narcotics is rather due to specific protein targets (like ion channels) which have non-specific binding site (Franks & Lieb, 1978; Lugli et al., 2009).

E. Bourgart, C. Charmeau-Genevois, F. Larras , P. Thomas, G. Levet, F. Bauer, MechoA Premium model for human health and environment hazard assessment : a case study of cross-species extrapolation for narcosis, Poster presented by Dr Carole Charmeau-Genevois at the Eurotox 2023, Congress in Ljubljana. https://api.kreatis.eu/CDN/uploads/files/1694528853_d1ad36b79c01cd4cc21d.pdf.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

J.P. de Sousa, R. de A. Torres, G.A. de Azerêdo, R.C.B.Q. Figueiredo, M.A. da S. Vasconcelos, E.L. de Souza, Carvacrol and 1,8-cineole alone or in combination at sublethal concentrations induce changes in the cell morphology and membrane permeability of Pseudomonas fluorescens in a vegetable-based broth, International Journal of Food Microbiology 158 (2012) 9–13. https://doi.org/10.1016/j.ijfoodmicro.2012.06.008.

N.P. Franks, W.R. Lieb, Which molecular targets are most relevant to general anaesthesia?, Toxicology Letters 100–101 (1998) 1–8. https://doi.org/10.1016/S0378-4274(98)00158-1.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

A.K. Lugli, C.S. Yost, C.H. Kindler, Anaesthetic mechanisms: update on the challenge of unravelling the mystery of anaesthesia, Eur J Anaesthesiol 26 (2009) 807–820. https://doi.org/10.1097/EJA.0b013e32832d6b0f.

D. Richard, Biologie, 5e éd, Dunod, Malakoff, 2022.

J. Sikkema, J.A. de Bont, B. Poolman, Mechanisms of membrane toxicity of hydrocarbons., Microbiol Rev 59 (1995) 201–222.

H.J.M. Verhaar, C.J. van Leeuwen, J.L.M. Hermens, Classifying environmental pollutants, Chemosphere 25 (1992) 471–491. https://doi.org/10.1016/0045-6535(92)90280-5.

So-called “non-polar narcotics” include a wide range of chemical families. Those chemicals are not necessarily non polar (according to chemistry principle) and they are thought to account for more than 50% of the organic chemical universe (Ellison et al., 2015a). This can be further observed when running MechoA+ profiler on a large dataset of compounds (Levet et al., 2026).

Non-polar narcosis (MechoA 1.1), or "baseline toxicity", is assumed to be the least toxic of the known mechanisms of action for less hydrophobic substances (Verhaar et al., 1992). Generally, at comparable hydrophobicities or solubilities, non-polar narcotics are less toxic than substances with any other MechoA (as observed by Dearden, 2002). However, this does not hold true at higher hydrophobicity where non-polar narcosis can be as much or even more potent than other mechanisms. For instance, in aquatic toxicity studies, non-polar narcotic substances with high hydrophobicity have very low EC50s, i.e. a small concentration leads to strong toxicological effects, i.e. they have high toxicity. This is because such substances have very high affinity with lipid membranes and thus accumulate in them in great proportions. However, substances with very high hydrophobicity become non bioavailable and thus not toxic.

Figure 1.1.1: A) shows an asymptomatic state where the phospholipidic bilayer is stable. B) depicts non polar narcosis: it is the presence of a xenobiotic which interacts with intracellular matrices that induces changes in the internal composition of the cell membrane, which leads to toxicity: without further mechanism of action observed, the non-polar narcosis explains the most basic state of toxicity possibly triggered, thus it is regarded as the baseline toxicity of a compound. 

Adapted from Roberts et al. SAR QSAR Environ. Res., 2013, 24, 417–427.​ (R represents the hydrophilic part of the molecule)

Comment

J.C. Dearden, Prediction of environmental toxicity and fate using quantitative structure-activity relationships (QSARs), Journal of the Brazilian Chemical Society 13 (2002) 754–762. https://doi.org/10.1590/S0103-50532002000600005.

C.M. Ellison, J.C. Madden, M.T.D. Cronin, S.J. Enoch, Investigation of the Verhaar scheme for predicting acute aquatic toxicity: Improving predictions obtained from Toxtree ver. 2.6, Chemosphere 139 (2015) 146–154. https://doi.org/10.1016/j.chemosphere.2015.06.009.

G. Levet, F.J. Bauer, P.C. Thomas, M.T.D. Cronin, J. Roberts, S. Gutsell, B. Campos, G. Hodges, J. Firman, MechoA+: A Chemical Structure Profiler Raising the Bar for the Prediction of Mechanisms of Toxic Action for Chemical Safety Assessment, Environmental Science and Technology (2026). https://doi.org/10.1021/acs.est.5c18657.

D.W. Roberts, J.F. Roberts, G. Hodges, S. Gutsell, R.S. Ward, C. Llewellyn, Aquatic toxicity of cationic surfactants to Daphnia magna, SAR and QSAR in Environmental Research 24 (2013) 417–427. https://doi.org/10.1080/1062936X.2013.781538.

H.J.M. Verhaar, C.J. van Leeuwen, J.L.M. Hermens, Classifying environmental pollutants, Chemosphere 25 (1992) 471–491. https://doi.org/10.1016/0045-6535(92)90280-5.

Known to have narcotic effects (depending on taxa) are some chemical categories (hydrocarbons, ethers, aliphatic alcohols, ketones, amides, halogenated derivatives, etc):

Figure 1.1.2: Some examples of substances considered narcotics based on acute toxicity testing. Hydrocarbons, mononitroaromatics, pyridines (potentially chloro-alkyl substituted), heteroaromatic amines, halogenated hydrocarbons, ethers, aliphatic alcohols, alkyl alcohols with an aromatic ring, some thiols, ketones, aromatic ureas, nitroamines / nitroguanidines, sulfonate esters, dialkylsulfoxides, amides, carbamates (though none is present in our current MechoA dataset, linkages are considered as extremely stable bonds (Hermanson, 2013)), sugars.

Known to have narcotic effects (depending on taxa) are some chemical categories (hydrocarbons, ethers, aliphatic alcohols, ketones, amides, halogenated derivatives, etc):

Figure 1.1.2: Some examples of substances considered narcotics based on acute toxicity testing. Hydrocarbons, mononitroaromatics, pyridines (potentially chloro-alkyl substituted), heteroaromatic amines, halogenated hydrocarbons, ethers, aliphatic alcohols, alkyl alcohols with an aromatic ring, some thiols, ketones, aromatic ureas, nitroamines / nitroguanidines, sulfonate esters, dialkylsulfoxides, amides, carbamates (though none is present in our current MechoA dataset, linkages are considered as extremely stable bonds (Hermanson, 2013)), sugars.

Known to have narcotic effects (depending on taxa) are some chemical categories (hydrocarbons, ethers, aliphatic alcohols, ketones, amides, halogenated derivatives, etc):

Figure 1.1.2: Some examples of substances considered narcotics based on acute toxicity testing. Hydrocarbons, mononitroaromatics, pyridines (potentially chloro-alkyl substituted), heteroaromatic amines, halogenated hydrocarbons, ethers, aliphatic alcohols, alkyl alcohols with an aromatic ring, some thiols, ketones, aromatic ureas, nitroamines / nitroguanidines, sulfonate esters, dialkylsulfoxides, amides, carbamates (though none is present in our current MechoA dataset, linkages are considered as extremely stable bonds (Hermanson, 2013)), sugars.

Polar narcosis (MechoA 1.2) is very similar to non-polar narcosis despite the significant difference of toxicity, in aquatic toxicity studies, observed for compounds with equivalent hydrophobicity (Escher & Hermens, 2002; Verhaar et al., 1992). In practice, the distinction between non-polar (see MechoA 1.1) and polar narcosis (Figure 1) may be ambiguous.

Figure 1.2.1: A) shows an asymptomatic state where the phospholipidic bilayer is stable. B) show an example of polar narcosis: a polar compound (with a hydrophilic head) interacts with the phospholipidic bilayer to cause narcotic effects.
Adapted from Roberts et al. SAR QSAR Environ. Res., 2013, 24, 417–427.​ (R represents the hydrophobic part of the molecule)

B.I. Escher, J.L.M. Hermens, Modes of Action in Ecotoxicology:  Their Role in Body Burdens, Species Sensitivity, QSARs, and Mixture Effects, Environ. Sci. Technol. 36 (2002) 4201–4217. https://doi.org/10.1021/es015848h.

D.W. Roberts, J.F. Roberts, G. Hodges, S. Gutsell, R.S. Ward, C. Llewellyn, Aquatic toxicity of cationic surfactants to Daphnia magna, SAR and QSAR in Environmental Research 24 (2013) 417–427. https://doi.org/10.1080/1062936X.2013.781538.

H.J.M. Verhaar, C.J. van Leeuwen, J.L.M. Hermens, Classifying environmental pollutants, Chemosphere 25 (1992) 471–491. https://doi.org/10.1016/0045-6535(92)90280-5.

This mechanism encompasses mainly ‘simple’ phenols (de Sousa et al., 2012) and anilines (Janssens et al., 2011; Ramos et al., 1997), but also nitrogen heterocycles (Ellison et al., 2015b) as well as some surfactants such as long chain alkylsulfonates (Hodges et al., 2006). Additionally, in aquatic toxicity tests, hydroxylated Polycyclic Aromatic Hydrocarbons (PAHs) are expected to have this mechanism.

Figure 1.2.2: Examples of substance considered as polar narcotics

For instance, effects due to carvacrol (MechoA 1.2) are comparable to 1,8-cineole (MechoA 1.1) but they are observable at lower concentrations (de Sousa et al., 2012). Furthermore, some pyridines are considered to act with MechoA 1.1 while other pyridines are associated to MechoA 1.2 in aquatics species.

Some authors consider the separation between those two MechoA to be linked to polarizability of the molecules, or even their abilities to participate in strong hydrogen bonding (Roberts et al., 2013). Others have suggested the difference between MechoA 1.1 and 1.2 was a consequence of the use of the log KOW as a bad surrogate to quantify the equilibrium of the molecules between aqueous medium and cell membranes. With log KOW, octanol is assumed to represent membrane lipids.

Some experiments have been performed by replacing octanol with dimyristoylphosphatidylcholine (DMPC), thus normalising the toxic impact between so-called “polar narcotic” and “non-polar narcotic” compounds (Vaes et al., 1998).

Finally, other authors explained the difference of toxicity of these two groups by a difference of partitioning between different compartments, which is not taken into account with log KOW (Endo et al., 2013; Escher & Hermens, 2002).

J.P. de Sousa, R. de A. Torres, G.A. de Azerêdo, R.C.B.Q. Figueiredo, M.A. da S. Vasconcelos, E.L. de Souza, Carvacrol and 1,8-cineole alone or in combination at sublethal concentrations induce changes in the cell morphology and membrane permeability of Pseudomonas fluorescens in a vegetable-based broth, International Journal of Food Microbiology 158 (2012) 9–13. https://doi.org/10.1016/j.ijfoodmicro.2012.06.008.

S. Endo, T.N. Brown, K.-U. Goss, General Model for Estimating Partition Coefficients to Organisms and Their Tissues Using the Biological Compositions and Polyparameter Linear Free Energy Relationships, Environ. Sci. Technol. 47 (2013) 6630–6639. https://doi.org/10.1021/es401772m.

B.I. Escher, J.L.M. Hermens, Modes of Action in Ecotoxicology:  Their Role in Body Burdens, Species Sensitivity, QSARs, and Mixture Effects, Environ. Sci. Technol. 36 (2002) 4201–4217. https://doi.org/10.1021/es015848h.

Hodges, G., Roberts, D. W., Marshall, S. J., & Dearden, J. C. (2006). The aquatic toxicity of anionic surfactants to Daphnia magna—A comparative QSAR study of linear alkylbenzene sulphonates and ester sulphonates. Chemosphere, 63(9), 1443–1450. https://doi.org/10.1016/j.chemosphere.2005.10.001.

D.W. Roberts, J.F. Roberts, G. Hodges, S. Gutsell, R.S. Ward, C. Llewellyn, Aquatic toxicity of cationic surfactants to Daphnia magna, SAR and QSAR in Environmental Research 24 (2013) 417–427. https://doi.org/10.1080/1062936X.2013.781538.

W.H.J. Vaes, E. Urrestarazu Ramos, H.J.M. Verhaar, W. Seinen, J.L.M. Hermens, Measurement of the Free Concentration Using Solid-Phase Microextraction: Binding to Protein, Anal. Chem. 68 (1996) 4463–4467. https://doi.org/10.1021/ac960337c.

This mechanism encompasses mainly ‘simple’ phenols (de Sousa et al., 2012) and anilines (Janssens et al., 2011; Ramos et al., 1997), but also nitrogen heterocycles (Ellison et al., 2015b) as well as some surfactants such as long chain alkylsulfonates (Hodges et al., 2006). Additionally, in aquatic toxicity tests, hydroxylated Polycyclic Aromatic Hydrocarbons (PAHs) are expected to have this mechanism.

Figure 1.2.2: Examples of substance considered as polar narcotics

For instance, effects due to carvacrol (MechoA 1.2) are comparable to 1,8-cineole (MechoA 1.1) but they are observable at lower concentrations (de Sousa et al., 2012). Furthermore, some pyridines are considered to act with MechoA 1.1 while other pyridines are associated to MechoA 1.2 in aquatics species.

Some authors consider the separation between those two MechoA to be linked to polarizability of the molecules, or even their abilities to participate in strong hydrogen bonding (Roberts et al., 2013). Others have suggested the difference between MechoA 1.1 and 1.2 was a consequence of the use of the log KOW as a bad surrogate to quantify the equilibrium of the molecules between aqueous medium and cell membranes. With log KOW, octanol is assumed to represent membrane lipids.

Some experiments have been performed by replacing octanol with dimyristoylphosphatidylcholine (DMPC), thus normalising the toxic impact between so-called “polar narcotic” and “non-polar narcotic” compounds (Vaes et al., 1998).

Finally, other authors explained the difference of toxicity of these two groups by a difference of partitioning between different compartments, which is not taken into account with log KOW (Endo et al., 2013; Escher & Hermens, 2002).

J.P. de Sousa, R. de A. Torres, G.A. de Azerêdo, R.C.B.Q. Figueiredo, M.A. da S. Vasconcelos, E.L. de Souza, Carvacrol and 1,8-cineole alone or in combination at sublethal concentrations induce changes in the cell morphology and membrane permeability of Pseudomonas fluorescens in a vegetable-based broth, International Journal of Food Microbiology 158 (2012) 9–13. https://doi.org/10.1016/j.ijfoodmicro.2012.06.008.

S. Endo, T.N. Brown, K.-U. Goss, General Model for Estimating Partition Coefficients to Organisms and Their Tissues Using the Biological Compositions and Polyparameter Linear Free Energy Relationships, Environ. Sci. Technol. 47 (2013) 6630–6639. https://doi.org/10.1021/es401772m.

B.I. Escher, J.L.M. Hermens, Modes of Action in Ecotoxicology:  Their Role in Body Burdens, Species Sensitivity, QSARs, and Mixture Effects, Environ. Sci. Technol. 36 (2002) 4201–4217. https://doi.org/10.1021/es015848h.

Hodges, G., Roberts, D. W., Marshall, S. J., & Dearden, J. C. (2006). The aquatic toxicity of anionic surfactants to Daphnia magna—A comparative QSAR study of linear alkylbenzene sulphonates and ester sulphonates. Chemosphere, 63(9), 1443–1450. https://doi.org/10.1016/j.chemosphere.2005.10.001.

D.W. Roberts, J.F. Roberts, G. Hodges, S. Gutsell, R.S. Ward, C. Llewellyn, Aquatic toxicity of cationic surfactants to Daphnia magna, SAR and QSAR in Environmental Research 24 (2013) 417–427. https://doi.org/10.1080/1062936X.2013.781538.

W.H.J. Vaes, E. Urrestarazu Ramos, H.J.M. Verhaar, W. Seinen, J.L.M. Hermens, Measurement of the Free Concentration Using Solid-Phase Microextraction: Binding to Protein, Anal. Chem. 68 (1996) 4463–4467. https://doi.org/10.1021/ac960337c.

This mechanism encompasses mainly ‘simple’ phenols (de Sousa et al., 2012) and anilines (Janssens et al., 2011; Ramos et al., 1997), but also nitrogen heterocycles (Ellison et al., 2015b) as well as some surfactants such as long chain alkylsulfonates (Hodges et al., 2006). Additionally, in aquatic toxicity tests, hydroxylated Polycyclic Aromatic Hydrocarbons (PAHs) are expected to have this mechanism.

Figure 1.2.2: Examples of substance considered as polar narcotics

For instance, effects due to carvacrol (MechoA 1.2) are comparable to 1,8-cineole (MechoA 1.1) but they are observable at lower concentrations (de Sousa et al., 2012). Furthermore, some pyridines are considered to act with MechoA 1.1 while other pyridines are associated to MechoA 1.2 in aquatics species.

Some authors consider the separation between those two MechoA to be linked to polarizability of the molecules, or even their abilities to participate in strong hydrogen bonding (Roberts et al., 2013). Others have suggested the difference between MechoA 1.1 and 1.2 was a consequence of the use of the log KOW as a bad surrogate to quantify the equilibrium of the molecules between aqueous medium and cell membranes. With log KOW, octanol is assumed to represent membrane lipids.

Some experiments have been performed by replacing octanol with dimyristoylphosphatidylcholine (DMPC), thus normalising the toxic impact between so-called “polar narcotic” and “non-polar narcotic” compounds (Vaes et al., 1998).

Finally, other authors explained the difference of toxicity of these two groups by a difference of partitioning between different compartments, which is not taken into account with log KOW (Endo et al., 2013; Escher & Hermens, 2002).

J.P. de Sousa, R. de A. Torres, G.A. de Azerêdo, R.C.B.Q. Figueiredo, M.A. da S. Vasconcelos, E.L. de Souza, Carvacrol and 1,8-cineole alone or in combination at sublethal concentrations induce changes in the cell morphology and membrane permeability of Pseudomonas fluorescens in a vegetable-based broth, International Journal of Food Microbiology 158 (2012) 9–13. https://doi.org/10.1016/j.ijfoodmicro.2012.06.008.

S. Endo, T.N. Brown, K.-U. Goss, General Model for Estimating Partition Coefficients to Organisms and Their Tissues Using the Biological Compositions and Polyparameter Linear Free Energy Relationships, Environ. Sci. Technol. 47 (2013) 6630–6639. https://doi.org/10.1021/es401772m.

B.I. Escher, J.L.M. Hermens, Modes of Action in Ecotoxicology:  Their Role in Body Burdens, Species Sensitivity, QSARs, and Mixture Effects, Environ. Sci. Technol. 36 (2002) 4201–4217. https://doi.org/10.1021/es015848h.

Hodges, G., Roberts, D. W., Marshall, S. J., & Dearden, J. C. (2006). The aquatic toxicity of anionic surfactants to Daphnia magna—A comparative QSAR study of linear alkylbenzene sulphonates and ester sulphonates. Chemosphere, 63(9), 1443–1450. https://doi.org/10.1016/j.chemosphere.2005.10.001.

D.W. Roberts, J.F. Roberts, G. Hodges, S. Gutsell, R.S. Ward, C. Llewellyn, Aquatic toxicity of cationic surfactants to Daphnia magna, SAR and QSAR in Environmental Research 24 (2013) 417–427. https://doi.org/10.1080/1062936X.2013.781538.

W.H.J. Vaes, E. Urrestarazu Ramos, H.J.M. Verhaar, W. Seinen, J.L.M. Hermens, Measurement of the Free Concentration Using Solid-Phase Microextraction: Binding to Protein, Anal. Chem. 68 (1996) 4463–4467. https://doi.org/10.1021/ac960337c.

This mechanism encompasses mainly ‘simple’ phenols (de Sousa et al., 2012) and anilines (Janssens et al., 2011; Ramos et al., 1997), but also nitrogen heterocycles (Ellison et al., 2015b) as well as some surfactants such as long chain alkylsulfonates (Hodges et al., 2006). Additionally, in aquatic toxicity tests, hydroxylated Polycyclic Aromatic Hydrocarbons (PAHs) are expected to have this mechanism.

Figure 1.2.2: Examples of substance considered as polar narcotics

For instance, effects due to carvacrol (MechoA 1.2) are comparable to 1,8-cineole (MechoA 1.1) but they are observable at lower concentrations (de Sousa et al., 2012). Furthermore, some pyridines are considered to act with MechoA 1.1 while other pyridines are associated to MechoA 1.2 in aquatics species.

Some authors consider the separation between those two MechoA to be linked to polarizability of the molecules, or even their abilities to participate in strong hydrogen bonding (Roberts et al., 2013). Others have suggested the difference between MechoA 1.1 and 1.2 was a consequence of the use of the log KOW as a bad surrogate to quantify the equilibrium of the molecules between aqueous medium and cell membranes. With log KOW, octanol is assumed to represent membrane lipids.

Some experiments have been performed by replacing octanol with dimyristoylphosphatidylcholine (DMPC), thus normalising the toxic impact between so-called “polar narcotic” and “non-polar narcotic” compounds (Vaes et al., 1998).

Finally, other authors explained the difference of toxicity of these two groups by a difference of partitioning between different compartments, which is not taken into account with log KOW (Endo et al., 2013; Escher & Hermens, 2002).

J.P. de Sousa, R. de A. Torres, G.A. de Azerêdo, R.C.B.Q. Figueiredo, M.A. da S. Vasconcelos, E.L. de Souza, Carvacrol and 1,8-cineole alone or in combination at sublethal concentrations induce changes in the cell morphology and membrane permeability of Pseudomonas fluorescens in a vegetable-based broth, International Journal of Food Microbiology 158 (2012) 9–13. https://doi.org/10.1016/j.ijfoodmicro.2012.06.008.

S. Endo, T.N. Brown, K.-U. Goss, General Model for Estimating Partition Coefficients to Organisms and Their Tissues Using the Biological Compositions and Polyparameter Linear Free Energy Relationships, Environ. Sci. Technol. 47 (2013) 6630–6639. https://doi.org/10.1021/es401772m.

B.I. Escher, J.L.M. Hermens, Modes of Action in Ecotoxicology:  Their Role in Body Burdens, Species Sensitivity, QSARs, and Mixture Effects, Environ. Sci. Technol. 36 (2002) 4201–4217. https://doi.org/10.1021/es015848h.

Hodges, G., Roberts, D. W., Marshall, S. J., & Dearden, J. C. (2006). The aquatic toxicity of anionic surfactants to Daphnia magna—A comparative QSAR study of linear alkylbenzene sulphonates and ester sulphonates. Chemosphere, 63(9), 1443–1450. https://doi.org/10.1016/j.chemosphere.2005.10.001.

D.W. Roberts, J.F. Roberts, G. Hodges, S. Gutsell, R.S. Ward, C. Llewellyn, Aquatic toxicity of cationic surfactants to Daphnia magna, SAR and QSAR in Environmental Research 24 (2013) 417–427. https://doi.org/10.1080/1062936X.2013.781538.

W.H.J. Vaes, E. Urrestarazu Ramos, H.J.M. Verhaar, W. Seinen, J.L.M. Hermens, Measurement of the Free Concentration Using Solid-Phase Microextraction: Binding to Protein, Anal. Chem. 68 (1996) 4463–4467. https://doi.org/10.1021/ac960337c.

Permanently cationic narcosis (MechoA 1.3) is based on the same principle as polar narcosis. However, the xenobiotic potency is often higher because the intermolecular bond energies involved are stronger and more favourable for interactions between cationic compounds and phospholipids of the cell membranes.

The positively charged polar head of cationic molecules associates with the membrane surface, potentially altering membrane charge distribution (Roberts et al., 2013).

Figure 1.3.1: A) shows an asymptomatic state where the phospholipidic bilayer is stable. B) depicts a charge repulsion induced by a polarity mismatch, hence disrupting the phospholipidic bilayer’s integrity even more. This situation is deemed to happen for molecules with a cationic head and hydrophobic tail, like alkylammonium compounds.
Adapted scheme from Roberts et al. SAR QSAR Environ. Res., 2013, 24, 417–427.​ (R represents the hydrophilic part of the molecule)

D.W. Roberts, J.F. Roberts, G. Hodges, S. Gutsell, R.S. Ward, C. Llewellyn, Aquatic toxicity of cationic surfactants to Daphnia magna, SAR and QSAR in Environmental Research 24 (2013) 417–427. https://doi.org/10.1080/1062936X.2013.781538.

This mechanism especially concerns quaternary ammonium compounds. Due to their relative high toxicity to unicellular organisms compared to other narcotics, they are often used as antimicrobials with a broad-spectrum activity (Wessels & Ingmer, 2013).

Figure 1.3.2: Examples of a quaternary ammonium molecules classified as MechoA 1.3

Biological process description under ‘normal’ circumstances

After a xenobiotic substance is absorbed by an organism, metabolism can occur. Often, the xenobiotic, if hydrophobic, is broken down into smaller more hydrophilic pieces and/or conjugated (e.g. glucuronidation) to favour the elimination by the organism (Nelson et al., 2013).

Among the diversity of metabolic pathways, enzymatic hydrolysis, typically performed by enzymes named esterases, plays a significant role. The parent compound is often cleaved in two. Esters, phosphates, carbonates, polysaccharides and pyrophosphates are subject to this hydrolysis, which is mostly enzymatically driven.

MIE description

Toxic effects of a given compound can be the result of the exposure to both the parent and its metabolites. In this particular case, the hydrolysis reaction produces on one part a metabolite which is expected to generate acidity. This excess of acidity is expected to damage cells only if the amount of hydrolysed parent is high enough to exceed the buffer provided by biological media.

Taxonomy application

Depending on the species, the enzymatic hydrolysis rate of compounds may differ. Generally, the more complex the organism regarding the number of cell types, the better the metabolic capacity (McCarthy & Enquist, 2005).

M.C. McCarthy, B. Enquist, Organismal size, metabolism and the evolution of complexity in metazoans, Evolutionary Ecology Research 7 (2005) 681–696.

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

Veith and Broderius (1987) identified that simple esters (ester with only alkyl chain) lead to symptoms similar to those of narcosis. Moreover, these compounds are not very reactive in aqueous medium at neutral pH.

Since they observed an excess of toxicity compared to narcotic compounds, these authors then described the toxicity of esters as another form of narcosis they termed "esters narcosis". Russom et al. (1997) also used this category in their classification.

Nevertheless, in 1995, Jaworska et al. reported that, while esters behave as non-polar narcotic compounds to protozoa (Tetrahymena pyriformis), they were actually more toxic than non-polar narcotics to fish (Pimephales promelas). The authors explained this observation by assuming that protozoa do not, or barely, metabolise esters while fish esterase enzymes can hydrolyse esters very efficiently (Figure below).

Figure 2.1.1: Enzymatic hydrolysis of esters. R1=H or alkyl, R2=alkyl.

This hydrolysis changes the equilibrium concentration between the external aqueous medium and the internal fish concentration since the ester internal concentration is decreased by the hydrolysis reaction. Thus, due to thermodynamic equilibrium, more esters will be transferred from external medium to fish tissues. Consequently, the fish might potentially be exposed to a higher amount of ester than if it wasn't metabolised, although this equilibrium is also determined by the hydrophobicity of the ester itself.

Uptake of more hydrophobic substances by aquatic organisms may occur less rapidly than their metabolization once inside the body, giving an impression of reduced substance toxicity. Moreover, the hydrolysis mechanism favours ester depuration from aquatic organisms since the hydrolysis products are less hydrophobic than the parent ester.

The metabolites are also less toxic than the parent ester substance. For less hydrophobic esters, accumulation of the alcohol generated after hydrolysis combined with rapid uptake of the parent compound may exert a significant narcotic effect. Besides, the hydrolysis reaction produces acidity which may contribute to the disturbance of normal cell functioning (for carboxylic acid behaviour, see MechoA 5.2 section).

While being largely studied for fish, the toxicity of some esters on mammals has also been studied (Low et al., 2021a), and an in silico model was recently developed by KREATiS (KREATiS, 2024), showing that ‘simple’ esters are not classified for the acute oral toxicity (AOT) according to CLP, based on a training set of 51 substances with experimental studies performed on rats according to relevant OECD test guidelines. This concerns aliphatic esters not containing any other heteroatoms, excluding a,b-unsaturated esters, aromatic esters, phthalates, but also lactones.

Lactones are well-known additive and flavouring agents. Aliphatic lactones, for instance, appeared in several hydroxy fatty acids metabolic pathway, and their hydrolysis by the human serum enzyme paraoxonase (PON1) was observed (Billecke et al., 2000). With a variety of substrates including lactones, the enzyme opens the lactone ring to give a hydroxylic-substituted carboxylic acid. Some lactones also shown reactivity (further details provided on MechoA 3.1). Apart from particular structures identified with a MechoA 3.1, lactones are associated with the MechoA 2.1 in iSafeRat® QSARs for aquatic toxicity.

In addition to carboxylic esters, families of compounds bearing other chemical moieties may be hydrolysed and form similar metabolites. It was especially observed for carbonates, alkyl(thio)phosphates, alkyl phosphonates.

Carbonates are diester derivatives of carbonic acid. Further studies reported that carbonate moiety in substrates lowered the enzymatic hydrolytic rates and that hydrophobic interactions played a role in the capacity of human, rat and mouse carboxylesterases (Huang et al., 1993).

Some carbamates are known pesticides that act as neurotoxic chemicals (Gupta, 2014) and disrupt the acetylcholinesterase (see MechoA 6.1) but in general carbamates can be structurally considered as “amide-ester” hybrids with chemical reactivity comparable to these two functional groups.

Alkyl(thio)phosphates esters and alkyl phosphonates esters have been identified to bind covalently to proteins (Von Der Ohe et al., 2005; Enoch et al., 2011). This was mostly observed with the organophosphates which bind within acetylcholinesterase active site (see MechoA 6.1). However, following the MechoA 2.1 mechanism, further action of water as a nucleophile can liberate the corresponding dealkylated phosphates esters.

V.E. Bel’skii, Kinetics of the Hydrolysis of Phosphate Esters, Russ. Chem. Rev. 46 (1977) 828–841. https://doi.org/10.1070/RC1977v046n09ABEH002175.

Billecke, D. Draganov, R. Counsell, P. Stetson, C. Watson, C. Hsu, B.N.L. Du, Human Serum Paraoxonase (pon1) Isozymes Q and R Hydrolyze Lactones and Cyclic Carbonate Esters, Drug Metabolism and Disposition 28 (2000) 1335–1342. https://doi.org/10.1016/S0090-9556(24)15082-9

S.J. Enoch, C.M. Ellison, T.W. Schultz, M.T.D. Cronin, A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity, Critical Reviews in Toxicology 41 (2011) 783–802. https://doi.org/10.3109/10408444.2011.598141.

P.K. Gupta, Herbicides and fungicides, in: Biomarkers in Toxicology, Elsevier, 2014: pp. 409–431. https://doi.org/10.1016/B978-0-12-404630-6.00024-5.

H.A. Hassan, S. Rani, F.A. Kiani, S. Fischer, S. Aslam, A. Sik, ar, A Comprehensive Theoretical Model for the Hydrolysis Reactions of Phosphate Containing Compounds, Journal of Theoretical & Computational Science 9 (2023) 1–10.

T.L. Huang, A. Székács, T. Uematsu, E. Kuwano, A. Parkinson, B.D. Hammock, Hydrolysis of Carbonates, Thiocarbonates, Carbamates, and Carboxylic Esters of α-Naphthol, β-Naphthol, and p-Nitrophenol by Human, Rat, and Mouse Liver Carboxylesterases, Pharm Res 10 (1993) 639–648. https://doi.org/10.1023/A:1018987111362.

J.S. Jaworska, R.S. Hunter, T.W. Schultz, Quantitative structure-toxicity relationships and volume fraction analyses for selected esters, Arch. Environ. Contam. Toxicol. 29 (1995) 86–93. https://doi.org/10.1007/BF00213091.

S.C.L. Kamerlin, P.K. Sharma, R.B. Prasad, A. Warshel, Why nature really chose phosphate, Q Rev Biophys 46 (2013) 1–132. https://doi.org/10.1017/S0033583512000157.

KREATiS, QSAR Model Reporting Format (QMRF) for Acute Oral Toxicity Alerts (AOrTA) – Local model for non-reactive esters v1.0 in acute oral toxicity (AOT), 2024.

Y.S. Low, M.D. Garcia, T. Lonhienne, J.A. Fraser, G. Schenk, L.W. Guddat, Triazolopyrimidine herbicides are potent inhibitors of Aspergillus fumigatus acetohydroxyacid synthase and potential antifungal drug leads, Sci Rep 11 (2021) 21055. https://doi.org/10.1038/s41598-021-00349-9.

C.L. Russom, S.P. Bradbury, S.J. Broderius, D.E. Hammermeister, R.A. Drummond, Predicting modes of toxic action from chemical structure: Acute toxicity in the fathead minnow (Pimephales promelas), Environmental Toxicology and Chemistry 16 (1997) 948–967. https://doi.org/10.1002/etc.5620160514.

P.C. Von Der Ohe, R. Kühne, R.-U. Ebert, R. Altenburger, M. Liess, G. Schüürmann, Structural AlertsA New Classification Model to Discriminate Excess Toxicity from Narcotic Effect Levels of Organic Compounds in the Acute Daphnid Assay, Chem. Res. Toxicol. 18 (2005) 536–555. https://doi.org/10.1021/tx0497954.

G.D. Veith, S.J. Broderius, Structure-Toxicity Relationships for Industrial Chemicals Causing Type (II) Narcosis Syndrome, in: K.L.E. Kaiser (Ed.), QSAR in Environmental Toxicology - II, Springer Netherlands, 1987: pp. 385–391. https://doi.org/10.1007/978-94-009-3937-0_29.

[MechoA+ Alert 149] O-aryl ester, phosphate, carbonate or carbamate or 3-hydroxy-2-naphthanilide

The hydrolysis product of esters and the like is often an alcohol but not always. For a phenyl ester, for example, the degradation product will be a phenol with a more toxic MechoA than MechoA 1.1, that is polar narcosis (see MechoA 1.2). Though depending on its substitution, the released phenol can also have other MechoAs than polar narcosis (see section MechoA 2.3 below). Polar narcotics resulting from enzymatic hydrolysis are often aniline- or phenol-like compounds.

The hydrolysis product of esters and the like is often an alcohol but not always. For a phenyl ester, for example, the degradation product will be a phenol with a more toxic MechoA than MechoA 1.1, that is polar narcosis (see MechoA 1.2). Though depending on its substitution, the released phenol can also have other MechoAs than polar narcosis (see section MechoA 2.3 below). Polar narcotics resulting from enzymatic hydrolysis are often aniline- or phenol-like compounds.

On the other hand, ortho-phthalates, as aromatic diesters, are particular cases because the hydrolysis of one of the two ester moieties produces a monoester compound with recognised endocrine disruption properties (see MechoA 6.8).

O-phthalates (o-DAP) are often used as plasticizers and solvents, mostly for medical and cosmetics fields (Babich, 2010). Research studies have been conducted to characterize the hydrolysis in the digestive system of rats of the following phthalates (Peraza et al., 2006; Rowland et al., 1977):

Figure 2.3.1: Example structure of phthalates (Dimethyl phthalate (DMP), Dibutyl phthalate (DBP)), Dicyclohexyl phthalate (DCHP)

The main product of this hydrolysis reaction was the corresponding monoester of each phthalate diester (Rowland et al., 1977).

This monoester induces the PPARα (Peroxisome proliferator-activated receptor alpha) activation that trigger peroxisome proliferation which plays a central role in hepatocarcinogenesis of O-DAPs (Babich, 2010).

M.A. Babich, Overview of Phthalates Toxicity, United States Consumer Product Safety Commission, 2010. http://www.cpsc.gov/PageFiles/126521/phthalover.pdf (accessed March 31, 2015).

M.A. Peraza, A.D. Burdick, H.E. Marin, F.J. Gonzalez, J.M. Peters, The Toxicology of Ligands for Peroxisome Proliferator-Activated Receptors (PPAR), Toxicological Sciences 90 (2006) 269–295. https://doi.org/10.1093/toxsci/kfj062.

I.R. Rowland, R.C. Cottrell, J.C. Phillips, Hydrolysis of phthalate esters by the gastro-intestinal contents of the rat, Food and Cosmetics Toxicology 15 (1977) 17–21. https://doi.org/10.1016/S0015-6264(77)80257-5.

X.-J. Wang, G.-P. Xiong, X.-M. Luo, S.-Z. Huang, J. Liu, X.-L. Huang, Y.-Z. Xie, W.-P. Lin, Dibutyl Phthalate Inhibits the Effects of Follicle-Stimulating Hormone on Rat Granulosa Cells Through Down-Regulation of Follicle-Stimulating Hormone Receptor1, Biology of Reproduction 94 (2016) 144, 1–13. https://doi.org/10.1095/biolreprod.115.136002.

Enzymatic hydrolysis of esters of 2,4-dichlorophenoxyacetic acid (2,4-D) generates 2,4-D acid (Schulze, 1985), which is an antagonist of phytohormone auxin, overstimulating plant growth, more than it can afford (Song, 2014). Further details are provided on MechoA 6.9 (Inhibition of auxin hormone).

Figure 2.3.2: Structures of 2,4-D butoxyethanol ester (left) and 2,4-D 2-ethylhexyl ester (right)

Song, Y. (2014). Insight into the mode of action of 2,4-dichlorophenoxyacetic acid (2,4-D) as an herbicide. Journal of Integrative Plant Biology, 56(2), 106–113. https://doi.org/10.1111/jipb.12131.

Biological process description under ‘normal’ circumstances

All living organisms perform biological processes by the use of specialised proteins, which are made of building blocks called amino-acids. The varied structures of the side chains of these amino-acids allow the proteins to have different shapes and function based on their amino-acid sequence. All living organisms also use DNA to code for genes, which effectively serve as recipes to build the above-mentioned proteins.

Gan J., Zhang H., Humphreys W.G. (2016)  Drug-Protein Adducts—Chemistry, Mechanisms of Toxicity, and Methods of Characterization. Chem. Res. Toxicol. 29(12). DOI: 10.1021/acs.chemrestox.6b00274

Jacobs A., Hard and soft nucleophiles, in: Understanding Organic Reaction Mechanisms, Cambridge University Press, 1997: pp. 45–46.

Hard electrophiles sites have a significant lack of electrons due to the presence of an electron-withdrawing group at their proximity. Often it can be a good leaving group (nucleofuge).

Substances with MechoA 3.1 can form adducts with residues of proteins and genetic material (DNA and RNA).

Enoch et al. (2010) and Enoch et al. (2011) characterized several structural alerts with reactive MIEs based on chemical knowledge and experimental studies in various endpoints where adduct formation could be associated with distinct toxicity profiles.

Typical substances with MechoA 3.1 are aldehydes, epoxides and some other compounds which have in general an electrophilic site which can easily react with side chain of amino acids of proteins. In general, these substances may also have a good leaving group close to the electrophilic site that also favour the reactivity of the substance.

Electrophilic adducts form preferentially at thiol (cysteine) and amine (lysine, histidine) residues and N-terminal amine on proteins (Chipinda et al., 2011) although sometimes it can also react with serine (Santoni et al., 2021). Reaction with DNA is also possible, the most common sites for DNA adducts formation are N7, N2 and O6 of guanine bases (Pavanello & Lotti, 2014).

I. Chipinda, J.M. Hettick, P.D. Siegel, Haptenation: Chemical Reactivity and Protein Binding, Journal of Allergy 2011 (2011) 1–11. https://doi.org/10.1155/2011/839682.

S.J. Enoch, C.M. Ellison, T.W. Schultz, M.T.D. Cronin, A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity, Critical Reviews in Toxicology 41 (2011) 783–802. https://doi.org/10.3109/10408444.2011.598141.

S.J. Enoch, M.T.D. Cronin, A review of the electrophilic reaction chemistry involved in covalent DNA binding, Critical Reviews in Toxicology 40 (2010) 728–748. https://doi.org/10.3109/10408444.2010.494175.

S. Pavanello, M. Lotti, Biomonitoring exposures to carcinogens. In: Biomarkers in Toxicology (2014) 785-798. https://doi.org/10.1016/B978-0-12-404630-6.00047-6.

I. Santoni, B. Pizzo, Improvement of Water Resistance of Vegetable Proteins by the Use of Synthetic Origin Additives: Trials with Resins and Metal Ions, Coatings 11 (2021) 859. https://doi.org/10.3390/coatings11070859.

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Some well known reactions which can occur in this MechoA are illustrated in the Figure below.

Figure 3.1.1: example of adducts formation by hard electrophiles, example of reaction with lysine residues. The same kind of reaction might be expected for other nucleophilic amino-acids (e.g. serine, cysteine) and DNA bases. X can be any good leaving group, R1, R2,...can be any substituents, Y1,Y2,... are electron withdrawing groups

Reaction A and B are related to aldehydes, and to epoxides, thiiranes or aziridines. Moreover, reaction A describes Schiff base formation where the reaction between an aldehyde and an amine results in a carbon-nitrogen double bond (C=N, imine), thus forming adducts, potentially resulting in toxic effects.

These reactive compounds (aldehydes, epoxides, thiiranes or aziridines) often occur as intermediates in normal metabolic processes, such as oxidation and reduction reactions (Dunn et al., 2009). Indeed, both have a medium degree of oxidation.

Aldehydes can be an intermediate step in the detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be more easily eliminated.

Epoxides are often observed as intermediates in metabolic process. They are transiently used when hydrocarbons are transformed into diols to be eliminated as is the case for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism, but this is always a transient reaction. They are rapidly transformed by enzymes in order to prevent their concentration in the tissues reaching toxic levels.

In reaction C (see figure above), X typically corresponds to a halogen, therefore, in this case, a strong acid will be released (hydrofluoric, hydrochloric acid, etc.) while the carbonyl is attached to the protein (acylation). The same reaction also rapidly happens with water as a nucleophile instead of biological material which protects against acylation but not against the production of acidity (see MechoA 3.4). When substance are acid anhydrides, similar mechanism is also expected.

Reaction D (see figure above) is possible for compounds with a good leaving group branched to a molecule with a favoured transition state, thanks to a mesomeric effect for instance. The leaving group must be stabilised once separated from the "parent" molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl or allyl group, such as benzyl chloride.

Reaction E is called a nucleophilic aromatic substitution and typically occurs with X as a halogen (though other leaving groups are possible) and requires the aromatic cycle to be electron-deficient through the substitution by electron-withdrawing groups such as nitros.

F is another example of hard-electrophile reactivity mechanism that happens for isocyanates (Slatter, 1991).

Additionally to these common examples, many other kind of hard electrophile exists.

Some γ-diketones, with specific structural conditions, can form a pyrrole ring with amino residues of proteins and lead to protein cross-linking. The prototypical example of this category is hexane-2,5-dione (see section MechoA 4.3, iii.).

Dunn, M. F., Ramírez-Trujillo, J. A., & Hernández-Lucas, I. (2009). Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology, 155(10), 3166‑3175. https://doi.org/10.1099/mic.0.030858-0.

J.G. Slatter, M.S. Rashed, P.G. Pearson, D.H. Han, T.A. Baillie, Biotransformation of methyl isocyanate in the rat. Evidence for glutathione conjugation as a major pathway of metabolism and implications for isocyanate-mediated toxicities, Chem. Res. Toxicol. 4 (1991) 157–161. https://doi.org/10.1021/tx00020a006. 

Isothiazolinones are compounds used in cosmetics and more widely in chemical additives found in antifouling products due to their bacteriostatic and fungistatic properties. Isothiazolinones are proved to react with thiol-containing biomolecules of living organisms. They also inhibit the aldose reductase and act as antagonists of the 5-hydroxytryptamine receptors, commonly known as serotonin receptors (Silva et al., 2020).

Figure 3.1.2: Chemical structures of isothiazole and of the most frequently used isothiazolinone biocides in consumer products (Silva et al., 2020).

The toxicity of isothiazolinones compounds is explained by the presence of an electron-deficient sulfur at the N–S bond within these compounds which react with the nucleophilic groups of the cellular components (such as the thiols from cysteine residues of proteins active sites), blocking their enzymatic activity (Silva et al., 2020).

Most remarkable example is observed with a thiol-containing peptide known as glutathione (GSH) which reacts with this biocide to form disulfide derivatives (Silva et al., 2020) :

Figure 3.1.3: Reaction pathway for the interaction of isothiazolinones and thiols from cellular components (e.g., glutathione (GSH)) (Cóllier et al., 1990)).

P.J. Cóllier, A. Ramsey, R.D. Waigh, K.T. Douglas, P. Austin, P. Gilbert, Chemical reactivity of some isothiazolone biocides, J Appl Bacteriol 69 (1990) 578–584. https://doi.org/10.1111/j.1365-2672.1990.tb01551.x.

V. Silva, C. Silva, P. Soares, E.M. Garrido, F. Borges, J. Garrido, Isothiazolinone Biocides: Chemistry, Biological, and Toxicity Profiles, Molecules 25 (2020) 991. https://doi.org/10.3390/molecules25040991.

Mustard gas is a potent alkylating agent whose toxicity arises from the formation of reactive electrophilic intermediates. This molecule holds beta-chloroethyl groups attached to a sulphur atom. Similar molecules, which can have a nitrogen in place of the sulphur also display the same mechanism of toxicity: upon exposure to water or within biological environments, an intramolecular cyclization occurs, displacing a chloride ion and forming a three-membered cyclic episulfonium (e.g. C1[S⁺]C1) or aziridinium ion (e.g. C1[N⁺]C1) (Q.-Q. Wang et al., 2012).

They readily react with nucleophiles leading to a cross-linking process with DNA strands (e.g. aromatic amine of guanine). When bypassing the mechanism of reparation of DNA, such adducts interfere with replication and transcription, causing cytotoxicity (Benigni et al., 2000; Sharma et al., 2010). More recently, further studies have also assessed the formation of adducts with others endogenous biomolecules (Bielmann et al., 2018). Scientists are now considering less potent mustard gas derivatives as interesting biomarkers in several pathologies.

Figure 3.1.4: Formation of the reactive episulfonium ion and examples of bioadducts formed (DNA is the most common one but it is also observed with Glutathione (GSH) and albumin) (Bielmann et al., 2018).

R. Benigni, A. Giuliani, R. Franke, A. Gruska, Quantitative structure-activity relationships of mutagenic and carcinogenic aromatic amines, Chem Rev 100 (2000) 3697–3714. https://doi.org/10.1021/cr9901079.

A. Bielmann, N. Sambiagio, N. Wehr, S. Gerber-Lemaire, C.G. Bochet, C. Curty, Synthesis of different glutathione–sulfur mustard adducts of verified and potential biomarkers, RSC Adv. 8 (2018) 23881–23890. https://doi.org/10.1039/c8ra03360a.

M. Sharma, R. Vijayaraghavan, O.P. Agrawal, Comparative Toxic Effect of Nitrogen Mustards (HN-1, HN-2, and HN-3) and Sulfur Mustard on Hematological and Biochemical Variables and Their Protection by DRDE-07 and Its Analogues, Int J Toxicol 29 (2010) 391–401. https://doi.org/10.1177/1091581810365730.

Q.-Q. Wang, R.A. Begum, V.W. Day, K. Bowman-James, Sulfur, oxygen, and nitrogen mustards: stability and reactivity, Org. Biomol. Chem. 10 (2012) 8786–8793. https://doi.org/10.1039/C2OB26482J.

While β-lactams act as antibiotics by primarily targeting bacterial enzymes, some studies show they can also form adducts with DNA, though this is not their main mode of action (see MechoA 3.1).

β-lactams have enhanced electrophilicity because of their strained 4-membered cycle, easily reacting with nucleophilic amino acid side chain, including those essential to proper enzyme function, and DNA bases (Konaklieva, 2014).

According to studies, two major hypotheses emerge concerning beta lactams and lactones electrophilic reactivity:

-1^st hypothesis: nucleophilic attack at the beta carbon

-2^nd hypothesis: nucleophilic attack at the carbonyl. This mechanism is exemplified with the mechanism of inhibition of bacterial transpeptidase by a b-lactam antibiotic, or degradation of that antibiotic by the beta-lactamase enzyme.

Figure 3.1.5: 1^st hypothesis: Ring opening reaction for the four membered ring system of a beta-propiolactone (Enoch et al., 2011); 2^nd hypothesis: Beta-lactams antibiotics as substrates for transpeptidases and beta lactamases (van Bambeke et al., 2017)

Such interactions have been observed in vitro and may contribute to genotoxicity at high concentrations. Some β-lactam-derived metabolites (e.g., phthalazine substituted β-lactam derivatives) are more reactive and may contribute to low-level DNA damage (Aygün et al., 2022).

However, primary electrophilic activity of β-lactams remains protein acylation, not DNA alkylation.

S.J. Enoch, C.M. Ellison, T.W. Schultz, M.T.D. Cronin, A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity, Critical Reviews in Toxicology 41 (2011) 783–802. https://doi.org/10.3109/10408444.2011.598141.

Aygün, B., Berber, A. A., Doganci, M. A., Berber, N., Şen, S., Yildiz, E., & Aksoy, H. (2022). Genotoxicity evaluation of a new phthalazine substituted β-lactam derivative in human lymphocytes. Anais da Academia Brasileira de Ciências, 94(1). https://doi.org/10.1590/0001-3765202120191476.

Konaklieva, M. I. (2014). Molecular Targets of β-Lactam-Based Antimicrobials : Beyond the Usual Suspects. Antibiotics, 3(2), 128‑142. https://doi.org/10.3390/antibiotics3020128.

van Bambeke, F., Mingeot-Leclercq, M.-P., Glupczynski, Y., & Tulkens, P. M. (2017). Mechanisms of Action. In Infectious Diseases (p. 1162-1180.e1). Elsevier. https://doi.org/10.1016/B978-0-7020-6285-8.00137-4.

N-acyloxy-N-alkoxyamides (NAAs) are highly electrophilic due to their pyramidal nitrogen, bonded to two oxygen atoms. This electrophilicity facilitates direct alkylation of DNA bases, without need of metabolic activation either through the formation of a nitrenium ion intermediate via an autocatalytic process (SN1 reaction), or through a SN2 reaction (Bonin et al., 2001).

Figure 3.1.6: Formation of nitrenium ions by an autocatalytic process in acidic medium and further substitution of the nitrenium ion (via SN1 mechanism) (Bonin et al., 2001)

NAAs mutagenicity arises from covalent DNA adduct formation, especially at nucleophilic sites like guanine (attached to C8 of the guanine). The electrophilic centre is stabilized by mesomeric effects, enhancing reactivity toward DNA. Hydrophobicity aids cellular uptake but is secondary to electrophilic strength in determining mutagenicity (Bonin et al., 2001).

Some N-acyloxy-N-alkoxyamides (NAA), like N-acetoxy-N-butoxynaphthamide, show enhanced mutagenicity due to additional DNA intercalation. The electrophilic moiety is thus positioned near DNA bases, increasing adduct formation likelihood. Steric hindrance around the electrophilic centre can reduce DNA reactivity and mutagenicity (Bonin et al., 2001).

A.M. Bonin, T.M. Banks, J.J. Campbell, S.A. Glover, G.P. Hammond, A.S. Prakash, C.A. Rowbottom, Mutagenicity of electrophilic N-acyloxy-N-alkoxyamides, Mutat Res 494 (2001) 115–134. https://doi.org/10.1016/s1383-5718(01)00189-9.

Nitrosoamines, nitrosoguanidines and nitrosoureas are also direct-acting alkylating agents. They are able to react as hard electrophiles with proteins or DNA (Chipinda, 2011; Enoch, 2011; Carlson, 2016).

Figure 3.1.7: Structure of nitrosamines, nitrosoamines, nitrosoguanidines and nitrosoureas (left to right)

E.S. Carlson, P. Upadhyaya, S.S. Hecht, Evaluation of Nitrosamide Formation in the Cytochrome P450-Mediated Metabolism of Tobacco-Specific Nitrosamines, Chem. Res. Toxicol. 29 (2016) 2194–2205. https://doi.org/10.1021/acs.chemrestox.6b00384.

I. Chipinda, J.M. Hettick, P.D. Siegel, Haptenation: Chemical Reactivity and Protein Binding, Journal of Allergy 2011 (2011) 1–11. https://doi.org/10.1155/2011/839682.

S.J. Enoch, C.M. Ellison, T.W. Schultz, M.T.D. Cronin, A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity, Critical Reviews in Toxicology 41 (2011) 783–802. https://doi.org/10.3109/10408444.2011.598141.

Contrarily to hard electrophiles, soft electrophiles have a spread out and deformable electron cloud. They preferentially react with soft nucleophiles such as thiol residues which often occur in biological content (e.g. as lateral chain of cysteine or in glutathione molecule (GSH)) (Schwöbel, 2010; Jackson, 2017; LoPachin, 2014; LoPachin, 2019; Liang, 2022; Obach & Kalgutkar, 2010).

P.A. Jackson, J.C. Widen, D.A. Harki, K.M. Brummond, Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions, J Med Chem 60 (2017) 839–885. https://doi.org/10.1021/acs.jmedchem.6b00788.

S.-T. Liang, C. Chen, R.-X. Chen, R. Li, W.-L. Chen, G.-H. Jiang, L.-L. Du, Michael acceptor molecules in natural products and their mechanism of action, Frontiers in Pharmacology 13 (2022). https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2022.1033003 (accessed February 26, 2024).

R.M. LoPachin, T. Gavin, Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective, Chem Res Toxicol 27 (2014) 1081–1091. https://doi.org/10.1021/tx5001046.

R.M. LoPachin, B.C. Geohagen, L.U. Nordstroem, Mechanisms of Soft and Hard Electrophile Toxicities, Toxicology 418 (2019) 62–69. https://doi.org/10.1016/j.tox.2019.02.005.

 R. S. Obach, A.S. Kalgutkar, 1.15 - Reactive Electrophiles and Metabolic Activation, in: C.A. McQueen (Ed.), Comprehensive Toxicology (Second Edition), Elsevier, Oxford, 2010: pp. 309–347. https://doi.org/10.1016/B978-0-08-046884-6.00115-9.

J.A.H. Schwöbel, Y.K. Koleva, S.J. Enoch, F. Bajot, M. Hewitt, J.C. Madden, D.W. Roberts, T.W. Schultz, M.T.D. Cronin, Measurement and Estimation of Electrophilic Reactivity for Predictive Toxicology, Chem. Rev. 111 (2011) 2562–2596. https://doi.org/10.1021/cr100098n.

J.A.H. Schwöbel, D. Wondrousch, Y.K. Koleva, J.C. Madden, M.T.D. Cronin, G. Schüürmann, Prediction of Michael-Type Acceptor Reactivity toward Glutathione, Chem. Res. Toxicol. 23 (2010) 1576–1585. https://doi.org/10.1021/tx100172x.

The most representative soft electrophiles have an α,β-unsaturated carbonyl group, most commonly esters and aldehydes but also quinones which are specific α,β-unsaturated ketones which could also act via another mechanism (see MechoA 4.4). Other electron-withdrawing groups conjugated to a double or triple bond, different from a carbonyl, may also have soft electrophile behaviour like, for instance, α,β-unsaturated nitriles.

Figure 3.2.1: General structure of α,β-unsaturated carbonyl-based compounds or α,β-unsaturated nitriles (R1,R2,R3=any atoms, R4=proton, alkyl or O-alkyl)

These substances and their dedicated reactivity mechanisms (typically by Michael addition (Enoch, 2011; Enoch & Roberts, 2013)) are shown in the figure below.

Figure 3.2.2: Example of adducts formation between proteins (typically cysteine residues, but others can react too) and soft electrophiles.

Often, this type of reactivity leads to strong skin sensitisation potential and also high toxicity in other endpoints such as acute toxicity to fish, probably because of the essential role of cysteine residues that are particularly targeted by these substances.

S.J. Enoch, C.M. Ellison, T.W. Schultz, M.T.D. Cronin, A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity, Critical Reviews in Toxicology 41 (2011) 783–802. https://doi.org/10.3109/10408444.2011.598141.

S.J. Enoch, D.W. Roberts, Predicting Skin Sensitization Potency for Michael Acceptors in the LLNA Using Quantum Mechanics Calculations, Chem. Res. Toxicol. 26 (2013) 767–774. https://doi.org/10.1021/tx4000655.

P.A. Jackson, J.C. Widen, D.A. Harki, K.M. Brummond, Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions, J Med Chem 60 (2017) 839–885. https://doi.org/10.1021/acs.jmedchem.6b00788.

The most representative soft electrophiles have an α,β-unsaturated carbonyl group, most commonly esters and aldehydes but also quinones which are specific α,β-unsaturated ketones which could also act via another mechanism (see MechoA 4.4). Other electron-withdrawing groups conjugated to a double or triple bond, different from a carbonyl, may also have soft electrophile behaviour like, for instance, α,β-unsaturated nitriles.

Figure 3.2.1: General structure of α,β-unsaturated carbonyl-based compounds or α,β-unsaturated nitriles (R1,R2,R3=any atoms, R4=proton, alkyl or O-alkyl)

These substances and their dedicated reactivity mechanisms (typically by Michael addition (Enoch, 2011; Enoch & Roberts, 2013)) are shown in the figure below.

Figure 3.2.2: Example of adducts formation between proteins (typically cysteine residues, but others can react too) and soft electrophiles.

Often, this type of reactivity leads to strong skin sensitisation potential and also high toxicity in other endpoints such as acute toxicity to fish, probably because of the essential role of cysteine residues that are particularly targeted by these substances.

S.J. Enoch, C.M. Ellison, T.W. Schultz, M.T.D. Cronin, A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity, Critical Reviews in Toxicology 41 (2011) 783–802. https://doi.org/10.3109/10408444.2011.598141.

S.J. Enoch, D.W. Roberts, Predicting Skin Sensitization Potency for Michael Acceptors in the LLNA Using Quantum Mechanics Calculations, Chem. Res. Toxicol. 26 (2013) 767–774. https://doi.org/10.1021/tx4000655.

P.A. Jackson, J.C. Widen, D.A. Harki, K.M. Brummond, Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions, J Med Chem 60 (2017) 839–885. https://doi.org/10.1021/acs.jmedchem.6b00788.

The most representative soft electrophiles have an α,β-unsaturated carbonyl group, most commonly esters and aldehydes but also quinones which are specific α,β-unsaturated ketones which could also act via another mechanism (see MechoA 4.4). Other electron-withdrawing groups conjugated to a double or triple bond, different from a carbonyl, may also have soft electrophile behaviour like, for instance, α,β-unsaturated nitriles.

Figure 3.2.1: General structure of α,β-unsaturated carbonyl-based compounds or α,β-unsaturated nitriles (R1,R2,R3=any atoms, R4=proton, alkyl or O-alkyl)

These substances and their dedicated reactivity mechanisms (typically by Michael addition (Enoch, 2011; Enoch & Roberts, 2013)) are shown in the figure below.

Figure 3.2.2: Example of adducts formation between proteins (typically cysteine residues, but others can react too) and soft electrophiles.

Often, this type of reactivity leads to strong skin sensitisation potential and also high toxicity in other endpoints such as acute toxicity to fish, probably because of the essential role of cysteine residues that are particularly targeted by these substances.

S.J. Enoch, C.M. Ellison, T.W. Schultz, M.T.D. Cronin, A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity, Critical Reviews in Toxicology 41 (2011) 783–802. https://doi.org/10.3109/10408444.2011.598141.

S.J. Enoch, D.W. Roberts, Predicting Skin Sensitization Potency for Michael Acceptors in the LLNA Using Quantum Mechanics Calculations, Chem. Res. Toxicol. 26 (2013) 767–774. https://doi.org/10.1021/tx4000655.

P.A. Jackson, J.C. Widen, D.A. Harki, K.M. Brummond, Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions, J Med Chem 60 (2017) 839–885. https://doi.org/10.1021/acs.jmedchem.6b00788.

The most representative soft electrophiles have an α,β-unsaturated carbonyl group, most commonly esters and aldehydes but also quinones which are specific α,β-unsaturated ketones which could also act via another mechanism (see MechoA 4.4). Other electron-withdrawing groups conjugated to a double or triple bond, different from a carbonyl, may also have soft electrophile behaviour like, for instance, α,β-unsaturated nitriles.

Figure 3.2.1: General structure of α,β-unsaturated carbonyl-based compounds or α,β-unsaturated nitriles (R1,R2,R3=any atoms, R4=proton, alkyl or O-alkyl)

These substances and their dedicated reactivity mechanisms (typically by Michael addition (Enoch, 2011; Enoch & Roberts, 2013)) are shown in the figure below.

Figure 3.2.2: Example of adducts formation between proteins (typically cysteine residues, but others can react too) and soft electrophiles.

Often, this type of reactivity leads to strong skin sensitisation potential and also high toxicity in other endpoints such as acute toxicity to fish, probably because of the essential role of cysteine residues that are particularly targeted by these substances.

S.J. Enoch, C.M. Ellison, T.W. Schultz, M.T.D. Cronin, A review of the electrophilic reaction chemistry involved in covalent protein binding relevant to toxicity, Critical Reviews in Toxicology 41 (2011) 783–802. https://doi.org/10.3109/10408444.2011.598141.

S.J. Enoch, D.W. Roberts, Predicting Skin Sensitization Potency for Michael Acceptors in the LLNA Using Quantum Mechanics Calculations, Chem. Res. Toxicol. 26 (2013) 767–774. https://doi.org/10.1021/tx4000655.

P.A. Jackson, J.C. Widen, D.A. Harki, K.M. Brummond, Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions, J Med Chem 60 (2017) 839–885. https://doi.org/10.1021/acs.jmedchem.6b00788.

Peroxides and disulfides are the main examples of radical generation.

These radical-generating compounds have a simple, weak oxygen-oxygen (or sulfur-sulfur) bond which can easily break in a homolytic way (i.e. leaving one single electron on each side and not giving the whole electron pair to just one of the products).

When this bond breaks, the electrons within the bond are separated thus creating two free radicals. These highly reactive entities can directly form adducts or rather be trapped by oxygen to form superoxide anion. This chemical species is highly harmful to organisms because it reacts with endogenous molecules (lipids, proteins and genetic material) to transform them into peroxides altering their function (Bialas, 2023; Clark, 2001; Kazius 2005; Munday, 1989; Wiley-VCH Verlag, 2002a).

Figure 3.3.1: Example of degradation of acyl peroxides leading to the formation of superoxide anion and damage to proteins.

I. Bialas, S. Zelent-Kraciuk, K. Jurowski, The Skin Sensitisation of Cosmetic Ingredients: Review of Actual Regulatory Status, Toxics 11 (2023) 392. https://doi.org/10.3390/toxics11040392.

D.E. Clark, Peroxides and peroxide-forming compounds, Chem. Health Saf. 8 (2001) 12–22. https://doi.org/10.1016/S1074-9098(01)00247-7.

J. Kazius, R. McGuire, R. Bursi, Derivation and Validation of Toxicophores for Mutagenicity Prediction, J. Med. Chem. 48 (2005) 312–320. https://doi.org/10.1021/jm040835a.

R. Munday, Toxicity of thiols and disulphides: Involvement of free-radical species, Free Radical Biology and Medicine 7 (1989) 659–673. https://doi.org/10.1016/0891-5849(89)90147-0.

Wiley-VCH Verlag. (2002a). Organic peroxides [MAK Value Documentation, 1992]. In The MAK-Collection for Occupational Health and Safety. Wiley-VCH Verlag GmbH & Co. KGaA. https://doi.org/10.1002/3527600418.mb0opexgrpe0003.

Peroxides and disulfides are the main examples of radical generation.

These radical-generating compounds have a simple, weak oxygen-oxygen (or sulfur-sulfur) bond which can easily break in a homolytic way (i.e. leaving one single electron on each side and not giving the whole electron pair to just one of the products).

When this bond breaks, the electrons within the bond are separated thus creating two free radicals. These highly reactive entities can directly form adducts or rather be trapped by oxygen to form superoxide anion. This chemical species is highly harmful to organisms because it reacts with endogenous molecules (lipids, proteins and genetic material) to transform them into peroxides altering their function (Bialas, 2023; Clark, 2001; Kazius 2005; Munday, 1989; Wiley-VCH Verlag, 2002a).

Figure 3.3.1: Example of degradation of acyl peroxides leading to the formation of superoxide anion and damage to proteins.

I. Bialas, S. Zelent-Kraciuk, K. Jurowski, The Skin Sensitisation of Cosmetic Ingredients: Review of Actual Regulatory Status, Toxics 11 (2023) 392. https://doi.org/10.3390/toxics11040392.

D.E. Clark, Peroxides and peroxide-forming compounds, Chem. Health Saf. 8 (2001) 12–22. https://doi.org/10.1016/S1074-9098(01)00247-7.

J. Kazius, R. McGuire, R. Bursi, Derivation and Validation of Toxicophores for Mutagenicity Prediction, J. Med. Chem. 48 (2005) 312–320. https://doi.org/10.1021/jm040835a.

R. Munday, Toxicity of thiols and disulphides: Involvement of free-radical species, Free Radical Biology and Medicine 7 (1989) 659–673. https://doi.org/10.1016/0891-5849(89)90147-0.

Wiley-VCH Verlag. (2002a). Organic peroxides [MAK Value Documentation, 1992]. In The MAK-Collection for Occupational Health and Safety. Wiley-VCH Verlag GmbH & Co. KGaA. https://doi.org/10.1002/3527600418.mb0opexgrpe0003.

This mechanism of action is exerted by dialkylsulfates, alkylsulfonates, acyl halides, acid anhydrides, possibly enones, among others.

Compared to other mechanisms, MechoA 3.4 describes substances that react with abiotic medium before interaction with biological matrices. More accurately, this mechanism is often observed for compounds which are unstable or extremely sensitive to air, light, water or traces of metals for example. In most cases, the products are formed after oxidation or hydrolysis (Goodman et al., 2008; Nelson et al., 2013).

Alkyl benzenesulfonate esters, as alkylating agents, react with nucleic acids via a nucleophilic substitution (Eder et al., 1989). However, they also react with water as a nucleophile, which is effectively a hydrolysis reaction, generating a sulfonic acid and an alcohol. Sulfonic acids are strong acids and are corrosive, so this hydrolysis in itself is already the source of some adverse effects, while the molecules that did not first react with water will further be able to alkylate proteins and DNA generating further adverse effects such as mutagenicity (Glowienke, 2005).

Figure 3.4.1: Mechanism of alkyl benzenesulfonates compounds.

Likely, some sulfonyl chloride (e.g. 4-Toluenesulfonyl chloride or methanesulfonyl chloride) have a similar behaviour as alkyl benzenesulfonate esters reacting with both water and nucleophilic moiety found in biological medium.

Acyl halides as well, acetyl chloride for instance, violently react with water generating strong acidity. Acid anhydrides also react in a similar manner.

Figure 3.4.2: Exothermic reaction observed when acetyl chloride is in contact with water (formed hydrochloric acid appears as a steamy gas)

Furthermore, because of its strong reactivity it is advised to avoid contact of acyl-chloride (e.g. acetyl chloride) with some chemicals such as alkaline earth metals, alcohols, alkali metals, strong oxidizing agents and non-metallic halides. It has even been proven that explosive reactions and toxic gas might occur (Sigma, 2017).

A few other substances are hypothesised to have a MechoA 3.4 based on observations in ecotoxicity tests, though no literature reference was found to support such hypotheses. Some of these substances are enones (a,b-unsaturated ketones) and propargylic tertiary alcohols.

Eder, E., Deininger, C., & Kütt, W. (1989). Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 211(1), 51‑64. https://doi.org/10.1016/0027-5107(89)90106-1.

S. Glowienke, W. Frieauff, T. Allmendinger, H.-J. Martus, W. Suter, L. Mueller, Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters, Mutat Res 581 (2005) 23–34. https://doi.org/10.1016/j.mrgentox.2004.10.004.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

Sigma-Aldrich, 2017; Safety Data Sheet for Acetyl chloride.

This mechanism of action is exerted by dialkylsulfates, alkylsulfonates, acyl halides, acid anhydrides, possibly enones, among others.

Compared to other mechanisms, MechoA 3.4 describes substances that react with abiotic medium before interaction with biological matrices. More accurately, this mechanism is often observed for compounds which are unstable or extremely sensitive to air, light, water or traces of metals for example. In most cases, the products are formed after oxidation or hydrolysis (Goodman et al., 2008; Nelson et al., 2013).

Alkyl benzenesulfonate esters, as alkylating agents, react with nucleic acids via a nucleophilic substitution (Eder et al., 1989). However, they also react with water as a nucleophile, which is effectively a hydrolysis reaction, generating a sulfonic acid and an alcohol. Sulfonic acids are strong acids and are corrosive, so this hydrolysis in itself is already the source of some adverse effects, while the molecules that did not first react with water will further be able to alkylate proteins and DNA generating further adverse effects such as mutagenicity (Glowienke, 2005).

Figure 3.4.1: Mechanism of alkyl benzenesulfonates compounds.

Likely, some sulfonyl chloride (e.g. 4-Toluenesulfonyl chloride or methanesulfonyl chloride) have a similar behaviour as alkyl benzenesulfonate esters reacting with both water and nucleophilic moiety found in biological medium.

Acyl halides as well, acetyl chloride for instance, violently react with water generating strong acidity. Acid anhydrides also react in a similar manner.

Figure 3.4.2: Exothermic reaction observed when acetyl chloride is in contact with water (formed hydrochloric acid appears as a steamy gas)

Furthermore, because of its strong reactivity it is advised to avoid contact of acyl-chloride (e.g. acetyl chloride) with some chemicals such as alkaline earth metals, alcohols, alkali metals, strong oxidizing agents and non-metallic halides. It has even been proven that explosive reactions and toxic gas might occur (Sigma, 2017).

A few other substances are hypothesised to have a MechoA 3.4 based on observations in ecotoxicity tests, though no literature reference was found to support such hypotheses. Some of these substances are enones (a,b-unsaturated ketones) and propargylic tertiary alcohols.

Eder, E., Deininger, C., & Kütt, W. (1989). Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 211(1), 51‑64. https://doi.org/10.1016/0027-5107(89)90106-1.

S. Glowienke, W. Frieauff, T. Allmendinger, H.-J. Martus, W. Suter, L. Mueller, Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters, Mutat Res 581 (2005) 23–34. https://doi.org/10.1016/j.mrgentox.2004.10.004.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

Sigma-Aldrich, 2017; Safety Data Sheet for Acetyl chloride.

This mechanism of action is exerted by dialkylsulfates, alkylsulfonates, acyl halides, acid anhydrides, possibly enones, among others.

Compared to other mechanisms, MechoA 3.4 describes substances that react with abiotic medium before interaction with biological matrices. More accurately, this mechanism is often observed for compounds which are unstable or extremely sensitive to air, light, water or traces of metals for example. In most cases, the products are formed after oxidation or hydrolysis (Goodman et al., 2008; Nelson et al., 2013).

Alkyl benzenesulfonate esters, as alkylating agents, react with nucleic acids via a nucleophilic substitution (Eder et al., 1989). However, they also react with water as a nucleophile, which is effectively a hydrolysis reaction, generating a sulfonic acid and an alcohol. Sulfonic acids are strong acids and are corrosive, so this hydrolysis in itself is already the source of some adverse effects, while the molecules that did not first react with water will further be able to alkylate proteins and DNA generating further adverse effects such as mutagenicity (Glowienke, 2005).

Figure 3.4.1: Mechanism of alkyl benzenesulfonates compounds.

Likely, some sulfonyl chloride (e.g. 4-Toluenesulfonyl chloride or methanesulfonyl chloride) have a similar behaviour as alkyl benzenesulfonate esters reacting with both water and nucleophilic moiety found in biological medium.

Acyl halides as well, acetyl chloride for instance, violently react with water generating strong acidity. Acid anhydrides also react in a similar manner.

Figure 3.4.2: Exothermic reaction observed when acetyl chloride is in contact with water (formed hydrochloric acid appears as a steamy gas)

Furthermore, because of its strong reactivity it is advised to avoid contact of acyl-chloride (e.g. acetyl chloride) with some chemicals such as alkaline earth metals, alcohols, alkali metals, strong oxidizing agents and non-metallic halides. It has even been proven that explosive reactions and toxic gas might occur (Sigma, 2017).

A few other substances are hypothesised to have a MechoA 3.4 based on observations in ecotoxicity tests, though no literature reference was found to support such hypotheses. Some of these substances are enones (a,b-unsaturated ketones) and propargylic tertiary alcohols.

Eder, E., Deininger, C., & Kütt, W. (1989). Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 211(1), 51‑64. https://doi.org/10.1016/0027-5107(89)90106-1.

S. Glowienke, W. Frieauff, T. Allmendinger, H.-J. Martus, W. Suter, L. Mueller, Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters, Mutat Res 581 (2005) 23–34. https://doi.org/10.1016/j.mrgentox.2004.10.004.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

Sigma-Aldrich, 2017; Safety Data Sheet for Acetyl chloride.

This mechanism of action is exerted by dialkylsulfates, alkylsulfonates, acyl halides, acid anhydrides, possibly enones, among others.

Compared to other mechanisms, MechoA 3.4 describes substances that react with abiotic medium before interaction with biological matrices. More accurately, this mechanism is often observed for compounds which are unstable or extremely sensitive to air, light, water or traces of metals for example. In most cases, the products are formed after oxidation or hydrolysis (Goodman et al., 2008; Nelson et al., 2013).

Alkyl benzenesulfonate esters, as alkylating agents, react with nucleic acids via a nucleophilic substitution (Eder et al., 1989). However, they also react with water as a nucleophile, which is effectively a hydrolysis reaction, generating a sulfonic acid and an alcohol. Sulfonic acids are strong acids and are corrosive, so this hydrolysis in itself is already the source of some adverse effects, while the molecules that did not first react with water will further be able to alkylate proteins and DNA generating further adverse effects such as mutagenicity (Glowienke, 2005).

Figure 3.4.1: Mechanism of alkyl benzenesulfonates compounds.

Likely, some sulfonyl chloride (e.g. 4-Toluenesulfonyl chloride or methanesulfonyl chloride) have a similar behaviour as alkyl benzenesulfonate esters reacting with both water and nucleophilic moiety found in biological medium.

Acyl halides as well, acetyl chloride for instance, violently react with water generating strong acidity. Acid anhydrides also react in a similar manner.

Figure 3.4.2: Exothermic reaction observed when acetyl chloride is in contact with water (formed hydrochloric acid appears as a steamy gas)

Furthermore, because of its strong reactivity it is advised to avoid contact of acyl-chloride (e.g. acetyl chloride) with some chemicals such as alkaline earth metals, alcohols, alkali metals, strong oxidizing agents and non-metallic halides. It has even been proven that explosive reactions and toxic gas might occur (Sigma, 2017).

A few other substances are hypothesised to have a MechoA 3.4 based on observations in ecotoxicity tests, though no literature reference was found to support such hypotheses. Some of these substances are enones (a,b-unsaturated ketones) and propargylic tertiary alcohols.

Eder, E., Deininger, C., & Kütt, W. (1989). Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 211(1), 51‑64. https://doi.org/10.1016/0027-5107(89)90106-1.

S. Glowienke, W. Frieauff, T. Allmendinger, H.-J. Martus, W. Suter, L. Mueller, Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters, Mutat Res 581 (2005) 23–34. https://doi.org/10.1016/j.mrgentox.2004.10.004.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

Sigma-Aldrich, 2017; Safety Data Sheet for Acetyl chloride.

This mechanism of action is exerted by dialkylsulfates, alkylsulfonates, acyl halides, acid anhydrides, possibly enones, among others.

Compared to other mechanisms, MechoA 3.4 describes substances that react with abiotic medium before interaction with biological matrices. More accurately, this mechanism is often observed for compounds which are unstable or extremely sensitive to air, light, water or traces of metals for example. In most cases, the products are formed after oxidation or hydrolysis (Goodman et al., 2008; Nelson et al., 2013).

Alkyl benzenesulfonate esters, as alkylating agents, react with nucleic acids via a nucleophilic substitution (Eder et al., 1989). However, they also react with water as a nucleophile, which is effectively a hydrolysis reaction, generating a sulfonic acid and an alcohol. Sulfonic acids are strong acids and are corrosive, so this hydrolysis in itself is already the source of some adverse effects, while the molecules that did not first react with water will further be able to alkylate proteins and DNA generating further adverse effects such as mutagenicity (Glowienke, 2005).

Figure 3.4.1: Mechanism of alkyl benzenesulfonates compounds.

Likely, some sulfonyl chloride (e.g. 4-Toluenesulfonyl chloride or methanesulfonyl chloride) have a similar behaviour as alkyl benzenesulfonate esters reacting with both water and nucleophilic moiety found in biological medium.

Acyl halides as well, acetyl chloride for instance, violently react with water generating strong acidity. Acid anhydrides also react in a similar manner.

Figure 3.4.2: Exothermic reaction observed when acetyl chloride is in contact with water (formed hydrochloric acid appears as a steamy gas)

Furthermore, because of its strong reactivity it is advised to avoid contact of acyl-chloride (e.g. acetyl chloride) with some chemicals such as alkaline earth metals, alcohols, alkali metals, strong oxidizing agents and non-metallic halides. It has even been proven that explosive reactions and toxic gas might occur (Sigma, 2017).

A few other substances are hypothesised to have a MechoA 3.4 based on observations in ecotoxicity tests, though no literature reference was found to support such hypotheses. Some of these substances are enones (a,b-unsaturated ketones) and propargylic tertiary alcohols.

Eder, E., Deininger, C., & Kütt, W. (1989). Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 211(1), 51‑64. https://doi.org/10.1016/0027-5107(89)90106-1.

S. Glowienke, W. Frieauff, T. Allmendinger, H.-J. Martus, W. Suter, L. Mueller, Structure-activity considerations and in vitro approaches to assess the genotoxicity of 19 methane-, benzene- and toluenesulfonic acid esters, Mutat Res 581 (2005) 23–34. https://doi.org/10.1016/j.mrgentox.2004.10.004.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

Sigma-Aldrich, 2017; Safety Data Sheet for Acetyl chloride.

This mechanism involves a metabolism step, typically an oxidation, a reduction or a conjugation with an endogenic molecule.

Biological process description under ‘normal’ circumstances

The organism either aims to use xenobiotic substances within metabolic processes (e.g. recovering energy by oxidising carbohydrates and lipids, or production of amino acids from proteins hydrolysis, etc.) or to detoxify and eliminate them by making them more hydrophilic (and generally thereby also less toxic). Xenobiotics become more hydrophilic after oxidation or hydrolysis, and thereafter they can be conjugated with hydrophilic endogenic molecules, such as glutathione, glucuronide or glycine (Goodman et al., 2008; Nelson 2013).

Hydrophilic compounds can be more readily eliminated than hydrophobic ones because they accumulate less in membranes and adipose tissue and will preferentially circulate in more aqueous biological media ultimately being depurated via urine (Goodman et al., 2008; Nelson et al., 2013).

MIE description

Benzyl alcohol is an example of a readily metabolised compound fitting in this MechoA class. It is first oxidised to an acid, then conjugated with glycine. The product is depurated more easily than the parent (Nair, 2001).

However, in certain cases these metabolic pathways may generate compounds that are more toxic than the parent as exemplified in by Kalgutkar et al. (2005). Therefore, the MechoA related to pro-activation have been split into different sub-categories of MechoA to cover a wide variety of metabolic product types.

Taxonomy applicability

These mechanisms of action are often species-dependant because they involve metabolism which varies in path, degree and kinetics between species. For instance, mammals can metabolise 3-methoxyphenol into a catechol derivative which will be toxic through RedOx cycling (Moridani et al., 2003), while fish and protozoa apparently cannot effectively metabolise this substance as no toxic effect from the putative catechol metabolite could be observed in aquatic toxicity tests on 3-methoxyphenol. For these latter species, polar narcosis is expected for 3-methoxyphenol (Ellison et al., 2015a) (see MechoA 4.3 to learn more about this case).

The subclasses of this MechoA class 4 are numbered based on the MechoA of the major metabolite generated. Thus, if the MechoA of the metabolite is a membrane destabiliser (MechoA 1), then the parent is classified as a MechoA 4.1, while if the metabolite is a reactive substance (MechoA 3), then the parent is classified as a MechoA 4.3. The particular case of RedOx cycling where oxidations and reductions of the xenobiotic come one after another in a cyclic way is assigned the subclass number 4.4, which makes sense since there is a continuous involvement of metabolism in this mechanism.

Ellison, C. M., Madden, J. C., Cronin, M. T. D., & Enoch, S. J. (2015a). Investigation of the Verhaar scheme for predicting acute aquatic toxicity : Improving predictions obtained from Toxtree ver. 2.6. Chemosphere, 139, 146‑154. https://doi.org/10.1016/j.chemosphere.2015.06.009

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

A. Kalgutkar, I. Gardner, R. Obach, C. Shaffer, E. Callegari, K. Henne, A. Mutlib, D. Dalvie, J. Lee, Y. Nakai, J. O’Donnell, J. Boer, S. Harriman, A Comprehensive Listing of Bioactivation Pathways of Organic Functional Groups, CDM 6 (2005) 161–225. https://doi.org/10.2174/1389200054021799.

M.Y. Moridani, S.S. Cheon, S. Khan, P.J. O’Brien, Metabolic activation of 3-hydroxyanisole by isolated rat hepatocytes, Chemico-Biological Interactions 142 (2003) 317–333. https://doi.org/10.1016/S0009-2797(02)00125-4.

B. Nair, Final report on the safety assessment of Benzyl Alcohol, Benzoic Acid, and Sodium Benzoate, Int. J. Toxicol. 20 Suppl 3 (2001) 23–50.

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

More complex organisms tend to possess more efficient detoxification processes. It allows for a better protection against a wider range of substances. It protects them from damaging effects mostly from reactive compounds. Glutathione (GSH) and associated enzymes, aka glutathione-S-transferase (GST) and glutathione peroxidase (GPx) are present in higher organisms for this purpose. GSH can be oxidised into glutathione disulfide (GSSG) in order to reduce toxic xenobiotics (Goodman et al., 2008). Specifically, GPx can reduce peroxides while oxidising GSH to GSSG.

Then, GSSG can be converted back into GSH thanks to glutathione reductase and NADPH cofactor consumption (Li, 2009; Liska, 1998). Alternatively, GST catalyses the process of GSH conjugation with reactive molecules. Conjugated molecules are easier to eliminate because they have become more hydrophilic and are no longer as reactive.

Other molecules (amino acids, glucuronic acid, sulphate, acetate, methyl) can be conjugated to xenobiotics to reduce their toxicity or modify their solubility, helping their elimination. Note that conjugation processes are only possible if the xenobiotic already possesses a chemical group that is reactive enough (e.g. epoxide, leaving group like halide, phenol, carboxylic acid, etc.). More inert compounds need to be first activated by oxidation with cytochromes P450 (Liska, 1998). 

These detoxification mechanisms consume limited organism resources like GSH, glucuronic acid, and NAD(P)H. In the case of an exposure to high or chronic doses of xenobiotics, these resources may be drained, limiting the extent of detoxification or preventing it altogether.

Such mechanisms cover a wide range of structures.

Readily detoxified compounds are typically hard electrophiles with a good leaving group, electron-rich aromatic aldehydes, benzylic alcohols, methacrylates and lactic acids and lactates. Other compounds which are not cited here can also be readily detoxified (Nelson et al., 2013).

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

B.G. Lake, Coumarin Metabolism, Toxicity and Carcinogenicity: Relevance for Human Risk Assessment, Food and Chemical Toxicology 37 (1999) 423–453. https://doi.org/10.1016/S0278-6915(99)00010-1.

Li, X. (2009). Glutathione and Glutathione-S-Transferase in Detoxification Mechanisms. In General, Applied and Systems Toxicology. John Wiley & Sons, Ltd. https://doi.org/10.1002/9780470744307.gat166

Liska, D. J. (1998). The detoxification enzyme systems. Alternative Medicine Review: A Journal of Clinical Therapeutic, 3(3), 187‑198.

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

Coumarin and its derivatives are xenobiotics acting as oxidising compounds disrupting some metabolic proteins. The coumarin skeleton contains a polarized carbon-carbon double bond which is easily oxidised as an epoxide (MechoA 4.3). This epoxide can lead to adducts to proteins and DNA. However, primates efficiently metabolise coumarin into 7-hydroxycoumarin allowing its fast elimination before it generates toxic metabolites (Lake, 1999).

Figure 4.1.1: Detoxification of coumarin

B.G. Lake, Coumarin Metabolism, Toxicity and Carcinogenicity: Relevance for Human Risk Assessment, Food and Chemical Toxicology 37 (1999) 423–453. https://doi.org/10.1016/S0278-6915(99)00010-1.

Hypothetically, this mechanism of action is related to substances in which a chemical group initially not hydrolysable would be metabolised (MechoA 4, by oxidation or reduction) to a hydrolysable group (e.g. an ester) so that the metabolite would then be enzymatically hydrolysed (MechoA 2 metabolite).

To date no substance has been identified which corresponds to this definition, so this MechoA subclass just stands here as a placeholder.

M.M. Airaksinen, P.H. Rosenberg, T. Tammisto, A Possible Mechanism of Toxicity of Trifluoroethanol and Other Halothane Metabolites, Acta Pharmacologica et Toxicologica 28 (1970) 299–304. https://doi.org/10.1111/j.1600-0773.1970.tb00556.x.

Y. Koleva, I. Barzilov, Comparative study of mechanism of action of allyl alcohols for different endpoints, 49 (2010) 55–59.

Aromatic hydrocarbons like Polycyclic Aromatic Hydrocarbons (PAHs) are converted into epoxides as intermediate compounds (and later quinones) which are able to cause adverse effects like DNA adducts if the elimination rate is not high enough. Note that an excess of toxicity compared to non-polar narcosis was not observed for fish, daphnids and algae in both acute and chronic exposure to PAHs, based on experimental studies available from ECHA. That may be due to a lack of CYP2E1 in these organisms (Ioannides, 1996). Additionally, when looking at hydroxylated PAHs, toxicity related to polar narcosis might be expected. Thus hydroxylated PAHs are expected to behave the same as polar narcotic phenols.

Figure 4.3.1: Bioconversion of benzo[a]pyrene, a PAH associated with DNA adducts formation inducing genotoxicity (Mesquita et al., 2014)

C. Ioannides, The CYP2E Subfamily - Evolution, in: Cytochromes P450: Metabolic and Toxicological Aspects, CRC Press, 1996: p. 213.

S.R. Mesquita, B. L. Van Drooge, C. Barata, N. Vieira, L. Guimarães, B. Piña, Toxicity of atmospheric particle-bound PAHs: an environmental perspective, Environ Sci Pollut Res 21 (2014) 11623–11633. https://doi.org/10.1007/s11356-014-2628-y.

Aromatic hydrocarbons like Polycyclic Aromatic Hydrocarbons (PAHs) are converted into epoxides as intermediate compounds (and later quinones) which are able to cause adverse effects like DNA adducts if the elimination rate is not high enough. Note that an excess of toxicity compared to non-polar narcosis was not observed for fish, daphnids and algae in both acute and chronic exposure to PAHs, based on experimental studies available from ECHA. That may be due to a lack of CYP2E1 in these organisms (Ioannides, 1996). Additionally, when looking at hydroxylated PAHs, toxicity related to polar narcosis might be expected. Thus hydroxylated PAHs are expected to behave the same as polar narcotic phenols.

Figure 4.3.1: Bioconversion of benzo[a]pyrene, a PAH associated with DNA adducts formation inducing genotoxicity (Mesquita et al., 2014)

Aromatic nitros and aromatic amines (a.k.a. anilines (Begnini et al., 2000)) are respectively reduced and oxidised in mammals to generate nitroso, nitrenium or hydroxylamine compounds, which in turn are reactive (MechoA 3.1) and generate adducts with proteins and DNA:

Figure 4.3.2: Aromatic nitro and aromatic amines both lead to the same reactive metabolites by several oxidoreduction steps.

R. Benigni, A. Giuliani, R. Franke, A. Gruska, Quantitative structure-activity relationships of mutagenic and carcinogenic aromatic amines, Chem Rev 100 (2000) 3697–3714. https://doi.org/10.1021/cr9901079.

M. Mohammed, L.P. Mekala, S. Chintalapati, V.R. Chintalapati, New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2, Journal of Hazardous Materials 385 (2020) 121571. https://doi.org/10.1016/j.jhazmat.2019.121571.

Figure 4.3.2: Aromatic nitro and aromatic amines both lead to the same reactive metabolites by several oxidoreduction steps.

R. Benigni, A. Giuliani, R. Franke, A. Gruska, Quantitative structure-activity relationships of mutagenic and carcinogenic aromatic amines, Chem Rev 100 (2000) 3697–3714. https://doi.org/10.1021/cr9901079.

M. Mohammed, L.P. Mekala, S. Chintalapati, V.R. Chintalapati, New insights into aniline toxicity: Aniline exposure triggers envelope stress and extracellular polymeric substance formation in Rubrivivax benzoatilyticus JA2, Journal of Hazardous Materials 385 (2020) 121571. https://doi.org/10.1016/j.jhazmat.2019.121571.

Hexane is a particular alkane which is specifically metabolised into hexan-2-one, then hexane-2,5-dione in mammals. Hexane-2,5-dione can react with lysine residues of proteins leading to protein cross-linking. These reactions occur more specifically in neurons where they cause neuropathology (Goodman et al., 2008).

Figure 4.3.3: 2,5-hexanedione (or hexane-2,5-dione) binding to lysine residues, leading to pyrrolated proteins formation that are neurotoxic (Spencer & Chen, 2021).

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

P. Spencer, X. Chen, The Role of Protein Adduction in Toxic Neuropathies of Exogenous and Endogenous Origin, Toxics 9 (2021) 98. https://doi.org/10.3390/toxics9050098.

Furthermore, haloalkanes (mentioned in MechoA 1.1) such as halothane can be bioactivated by cytochromes P450 to form adducts to DNA and proteins and generate reactive oxygen species (ROS), leading to lipid peroxidation.

Figure 4.3.4: Halothane and its deleterious effects regarding the oxidation and reduction of two cychromes (Atkinson, 2007)

Another example is chloramphenicol which enables the formation of oxamyl chloride inducing macromolecular adducts (Kalgutkar et al., 2005). 

A.J. Atkinson, ed., Principles of clinical pharmacology, 2nd ed, Academic Press, Amsterdam ; Boston, 2007.

A. Kalgutkar, I. Gardner, R. Obach, C. Shaffer, E. Callegari, K. Henne, A. Mutlib, D. Dalvie, J. Lee, Y. Nakai, J. O’Donnell, J. Boer, S. Harriman, A Comprehensive Listing of Bioactivation Pathways of Organic Functional Groups, CDM 6 (2005) 161–225. https://doi.org/10.2174/1389200054021799.

Some vicinal haloalkanes (i.e. halide atoms substituted on two adjacent carbons, e.g. DCE and DBE, see figure below) use the natural detoxifying process of glutathione to actually generate reactive, toxic intermediates which cause DNA adducts with the N⁷ of guanine residues.

Figure 4.3.5: Structures of dichloroethane (DCE) and dibromoethane (DBE)

This disruption is quite difficult to counter by the organism whereas the only action available would be the modification of gluthatione (GSH/GSH-) transferase concentration putting the organism at greater risk. For 1,2-dibromoethane and 1,2-dichloroethane DNA adducts by glutathione conjugation is indeed observed (Guengerich et al., 1987).

F.P. Guengerich, L.A. Peterson, J.L. Cmarik, N. Koga, P.B. Inskeep, Activation of dihaloalkanes by glutathione conjugation and formation of DNA adducts., Environ Health Perspect 76 (1987) 15–18. https://doi.org/10.1289/ehp.877615.

Furans which are not substituted in both positions 2 and 5, can be oxidised to epoxides, then into α,β-unsaturated dialdehydes. These very reactive compounds form DNA and proteins adducts leading to carcinogenic effects (Food and Drug Administration, 2004; Smith, 2011).

Figure 4.3.6: Potential genotoxic mechanism of furan-induced carcinogenesis (Byrns et al., 2006)

M.C. Byrns, C.C. Vu, J.W. Neidigh, J.-L. Abad, R.A. Jones, L.A. Peterson, Detection of DNA adducts derived from the reactive metabolite of furan, cis-2-butene-1,4-dial, Chem Res Toxicol 19 (2006) 414–420. https://doi.org/10.1021/tx050302k.

Furan in Food, Thermal Treatment; Request for Data and Information, Federal Register (2004). https://www.federalregister.gov/documents/2004/05/10/04-10588/furan-in-food-thermal-treatment-request-for-data-and-information (accessed July 31, 2024).

G.F. Smith, Designing Drugs to Avoid Toxicity, in: G. Lawton, D.R. Witty (Eds.), Progress in Medicinal Chemistry, Elsevier, 2011: pp. 1–47. http://www.sciencedirect.com/science/article/pii/B978012381290200001X (accessed July 24, 2015).

Concerning thiophene cycles, if a substituent in ortho of the sulfur makes it favourable, the ring can be metabolized in a similar way as for furans by mammals, generating proteins & DNA adducts (Smith, 2011). However, current experimental data found point out that this happened only in cases when the thiophene is activated, by a carbonyl for example.

Figure 4.3.7: Metabolism of some substituted thiophenes. Figure adapted from (Smith, 2011). Note: the aldehyde on the right is very reactive and will also generate protein adducts with nucleophiles.

G.F. Smith, Designing Drugs to Avoid Toxicity, in: G. Lawton, D.R. Witty (Eds.), Progress in Medicinal Chemistry, Elsevier, 2011: pp. 1–47. http://www.sciencedirect.com/science/article/pii/B978012381290200001X (accessed July 24, 2015).

Vinyl alkoxy compounds (i.e. compounds with a terminal carbon-carbon double bond linked to an oxygen from e.g. an ester or an ether) have been identified to generate epoxide residues which are known compounds that can induce mutagenicity (Gervasi et al., 1985).

Figure 4.3.8: Proposed scheme showing epoxides groups mutagenicity of vinyl alkoxy compounds that forms DNA adducts inducing mutations (Hernandez & Forkert, 2007)

P.G. Gervasi, L. Citti, M. Del Monte, V. Longo, D. Benetti, Mutagenicity and chemical reactivity of epoxidic intermediates of the isoprene metabolism and other structurally related compounds, Mutation Research/Genetic Toxicology 156 (1985) 77–82. https://doi.org/10.1016/0165-1218(85)90009-6.

L.G. Hernandez, P.-G. Forkert, Inhibition of vinyl carbamate-induced mutagenicity and clastogenicity by the garlic constituent diallyl sulfone in F1 (Big Blue® × A/J) transgenic mice, Carcinogenesis 28 (2007) 1824–1830. https://doi.org/10.1093/carcin/bgm051.

It was reported that alkynes can degrade hemes of CYP450 after being oxidised as described by Smith (2011). Little information is available about the kind of alkynes capable to exert this mechanism of action. However, but-3-yn-1-ol has been clearly identified to be reactive and toxic in some aquatic species by Enoch et al. (2008).

Figure 4.3.9: Structure of but-3-yn-1-ol

S.J. Enoch, J.C. Madden, M.T.D. Cronin, Identification of mechanisms of toxic action for skin sensitisation using a SMARTS pattern based approach, SAR QSAR Environ Res 19 (2008) 555–578. https://doi.org/10.1080/10629360802348985.

G.F. Smith, Designing Drugs to Avoid Toxicity, in: G. Lawton, D.R. Witty (Eds.), Progress in Medicinal Chemistry, Elsevier, 2011: pp. 1–47. http://www.sciencedirect.com/science/article/pii/B978012381290200001X (accessed July 24, 2015).

Small organo-halogenated compounds can generate reactive species. The most studied cases are carbon tetrachloride (Recknagel et al., 1989), chloroform (ATSDR, 1997a) and trichloroethylene (Brüning & Bolt, 2000). All of them are metabolised into very reactive compounds in mammals. For aquatic organisms no evidence was found for excess toxicity beyond non-polar narcosis to date.

Figure 4.3.10: Bioconversion of chloroform and carbon tetrachloride that induce the activation of lipid peroxidation inducing reactive species formation and other hazardous compounds (Wexler, 2024).

ATSDR, Toxicological Profile: Chloroform, (1997). https://www.atsdr.cdc.gov/toxprofiles/tp.asp?id=53&tid=16 (accessed October 26, 2016).

T. Brüning, H.M. Bolt, Renal toxicity and carcinogenicity of trichloroethylene: key results, mechanisms, and controversies, Crit. Rev. Toxicol. 30 (2000) 253–285. https://doi.org/10.1080/10408440091159202.

R.O. Recknagel, E.A. Glende Jr., J.A. Dolak, R.L. Waller, Mechanisms of carbon tetrachloride toxicity, Pharmacology & Therapeutics 43 (1989) 139–154. https://doi.org/10.1016/0163-7258(89)90050-8.

P. Wexler, ed., Encyclopedia of toxicology, Fourth edition, Elsevier, Amsterdam, Netherlands, 2024.

Hydrazines (and similar compounds with a simple N-N bond) are metabolised in several ways depending on their structure (Sinha, 2014), generating free radicals and thus oxidative stress. Besides these, diazonium metabolites are also produced (ATSDR, 1997b). As very reactive electrophiles, diazoniums are recognised as forming DNA adducts (Brown & Vito, 1993). These kinds of compounds have also been identified as inhibitors of aminotransferases (see MechoA 6.9 : Inhibition of aminotransferases- Aspartate aminotransferase inhibitors).

ATSDR, Toxicological Profile: Hydrazines, (1997). http://www.atsdr.cdc.gov/ToxProfiles/tp.asp?id=502&tid=89 (accessed July 24, 2015).

M.A. Brown, S.C.D. Vito, Predicting azo dye toxicity, Critical Reviews in Environmental Science and Technology 23 (1993) 249–324. https://doi.org/10.1080/10643389309388453.

Aromatic azo compounds are likely to be metabolised by enzymes capable of breaking the N=N bond thus forming two anilines which will follow the mechanism of action of anilines (explained above, in paragraph ii.). They can also be metabolised into reactive diazoniums (Brown & Vito, 1993).

The following figure illustrates the earlier statements:

Figure 4.3.11: Proposed azoreductase mechanism for Amaranth azo dye degradation (Misal & Gawai, 2018).

- Step 1 :  Upon oxidation of NADH, the hydride is transferred to FMN present in the active site of azoreductase. Substrate (Amaranth azo dye) binds to one of the active sites of azoreductase. Reduced FMN transfers the hydride to Amaranth azo dye.

- Step 2 is another cycle of hydride transfer from NADH to Amaranth azo dye via FMN.

M.A. Brown, S.C.D. Vito, Predicting azo dye toxicity, Critical Reviews in Environmental Science and Technology 23 (1993) 249–324. https://doi.org/10.1080/10643389309388453.

S.A. Misal, K.R. Gawai, Azoreductase: a key player of xenobiotic metabolism, Bioresources and Bioprocessing 5 (2018) 17. https://doi.org/10.1186/s40643-018-0206-8.

Benzenediols (in ortho/para) and their substituted methoxy ether derivatives have a specific MechoA. Mammals CYP450 can demethylate a methyl ether group of an alkoxyphenol to generate a diphenol (as an example: 4-methylanisole metabolism (Registration Dossier - ECHA)). This metabolic step is however not observed in aquatic organisms for which alkoxyphenols simply have toxicity comparable to polar narcotic compounds (MechoA 1.2) (Enoch, 2008; Schultz, 1987a, 1987b).

The generated metabolites are hydroquinone and catechol derivatives which are oxidised into corresponding quinones (respectively, p-benzoquinone and o-benzoquinone) which are soft electrophiles (MechoA 3.2) and can generate RedOx cycling (MechoA 4.4).

Figure 4.3.12: Methoxyphenol and its derivatives after metabolization (adapted from Sepúlveda et al., 2011 and Avendaño & Menéndez, 2008)

Same mechanism is observed for hydroquinonimine (=aminophenol), or benzenediamines when the amines or phenols are in ortho or para of each other (Hinson, 1983):

Figure 4.3.13: Structures of aminophenol and benzenediamines

C. Avendaño, J.C. Menéndez, Chapter 5 - DNA Alkylating Agents, in: C. Avendaño, J.C. Menéndez (Eds.), Medicinal Chemistry of Anticancer Drugs, Elsevier, Amsterdam, 2008: pp. 139–176. https://doi.org/10.1016/B978-0-444-52824-7.00005-6.

S.J. Enoch, M. Hewitt, M.T.D. Cronin, S. Azam, J.C. Madden, Classification of chemicals according to mechanism of aquatic toxicity: An evaluation of the implementation of the Verhaar scheme in Toxtree, Chemosphere 73 (2008) 243–248. https://doi.org/10.1016/j.chemosphere.2008.06.052. 

J.A. Hinson, Reactive metabolites of phenacetin and acetaminophen: a review, Environ Health Perspect 49 (1983) 71–79.

T.W. Schultz, Relative toxicity of para-substituted phenols: Log KOW and pKa-dependent structure-activity relationships, Bulletin of Environmental Contamination and Toxicology 38 (1987a) 994–999. https://doi.org/10.1007/BF01609086.

T.W. Schultz, The use of the ionization constant (pKa) in selecting models of toxicity in phenols, Ecotoxicol. Environ. Saf. 14 (1987b) 178–183.

C. Sepúlveda, K. Leiva, R. García, L.R. Radovic, I.T. Ghampson, W.J. DeSisto, J.L.G. Fierro, N. Escalona, Hydrodeoxygenation of 2-methoxyphenol over Mo2N catalysts supported on activated carbons, Catalysis Today 172 (2011) 232–239. https://doi.org/10.1016/j.cattod.2011.02.061.

Benzenediols (in ortho/para) and their substituted methoxy ether derivatives have a specific MechoA. Mammals CYP450 can demethylate a methyl ether group of an alkoxyphenol to generate a diphenol (as an example: 4-methylanisole metabolism (Registration Dossier - ECHA)). This metabolic step is however not observed in aquatic organisms for which alkoxyphenols simply have toxicity comparable to polar narcotic compounds (MechoA 1.2) (Enoch, 2008; Schultz, 1987a, 1987b).

The generated metabolites are hydroquinone and catechol derivatives which are oxidised into corresponding quinones (respectively, p-benzoquinone and o-benzoquinone) which are soft electrophiles (MechoA 3.2) and can generate RedOx cycling (MechoA 4.4).

Figure 4.3.12: Methoxyphenol and its derivatives after metabolization (adapted from Sepúlveda et al., 2011 and Avendaño & Menéndez, 2008)

Same mechanism is observed for hydroquinonimine (=aminophenol), or benzenediamines when the amines or phenols are in ortho or para of each other (Hinson, 1983):

Figure 4.3.13: Structures of aminophenol and benzenediamines

C. Avendaño, J.C. Menéndez, Chapter 5 - DNA Alkylating Agents, in: C. Avendaño, J.C. Menéndez (Eds.), Medicinal Chemistry of Anticancer Drugs, Elsevier, Amsterdam, 2008: pp. 139–176. https://doi.org/10.1016/B978-0-444-52824-7.00005-6.

S.J. Enoch, M. Hewitt, M.T.D. Cronin, S. Azam, J.C. Madden, Classification of chemicals according to mechanism of aquatic toxicity: An evaluation of the implementation of the Verhaar scheme in Toxtree, Chemosphere 73 (2008) 243–248. https://doi.org/10.1016/j.chemosphere.2008.06.052. 

J.A. Hinson, Reactive metabolites of phenacetin and acetaminophen: a review, Environ Health Perspect 49 (1983) 71–79.

T.W. Schultz, Relative toxicity of para-substituted phenols: Log KOW and pKa-dependent structure-activity relationships, Bulletin of Environmental Contamination and Toxicology 38 (1987a) 994–999. https://doi.org/10.1007/BF01609086.

T.W. Schultz, The use of the ionization constant (pKa) in selecting models of toxicity in phenols, Ecotoxicol. Environ. Saf. 14 (1987b) 178–183.

C. Sepúlveda, K. Leiva, R. García, L.R. Radovic, I.T. Ghampson, W.J. DeSisto, J.L.G. Fierro, N. Escalona, Hydrodeoxygenation of 2-methoxyphenol over Mo2N catalysts supported on activated carbons, Catalysis Today 172 (2011) 232–239. https://doi.org/10.1016/j.cattod.2011.02.061.

Aromatic diols (e.g. hydroquinone like substances) are oxidised into quinones in two steps, passing via semi-quinones which are radical compounds generating oxidative stress. This oxidation can occur slowly and spontaneously with oxygen or be catalysed by superoxide dismutase. However, the reverse reaction also occurs supported by other enzymes, mainly NADPH-CYP450 reductase. Therefore, these compounds can be interconverted between hydroquinone, semiquinone and quinone in a cyclic way, with superoxide anion generation for each oxidation step (see Figure below). Over time, the stock of NAD(P)H cofactors is consumed (Bolton et al., 2000; Di Francesco et al., 2004).

J.L. Bolton, M.A. Trush, T.M. Penning, G. Dryhurst, T.J. Monks, Role of Quinones in Toxicology, Chemical Research in Toxicology 13 (2000) 135–160. https://doi.org/10.1021/tx9902082.

A.M. Di Francesco, T.H. Ward, J. Butler, Diaziridinylbenzoquinones, in: Quinones and Quinone Enzymes, Academic Press, 2004: pp. 181–182.

Paraquat (1,1’–dimethyl-4,4’-bipyridinium) and other bipyridinium or phenylpyridinium analogues are other examples of MechoA 4.4. This MechoA (figure below) is the cause of their herbicidal properties, but these substances are also toxic to animals through the same mechanism (Blanco-Ayala et al., 2014).

Figure 4.4.2 : Paraquat RedOx cycle (Blanco-Ayala et al., 2014).
SOD = superoxide dismutase / GPx: glutathione peroxidase / GR: Glutathione Reductase / GSH and GSSG: reduced and oxidised forms of glutathione / Fenton reaction = a process that uses H₂O₂ and Fe²⁺ to generate hydroxyl radicals (organic contaminant degradation)

The reduction of paraquat ion is typically performed by either the photosystem I in plants, or the mitochondrial complex I. thus this RedOx cycle also directly affects the electron transport chain thus uncoupling the oxidative phosphorylation (Blanco-Ayala et al., 2014). See MechoA 6.3 section for more details on this.

T. Blanco-Ayala, A.C. Andérica-Romero, J. Pedraza-Chaverri, New insights into antioxidant strategies against paraquat toxicity, Free Radical Research 48 (2014) 623–640. https://doi.org/10.3109/10715762.2014.899694.

Rotenoids, such as rotenone, often used as pesticides, are RedOx cycling compounds. They bind to the complex I of the electron transport chain within the inner mitochondrial membrane where one electron is abstracted from the rotenoid (Heinz et al., 2017). (For more information on electron transport chain, see MechoA 6.3.)

Figure 4.4.3: Structure of rotenone

The radical thus formed can react with oxygen to recover its initial oxidised form while oxygen is transformed into superoxide anion. Then the rotenoid can once again initiate the cycle and generate more superoxide anions leading to important oxidative stress. Moreover, the complex I is inhibited preventing ATP production (Sherer et al., 2007).

S. Heinz, A. Freyberger, B. Lawrenz, L. Schladt, G. Schmuck, H. Ellinger-Ziegelbauer, Mechanistic Investigations of the Mitochondrial Complex I Inhibitor Rotenone in the Context of Pharmacological and Safety Evaluation, Scientific Reports 7 (2017) 45465. https://doi.org/10.1038/srep45465.

T.B. Sherer, J.R. Richardson, C.M. Testa, B.B. Seo, A.V. Panov, T. Yagi, A. Matsuno-Yagi, G.W. Miller, J.T. Greenamyre, Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson’s disease, Journal of Neurochemistry 100 (2007) 1469–1479. https://doi.org/10.1111/j.1471-4159.2006.04333.x.

Hydroxy- and amino-indoles reactive pathway was summarized by Kalgutkar et al., 2005.

Figure 4.4.4: 5-hydroxyindole metabolism formation of quinone-imine followed by conjugation (based on Kalgutkar, 2005)

The mechanism of bioactivation of indomethacin (a nonsteroidal anti-inflammatory drug) requires initial amide bond hydrolysis and O-dealkylation on the 5-methoxy substituent leading to the 5-hydroxyindole metabolite (in the following figure):

Figure 4.4.5: Bioactivation of indomethacin (based on Ju & Uetrecht, 1998)

5-Hydroxyindoles (such as the metabolite above) are bioactivated by the myeloperoxidase (MPO) and HOCl system which is considered as the major oxidant produced by activated neutrophils (Ju & Uetrecht, 1998) (figure above). This oxidation observed for indomethacin may be responsible for indomethacin-induced agranulocytosis, which is a state where the absolute neutrophil count (ANC) is less than 100 neutrophils per microlitre of the blood, causing a significant immunodeficiency (Sedhai et al., 2025).

Concerning the structure activity, hypothesis is that 5-alkoxyindoles bioactivation would be hindered if O-dealkylation reaction is difficult. For instance, this would probably be the case replacing the methoxy (of indomethacin) by bulky group substitution, or addition of electron withdrawing groups on the alkyl or 4- or 6-positions of the indole. It is has to be noted that hypothetically 3- and 7-alkoxyindoles could be similarly activated (Kalgutkar, 2005).

C. Ju, J.P. Uetrecht, Oxidation of a metabolite of indomethacin (Desmethyldeschlorobenzoylindomethacin) to reactive intermediates by activated neutrophils, hypochlorous acid, and the myeloperoxidase system, Drug Metab Dispos 26 (1998) 676–680.

A. Kalgutkar, I. Gardner, R. Obach, C. Shaffer, E. Callegari, K. Henne, A. Mutlib, D. Dalvie, J. Lee, Y. Nakai, J. O’Donnell, J. Boer, S. Harriman, A Comprehensive Listing of Bioactivation Pathways of Organic Functional Groups, CDM 6 (2005) 161–225. https://doi.org/10.2174/1389200054021799.

Y.R. Sedhai, A. Lamichhane, V. Gupta, Agranulocytosis, in: StatPearls, StatPearls Publishing, Treasure Island (FL), 2025. http://www.ncbi.nlm.nih.gov/books/NBK559275/ (accessed July 21, 2025).

Thiols and disulfides can also be the cause of RedOx cycling using the glutathione system as catalyst. Thiolates (basic form of thiols) are oxidised into thiyl radicals which can initiate RedOx cycling with the corresponding disulphide (Munday, 1989). This mechanism depends on stereo-electronic properties of the thiol. Indeed, tertiary thiols do not react the way described hereabove due to steric hindrance.

Furthermore, the reactivity of a thiophenol is lower because the electronic conjugation between the sulphur and the aromatic cycle stabilises the thiolate. If the aromatic cycle has groups with electron-withdrawing effect (e.g. halogens or a nitro group), thiolate will be further stabilised and less reactive. On the contrary, if the cycle has electron-donating groups (alkoxy for instance), the radical (reaction intermediate) is stabilised, and oxidation is fostered. For these particular cases, gluthathione, gluthathione-S-transferase and gluthathione peroxidase are not detoxifying, but they favour these toxicity-generating reactions by catalysing the RedOx cycle (Munday, 1989).

R. Munday, Toxicity of thiols and disulphides: Involvement of free-radical species, Free Radical Biology and Medicine 7 (1989) 659–673. https://doi.org/10.1016/0891-5849(89)90147-0.

This subcategory is related to substances which are metabolised into molecules disturbing biological systems in an indirect way. The final MechoA is explained in MechoA 5 section.

A priori this MechoA could be found for many substances. To date, only ethanol has been identified using this MechoA even if narcosis remains the main MechoA for ethanol.

The metabolic oxidation of ethanol into acetic acid consumes NAD+ to produce NADH. Thus, high and chronic exposure to ethanol leads to an impaired equilibrium of the cofactor NADH/NAD+. This, added to the induction of high levels of hepatic CYP2E1 and to the reactivity of the intermediate acetaldehyde, can lead to liver cancer development among other deleterious effects (King, 2015a).

Figure 4.5.1: Example of AOP implying the importance of CYP2E1 increased activity towards liver cancer’s development. Ethanol is here a chronic stressor that modulates CYP2E1’s activity (adapted from AOP 220 on AOP-Wiki)

The above description of the AOP shows what can be beyond MechoA's structure-activity aim: this example is well-described and referenced in the scientific community, hence being interesting enough to give further details due to its accuracy. 

Adverse Outcome Pathway on Cyp2E1 activation leading to liver cancer, 2021. https://doi.org/10.1787/56e9bbf0-en.

King, M. W. (2015). Ethanol (Alcohol) Metabolism : Acute and Chronic Toxicities. The Medical Biochemistry Page. http://themedicalbiochemistrypage.org/ethanol-metabolism.php

This subcategory is related to substances which are metabolised into molecules that can have specific interaction with endogen macromolecules (enzymes, receptors, ion channels, DNA, etc.). The effect of these metabolites is often related to a direct interaction with those. For instance, binding to active site or allosteric site of an enzyme can modulate activity of the protein (inhibition or activation).

The MechoA of the metabolic product produced further to the reaction is explained in MechoA 6 section.

Small organo-halogenated compounds, some nitriles, benzodioxoles and diphenylamines are the main examples of MechoA 4.6.

Small organo-halogenated compounds are metabolised into highly toxic compounds in mammals. In the case of trichloroethylene, the major metabolic pathway is the oxidation into chloral (MechoA 4.6) by cytochromes P450, then the reduction into 2,2,2-trichloroethanol (see MechoA 6.2) and its glucuronide (Brüning & Bolt, 2000) (see figure below). Another metabolite produced by mammals is carbon monoxide (see MechoA 6.9).

Figure 4.6.1: Metabolism of trichloroethylene (Brüning and Bolt, 2000).

Other haloalkanes, but also benzodioxoles can produce carbon monoxide upon metabolism. Carbon monoxide belongs to MechoA 6.9, competing with oxygen to bind hemes, it leads to oxygen deprivation in cells (see Inhibition of oxygen transport by haemoglobin in MechoA 6.9 section for more details).

T. Brüning, H.M. Bolt, Renal toxicity and carcinogenicity of trichloroethylene: key results, mechanisms, and controversies, Crit. Rev. Toxicol. 30 (2000) 253–285. https://doi.org/10.1080/10408440091159202.

All substances considered precursors of 2,2,2-trichloroethanol are considered part of MechoA 4.6, this would be the case for chloral hydrate after action alcohol (« Chloral Hydrate », 2012) or for example 2,2,2-trichloroacetaldehyde. This is explained by 2,2,2-trichloroethanol being an ion channel modulator (see MechoA 6.2). This compound can bind to GABA receptors controlling channels (e.g. chloride channels, sodium channel) involved in the action potential generation of neurons. Overall, it modulates various properties of these channels, resulting in the decreased excitability of nociceptive neurons (thus dampening pain signalling), contributing to peripheral analgesia (Kim et al., 2023).

Finally, diphenylamine is another particular case because it can be partially metabolised into indophenol in some mammals (IPCS Inchem, 1998).

Indophenol may also inhibit the mitochondrial electron transport chain while the molecular structure of the parent compound does not provide any indication of excess toxicity (Enoch et al., 2008). 

S.J. Enoch, M. Hewitt, M.T.D. Cronin, S. Azam, J.C. Madden, Classification of chemicals according to mechanism of aquatic toxicity: An evaluation of the implementation of the Verhaar scheme in Toxtree, Chemosphere 73 (2008) 243–248. https://doi.org/10.1016/j.chemosphere.2008.06.052.

IPCS Inchem, Diphenylamine (addendum), in: JMPR Evaluations, Rome, 1998. http://www.inchem.org/documents/jmpr/jmpmono/v098pr07.htm (accessed December 29, 2015).

G. Kim, H. Kim, I.-S. Jang, Trichloroethanol, an active metabolite of chloral hydrate, modulates tetrodotoxin-resistant Na+ channels in rat nociceptive neurons, BMC Anesthesiology 23 (2023) 145. https://doi.org/10.1186/s12871-023-02105-0.

NIH, National Institute of Diabetes and Digestive and Kidney Diseases, Chloral hydrate, in: LiverTox: Clinical and Research Information on Drug-Induced Liver Injury, , Bethesda (MD), 2012. http://www.ncbi.nlm.nih.gov/books/NBK548377/ (accessed July 24, 2025).

Cytochrome P450 (CYP450) monooxygenases are members of the cytochrome P450 family of enzymes, primarily involved in the metabolism of various molecules and chemicals, including medications, and is associated with genetic polymorphisms that can alter drug metabolism rates (Goodman et al., 2008).

Benzodioxoles, such as methylenedioxyphenyl compounds, are known to interact with cytochrome P450 monooxygenases in the liver.

These compounds can form metabolic intermediate complexes (MICs) with the heme iron of cytochrome P450. MIC formation involves oxidation of the methylenedioxy bridge, leading to a carbene intermediate (see figure below). This carbene tightly binds to the iron in the CYP450 active site, forming a quasi-irreversible complex, resulting in inhibition of enzyme activity, particularly affecting monooxygenases. This interaction can lead to enzyme inactivation, altered drug metabolism, and potential toxicity (Anders et al., 1984).

Figure 4.6.3: Proposed mechanism of benzodioxole metabolism (Kalgutkar, 2005)

M.W. Anders, J.M. Sunram, C.F. Wilkinson, Mechanism of the metabolism of 1,3-benzodioxoles to carbon monoxide, Biochemical Pharmacology 33 (1984) 577–580. https://doi.org/10.1016/0006-2952(84)90310-1.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

A. Kalgutkar, I. Gardner, R. Obach, C. Shaffer, E. Callegari, K. Henne, A. Mutlib, D. Dalvie, J. Lee, Y. Nakai, J. O’Donnell, J. Boer, S. Harriman, A Comprehensive Listing of Bioactivation Pathways of Organic Functional Groups, CDM 6 (2005) 161–225. https://doi.org/10.2174/1389200054021799.

Thiophanate and thiophanate-methyl are classified as benzimidazole fungicides due to their metabolic conversion into carbendazim, a benzimidazole derivative. Benzimidazole fungicides include benomyl, carbendazim, thiabendazole, albendazole, thiophanate, thiophanate-methyl, fuberidazole, methyl (1-{[(5-cyanopentyl)amino]carbonyl}-1H-benzimidazol-2-yl) carbamate, and carbendazim salicylate (Bai et al., 2024):

Figure 4.6.4: List of benzimidazole fungicides investigated by Bai et al., 2024.

These compounds often have benzodioxole-like structures, which undergo oxidative metabolism in plants and fungi. Additionally, the active metabolite carbendazim inhibits the polymerisation of fungal β-tubulin, disrupting microtubule formation. This interference blocks spindle fibre assembly during mitosis, halting fungal cell division. Result is abnormal germ tube development, cell wall distortion, and fungal death (Bai et al., 2024).

S. Bai, M. Zhang, S. Tang, M. Li, R. Wu, S. Wan, L. Chen, X. Wei, F. Li, Research Progress on Benzimidazole Fungicides: A Review, Molecules 29 (2024) 1218. https://doi.org/10.3390/molecules29061218.

Some nitriles can be oxidised at the alpha carbon by CYP450 to generate a cyanohydrin. This reactive compound spontaneously releases a cyanide anion on one hand, and an aldehyde or a ketone on the other, depending on the substitution of the parent (Grogan et al., 1992):

Figure 4.6.5: Oxidation of nitrile compounds by CYP450 (based on Grogan et al., 1992)

The cyanide anion (MechoA 6.9) inhibits cytochrome c oxidase, which is the 4^th complex within the mitochondrial electron transport chain, thus uncoupling oxidative phosphorylation, preventing production of ATP and generating oxidative stress (Way et al., 2007).

Grogan, S.C. DeVito, R.S. Pearlman, K.R. Korzekwa, Modeling cyanide release from nitriles: prediction of cytochrome P 450-mediated acute nitrile toxicity, Chem. Res. Toxicol. 5 (1992) 548–552. https://doi.org/10.1021/tx00028a014.

J.L. Way, P. Leung, E. Cannon, R. Morgan, C. Tamulinas, J. Leong-Way, L. Baxter, A. Nagi, C. Chui, The Mechanism of Cyanide Intoxication and its Antagonism - Ciba Foundation Symposium 140 - Cyanide Compounds in Biology - Way - Wiley Online Library, in: D. Evered and S. Hamett, 2007: pp. 232–248. http://onlinelibrary.wiley.com.scd-proxy.uha.fr/doi/10.1002/9780470513712.ch14/summary (accessed September 15, 2015).

Biological process description under ‘normal’ circumstances

To be able to function as intended, all enzymes, receptors, ion channels and other proteins need a specific environment, with a controlled pH, temperature, aqueous or lipidic medium, electrical potential, presence of cofactors in the right oxidation state, etc. Living organisms have systems in place for homeostasis, to maintain each local environment in its optimal range. For instance, ion channels control exchanges of key ions (such as calcium ions) between two compartments of the organism. However, these systems have their limits and can be overcome by the action of some xenobiotics if the dose is high enough and/or the exposure long enough.

MIE description

This general MechoA class is dedicated to molecules which indirectly disrupt the operation of enzymes, receptors or ion channels. In this case, xenobiotics do not directly bind to affected proteins but rather change their environment. Enzyme function can be disrupted by pH, electrical potential, oxidative state of cofactors, etc (Campbell, 1993; Goodman et al., 2008).

Taxonomy application

As this is a shared condition for all living organisms, MechoA 5 mechanisms typically apply for all species, unless otherwise noted.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

This mechanism is related to the disruption of a pH gradient across the inner mitochondrial membrane. This gradient is generated by the electron transport chain consisting of several membrane enzymes ultimately reducing molecular oxygen into water (Goodman et al., 2008).

The proton gradient is essential for the functioning of ATP-synthase enzyme which catalyses ATP production from ADP and inorganic phosphate while transporting one proton to the internal matrix of the mitochondria. Some substances are able to transport protons to the internal matrix by passive diffusion because they can cross the inner mitochondrial membrane in protonated form (acid form) in one direction and in deprotonated form (basic form) in the other direction. These substances thereby tend to neutralize the proton gradient that the electron transport chain is generating, until ATP-synthase can no longer function, and no more ATP is produced. This is called oxidative phosphorylation uncoupling. (Escher et al., 1999).

Organic substances concerned by this mechanism are acidic phenols and all examples currently in MechoA schemes are limited to these. A phenol is considered acidic when there are electron-withdrawing groups pulling the electrons away from the oxygen atom. Consequently, the oxygen will more easily release its proton thus forming a phenolate with negative charge stabilised by electron-withdrawing groups. Under normal conditions, the pH of the inner mitochondrial matrix is around 8 while it is 6.6 in the intermembrane space (Berg et al., 2002; Casey et al., 2010). 

Therefore, we assume the optimal pKa for a phenol to be an oxidative phosphorylation uncoupler is between 6.6 and 8. It should be noted that certain authors affirm the pKa should be less than 6.5 (Schultz, 1987) to achieve uncoupling.

In order to achieve significant uncoupling, the xenobiotic must meet several conditions (Schultz, 1987):

• sufficient hydrophobicity (related to log KOW)
• pKa within specific limits,
• a certain level of steric hindrance (related to the molecule bulkiness, i.e., the presence of large substituents close to the ionisable group that can prevent or slow down the molecule’s reaction rate).

All these factors make the uncoupling effect of phenols, and thus their toxicity, difficult to quantify.

Figure 5.1.1: Example phenol uncouplers of oxidative phosphorylation (Escher, 1995; Spycher, 2008)

Some substances are known to directly interact with the active site of one complex in the electron transport chain thereby disrupting oxidative phosphorylation. In this case, due to direct interaction of the xenobiotic with the complex, the uncoupling effect falls under MechoA 6.

J.M. Berg, J.L. Tymoczko, L. Stryer, A Proton Gradient Powers the Synthesis of ATP, in: Biochemistry, 5th ed., W.H. Freeman, New-York, 2002. https://www.ncbi.nlm.nih.gov/books/NBK22388/ (accessed June 26, 2017).

J.R. Casey, S. Grinstein, J. Orlowski, Sensors and regulators of intracellular pH, Nat Rev Mol Cell Biol 11 (2010) 50–61. https://doi.org/10.1038/nrm2820.

B.I. Escher, R. Hunziker, R.P. Schwarzenbach, J.C. Westall, Kinetic Model To Describe the Intrinsic Uncoupling Activity of Substituted Phenols in Energy Transducing Membranes, Environ. Sci. Technol. 33 (1999) 560–570. https://doi.org/10.1021/es980545h.

B.I. Escher. The relationship between membrane-water partitioning, uncoupling, and inhibitory activity of substituted phenols in chromatophores of Rhodobacter Sphaeroides. PhD thesis from the Swiss federal institute of technology Zürich (1995).

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York (2008).

S. Spycher, P. Smejtek, T.I. Netzeva, B.I. Escher, Toward a Class-Independent Quantitative Structure−Activity Relationship Model for Uncouplers of Oxidative Phosphorylation, Chem. Res. Toxicol. 21 (2008) 911–927. https://doi.org/10.1021/tx700391f.

T.W. Schultz, The use of the ionization constant (pKa) in selecting models of toxicity in phenols, Ecotoxicol. Environ. Saf. 14 (1987) 178–183.

This mechanism is related to the disruption of a pH gradient across the inner mitochondrial membrane. This gradient is generated by the electron transport chain consisting of several membrane enzymes ultimately reducing molecular oxygen into water (Goodman et al., 2008).

The proton gradient is essential for the functioning of ATP-synthase enzyme which catalyses ATP production from ADP and inorganic phosphate while transporting one proton to the internal matrix of the mitochondria. Some substances are able to transport protons to the internal matrix by passive diffusion because they can cross the inner mitochondrial membrane in protonated form (acid form) in one direction and in deprotonated form (basic form) in the other direction. These substances thereby tend to neutralize the proton gradient that the electron transport chain is generating, until ATP-synthase can no longer function, and no more ATP is produced. This is called oxidative phosphorylation uncoupling. (Escher et al., 1999).

Organic substances concerned by this mechanism are acidic phenols and all examples currently in MechoA schemes are limited to these. A phenol is considered acidic when there are electron-withdrawing groups pulling the electrons away from the oxygen atom. Consequently, the oxygen will more easily release its proton thus forming a phenolate with negative charge stabilised by electron-withdrawing groups. Under normal conditions, the pH of the inner mitochondrial matrix is around 8 while it is 6.6 in the intermembrane space (Berg et al., 2002; Casey et al., 2010). 

Therefore, we assume the optimal pKa for a phenol to be an oxidative phosphorylation uncoupler is between 6.6 and 8. It should be noted that certain authors affirm the pKa should be less than 6.5 (Schultz, 1987) to achieve uncoupling.

In order to achieve significant uncoupling, the xenobiotic must meet several conditions (Schultz, 1987):

• sufficient hydrophobicity (related to log KOW)
• pKa within specific limits,
• a certain level of steric hindrance (related to the molecule bulkiness, i.e., the presence of large substituents close to the ionisable group that can prevent or slow down the molecule’s reaction rate).

All these factors make the uncoupling effect of phenols, and thus their toxicity, difficult to quantify.

Figure 5.1.1: Example phenol uncouplers of oxidative phosphorylation (Escher, 1995; Spycher, 2008)

Some substances are known to directly interact with the active site of one complex in the electron transport chain thereby disrupting oxidative phosphorylation. In this case, due to direct interaction of the xenobiotic with the complex, the uncoupling effect falls under MechoA 6.

J.M. Berg, J.L. Tymoczko, L. Stryer, A Proton Gradient Powers the Synthesis of ATP, in: Biochemistry, 5th ed., W.H. Freeman, New-York, 2002. https://www.ncbi.nlm.nih.gov/books/NBK22388/ (accessed June 26, 2017).

J.R. Casey, S. Grinstein, J. Orlowski, Sensors and regulators of intracellular pH, Nat Rev Mol Cell Biol 11 (2010) 50–61. https://doi.org/10.1038/nrm2820.

B.I. Escher, R. Hunziker, R.P. Schwarzenbach, J.C. Westall, Kinetic Model To Describe the Intrinsic Uncoupling Activity of Substituted Phenols in Energy Transducing Membranes, Environ. Sci. Technol. 33 (1999) 560–570. https://doi.org/10.1021/es980545h.

B.I. Escher. The relationship between membrane-water partitioning, uncoupling, and inhibitory activity of substituted phenols in chromatophores of Rhodobacter Sphaeroides. PhD thesis from the Swiss federal institute of technology Zürich (1995).

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York (2008).

S. Spycher, P. Smejtek, T.I. Netzeva, B.I. Escher, Toward a Class-Independent Quantitative Structure−Activity Relationship Model for Uncouplers of Oxidative Phosphorylation, Chem. Res. Toxicol. 21 (2008) 911–927. https://doi.org/10.1021/tx700391f.

T.W. Schultz, The use of the ionization constant (pKa) in selecting models of toxicity in phenols, Ecotoxicol. Environ. Saf. 14 (1987) 178–183.

This mechanism is dedicated to acids with pKa < 5 and bases with pKa of conjugated acid > 9.

Most neutral chemical species can pass the cell membrane by passive diffusion, but ionic compounds cannot. For acids and bases that have pKa values at least 2 units below and above respectively, at an extracellular pH of around 7.4 and a cytosol pH of 7.2 (Casey et al., 2010), most of these molecules are ionised. Only a small portion of acids and bases exists in neutral form in both compartments. After one of the rare neutral molecules present in the external medium has crossed the membrane, it will release or catch electrons in the cytosol to return to its ionised form, driven by the acid-base equilibrium (Campbell, 1993; Goodman et al., 2008) (see figure below).

Figure 5.2.1: Example of cell acidification by acids.

In the figure, protonated acids (red) are neutral and can cross the membrane. When arriving in the cytosol, each molecule releases a proton which is recovered by a water molecule (blue) thereby increasing the acidity of the cytosol. Note that, after an acid molecule has passed into the cell, another acid in the extracellular medium becomes protonated because of the acid-base equilibrium (blue).

It is most probable that acidification or alkalinisation can also happen in other cell compartments than cytosol leading to general disruption of cell functions. This mechanism is partly limited by proton pumps which regulate cell pH. Nevertheless, proton pumps can be overwhelmed when acid or base concentrations become too high.

An example of this is the antimalarial effect of quinine and its analogues that has been explained by this alkalinization mechanism (Vallabh Minikel, 2016). The author explained that Plasmodium falciparum, the parasite responsible for the disease, is not able to degrade hemes which accumulate in acidic compartments where they spontaneously crystallise.

The basification of this compartment by quinine causes heme to be released. Furthermore, free hemes are highly toxic for the parasite because they catalyse oxidation reactions in an uncontrolled and unspecific way leading to radical production. The same author explains that bases, like quinine, can diffuse in the neutral form into more acidic compartments and capture protons.

It can be assume that the MechoA of bases is due to alkalinization of critical cell compartments like lysosomes. In this case proteases would be inhibited. Therefore, the cutaneous corrosivity of bases (e.g. pure bases or solutions with pH > 11) can be attributed to this alkalinization effect.

In parallel, the MechoA of acids must be similar to that of bases except that the critical targets would be the basic compartments such as the inner mitochondrial matrix.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

J.R. Casey, S. Grinstein, J. Orlowski, Sensors and regulators of intracellular pH, Nat Rev Mol Cell Biol 11 (2010) 50–61. https://doi.org/10.1038/nrm2820.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

E. Vallabh Minikel, A mechanism of action hypothesis, CureFFI.Org (2016). http://www.cureffi.org/2016/05/08/a-mechanism-of-action-hypothesis/ (accessed September 19, 2016).

This mechanism is dedicated to acids with pKa < 5 and bases with pKa of conjugated acid > 9.

Most neutral chemical species can pass the cell membrane by passive diffusion, but ionic compounds cannot. For acids and bases that have pKa values at least 2 units below and above respectively, at an extracellular pH of around 7.4 and a cytosol pH of 7.2 (Casey et al., 2010), most of these molecules are ionised. Only a small portion of acids and bases exists in neutral form in both compartments. After one of the rare neutral molecules present in the external medium has crossed the membrane, it will release or catch electrons in the cytosol to return to its ionised form, driven by the acid-base equilibrium (Campbell, 1993; Goodman et al., 2008) (see figure below).

Figure 5.2.1: Example of cell acidification by acids.

In the figure, protonated acids (red) are neutral and can cross the membrane. When arriving in the cytosol, each molecule releases a proton which is recovered by a water molecule (blue) thereby increasing the acidity of the cytosol. Note that, after an acid molecule has passed into the cell, another acid in the extracellular medium becomes protonated because of the acid-base equilibrium (blue).

It is most probable that acidification or alkalinisation can also happen in other cell compartments than cytosol leading to general disruption of cell functions. This mechanism is partly limited by proton pumps which regulate cell pH. Nevertheless, proton pumps can be overwhelmed when acid or base concentrations become too high.

An example of this is the antimalarial effect of quinine and its analogues that has been explained by this alkalinization mechanism (Vallabh Minikel, 2016). The author explained that Plasmodium falciparum, the parasite responsible for the disease, is not able to degrade hemes which accumulate in acidic compartments where they spontaneously crystallise.

The basification of this compartment by quinine causes heme to be released. Furthermore, free hemes are highly toxic for the parasite because they catalyse oxidation reactions in an uncontrolled and unspecific way leading to radical production. The same author explains that bases, like quinine, can diffuse in the neutral form into more acidic compartments and capture protons.

It can be assume that the MechoA of bases is due to alkalinization of critical cell compartments like lysosomes. In this case proteases would be inhibited. Therefore, the cutaneous corrosivity of bases (e.g. pure bases or solutions with pH > 11) can be attributed to this alkalinization effect.

In parallel, the MechoA of acids must be similar to that of bases except that the critical targets would be the basic compartments such as the inner mitochondrial matrix.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

J.R. Casey, S. Grinstein, J. Orlowski, Sensors and regulators of intracellular pH, Nat Rev Mol Cell Biol 11 (2010) 50–61. https://doi.org/10.1038/nrm2820.

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

E. Vallabh Minikel, A mechanism of action hypothesis, CureFFI.Org (2016). http://www.cureffi.org/2016/05/08/a-mechanism-of-action-hypothesis/ (accessed September 19, 2016).

[MechoA+ Alert 78] Azodicarbonamides

J. Arts, I. Kimber, Azodicarbonamide (ADCA): A reconsideration of classification as a respiratory sensitiser, Regulatory Toxicology and Pharmacology 89 (2017) 268–278. https://doi.org/10.1016/j.yrtph.2017.07.018.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

A. Hartwig, MAK Commission, Azodicarbonamide [MAK Value Documentation, 2017], in: Deutsche Forschungsgemeinschaft, Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (Eds.), The MAK-Collection for Occupational Health and Safety, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2018: pp. 1034–1074. https://doi.org/10.1002/3527600418.mb12377e6318.

M.W. King, Ethanol (Alcohol) Metabolism: Acute and Chronic Toxicities, The Medical Biochemistry Page (2015). http://themedicalbiochemistrypage.org/ethanol-metabolism.php (accessed September 15, 2015).

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

J. Arts, I. Kimber, Azodicarbonamide (ADCA): A reconsideration of classification as a respiratory sensitiser, Regulatory Toxicology and Pharmacology 89 (2017) 268–278. https://doi.org/10.1016/j.yrtph.2017.07.018.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

A. Hartwig, MAK Commission, Azodicarbonamide [MAK Value Documentation, 2017], in: Deutsche Forschungsgemeinschaft, Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (Eds.), The MAK-Collection for Occupational Health and Safety, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2018: pp. 1034–1074. https://doi.org/10.1002/3527600418.mb12377e6318.

M.W. King, Ethanol (Alcohol) Metabolism: Acute and Chronic Toxicities, The Medical Biochemistry Page (2015). http://themedicalbiochemistrypage.org/ethanol-metabolism.php (accessed September 15, 2015).

D.L. Nelson, M.M. Cox, A.L. Lehninger, Lehninger principles of biochemistry, 6. ed., [international ed.], Freeman, New York, NY, 2013.

Biological process description under ‘normal’ circumstances

Many biological processes work using a “lock and key” system, where a ligand specifically binds to a dedicated binding site in a protein, with stabilising interactions of different nature such as hydrogen bonds, ionic bonds (between an ion in the protein and ionic group in the ligand), hydrophobic interactions, Van der Waals interactions. These differences in shape and hydrophobic/hydrophilic regions in the binding sites allow for a good specificity of proteins for their natural ligands (Ben-Naim, 2018).

MIE description

However, there exist plenty of examples where xenobiotics also have an excellent affinity with one binding site of a given protein, modulating the activity of this protein.

This general MechoA class includes the largest number of MechoA subcategories and is related to any direct interaction with endogenous macromolecules such as enzymes, receptors, ion channels and/or DNA, among others. Some well-known MechoAs within this category are related to acetylcholinesterase inhibition, nicotinic or muscarinic acetylcholine receptor binding, ion channel binding (calcium, sodium, chloride, etc.), opioid receptor binding, heme binding, hormonal receptor binding, etc.

For mechanism related to protein interaction, the site of interaction of a xenobiotic can be localised at the active site of the protein (orthosteric) or at an allosteric site (Goodman et al., 2008; Nelson et al., 2013).

Taxonomy application

The MechoAs within this MechoA class 6 are often restricted to certain species, depending on the presence of proteins and even specific isoforms of proteins in different species. For instance, the MechoA of binding to acetylcholine receptors applies to the kingdom of animals, which all use this for nerve transmission, while other species (e.g. plants, fungi, bacteria etc.) don’t have neurons and no acetylcholine receptors, so this MechoA doesn’t apply to them.

[MechoA+ Alert 2] amiprophos derivative; [MechoA+ Alert 3] organophosphorous AChE inhibitors; [MechoA+ Alert 4] carbamate AChE inhibitors

This mechanism is responsible for the disruption of the acetylcholine (ACh)-dependant nervous transmission. ACh is a neurotransmitter which is released at the end of one neuron to the space between that neuron and the next cell (another neuron or a muscle cell) to transmit the signal. This inter-cell space is called the synapse, and the two neurons the presynaptic neuron and post-synaptic cell (e.i. neuron or muscle cell). ACh then binds to ACh receptors (two types: nicotinic or muscarinic) at the surface of the post-synaptic neuron, which triggers the opening of ions channels, which are either the receptors themselves for nicotinic receptors or separate ions channels for muscarinic receptors. The ion flux leads to depolarisation of the post-synaptic cell thus initiating the electric signal through it. Once the transmission of the signal is achieved, the activity due to ACh must be stopped in order to close ion channels and the cell to recover its resting state. This is the role of the acetylcholinesterase (AChE) which hydrolyses the ACh into choline and acetate in the synaptic cleft. AChE is therefore critical to ensure that the nervous transmission is executed. The choline molecules produced with AChE activity are recycled by transporting them back to the neuron which initially secreted the ACh (Campbell, 1993). 

Figure 6.1.1: acetylcholine-mediated nerve signal transmission

The inhibition of AChE rapidly leads to lethal effects for animals by arresting respiratory muscle functions (Elersek & Filipic, 2011; Fukuto, 1990). Nerve agents used in chemical weapons are typical examples of this mechanism (e.g. VX, sarin, tabun, etc.). They usually have a phosphorous atom attached to a double bond linked to a sulfur or oxygen atom, to a good leaving group (e.g. fluoride, cyano, alkyl-thioether, etc.) and to alkyl or alkyl-ether side chains.

At a molecular level, the hydrolysis of ACh includes several steps: binding of ACh to the serine residue at the AChE active site, followed by the formation of choline and acetate with action of water.

Inhibition of AChE is possible by imitating ACh binding to serine as presented in the figure below for neurotoxic organophosphates. The phosphate atom is attacked by the serine leading to the elimination of the leaving group in a second step. However, the elimination with the action of water of the phosphate now attached to the serine (Elersek & Filipic, 2011; Von Der Ohe et al., 2005) is in competition with another path: the formation of irreversible phosphorylated acetylcholine esterase by further dealkylation, thus blocking the AChE binding site (Elersek & Filipic, 2011; Hreljac & Filipič, 2009)). This induces muscular or neurologic issues and associated neuropathies (paralysis, heart attacks eventually leading to death).

Figure 6.1.2: Scheme of organophosphate binding regarding acetylcholine-esterase inhibition (adapted from Levet, 2021, otherwise coming from adapted scheme of Quinn, 2017 and Franjesevic, 2019)

Organophosphorus described above are known to have strong capacities to inhibit AChE. This would be the case for most species, from mammals to insects. There may be other less well-known enzymatic targets of organophosphorus compounds as suggested by Elersek & Filipic (2011) but because of the severity of this mechanism, priority was given to MechoA6.1 in the decision tree of MechoA scheme.

This is also a common mechanism of action used by various insecticides like azinphos-methyl, methomyl, aldicarb, malathion, etc.

Carbamates are another chemical family that can act as AChE inhibitors (Fukuto, 1990).

Some structure-activity relationships for these compounds have been established. Ester reactivity (for organophosphorus) and the strength of the leaving group (for carbamates) have been identified as having a strong impact on the inhibition efficiency. Nevertheless, reactivity is not the only parameter leading to inhibition. Steric hindrance also plays a significant role. For example, carbamates with a good leaving group such as a phenoxy or an oxime must have an adequate three-dimensional structure corresponding to the form and polarity of AChE active site (Fukuto, 1990).

This MechoA occurs only for organisms with neurons, i.e. most animals with a few exceptions. However, substances with MechoA 6.1 (notably organophosphorus compounds) are not necessarily inert for non-animal organisms because they can inhibit other esterases, especially in the family of serine hydrolases (which includes AChE) present in various organisms (Mangas et al., 2017).

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

T. Elersek, M. Filipic, T. Elersek, M. Filipic, Organophosphorous Pesticides - Mechanisms of Their Toxicity, in: Pesticides - The Impacts of Pesticides Exposure, IntechOpen, 2011. https://doi.org/10.5772/14020.

A.J. Franjesevic, S.B. Sillart, J.M. Beck, S. Vyas, C.S. Callam, C.M. Hadad, Resurrection and Reactivation of Acetylcholinesterase and Butyrylcholinesterase, Chemistry 25 (2019) 5337–5371. https://doi.org/10.1002/chem.201805075.

T.R. Fukuto, Mechanism of action of organophosphorus and carbamate insecticides., Environ Health Perspect 87 (1990) 245–254.

I. Hreljac, M. Filipič, Organophosphorus pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 671 (2009) 84–92. https://doi.org/10.1016/j.mrfmmm.2009.09.011.

G. Levet, Fonctionnalisation et utilisation de dérivés de cyclodextrine dans la lutte contre les agents neurotoxiques organophosphorés, thesis, Normandie, 2021. https://theses.fr/2021NORMR030 (accessed September 24, 2025).

D.M. Quinn, J. Topczewski, N. Yasapala, A. Lodge, Why is Aged Acetylcholinesterase So Difficult to Reactivate?, Molecules 22 (2017) 1464. https://doi.org/10.3390/molecules22091464.

P.C. Von Der Ohe, R. Kühne, R.-U. Ebert, R. Altenburger, M. Liess, G. Schüürmann, Structural Alerts-A New Classification Model to Discriminate Excess Toxicity from Narcotic Effect Levels of Organic Compounds in the Acute Daphnid Assay, Chem. Res. Toxicol. 18 (2005) 536–555. https://doi.org/10.1021/tx0497954.

This mechanism is responsible for the disruption of the acetylcholine (ACh)-dependant nervous transmission. ACh is a neurotransmitter which is released at the end of one neuron to the space between that neuron and the next cell (another neuron or a muscle cell) to transmit the signal. This inter-cell space is called the synapse, and the two neurons the presynaptic neuron and post-synaptic cell (e.i. neuron or muscle cell). ACh then binds to ACh receptors (two types: nicotinic or muscarinic) at the surface of the post-synaptic neuron, which triggers the opening of ions channels, which are either the receptors themselves for nicotinic receptors or separate ions channels for muscarinic receptors. The ion flux leads to depolarisation of the post-synaptic cell thus initiating the electric signal through it. Once the transmission of the signal is achieved, the activity due to ACh must be stopped in order to close ion channels and the cell to recover its resting state. This is the role of the acetylcholinesterase (AChE) which hydrolyses the ACh into choline and acetate in the synaptic cleft. AChE is therefore critical to ensure that the nervous transmission is executed. The choline molecules produced with AChE activity are recycled by transporting them back to the neuron which initially secreted the ACh (Campbell, 1993). 

Figure 6.1.1: acetylcholine-mediated nerve signal transmission

The inhibition of AChE rapidly leads to lethal effects for animals by arresting respiratory muscle functions (Elersek & Filipic, 2011; Fukuto, 1990). Nerve agents used in chemical weapons are typical examples of this mechanism (e.g. VX, sarin, tabun, etc.). They usually have a phosphorous atom attached to a double bond linked to a sulfur or oxygen atom, to a good leaving group (e.g. fluoride, cyano, alkyl-thioether, etc.) and to alkyl or alkyl-ether side chains.

At a molecular level, the hydrolysis of ACh includes several steps: binding of ACh to the serine residue at the AChE active site, followed by the formation of choline and acetate with action of water.

Inhibition of AChE is possible by imitating ACh binding to serine as presented in the figure below for neurotoxic organophosphates. The phosphate atom is attacked by the serine leading to the elimination of the leaving group in a second step. However, the elimination with the action of water of the phosphate now attached to the serine (Elersek & Filipic, 2011; Von Der Ohe et al., 2005) is in competition with another path: the formation of irreversible phosphorylated acetylcholine esterase by further dealkylation, thus blocking the AChE binding site (Elersek & Filipic, 2011; Hreljac & Filipič, 2009)). This induces muscular or neurologic issues and associated neuropathies (paralysis, heart attacks eventually leading to death).

Figure 6.1.2: Scheme of organophosphate binding regarding acetylcholine-esterase inhibition (adapted from Levet, 2021, otherwise coming from adapted scheme of Quinn, 2017 and Franjesevic, 2019)

Organophosphorus described above are known to have strong capacities to inhibit AChE. This would be the case for most species, from mammals to insects. There may be other less well-known enzymatic targets of organophosphorus compounds as suggested by Elersek & Filipic (2011) but because of the severity of this mechanism, priority was given to MechoA6.1 in the decision tree of MechoA scheme.

This is also a common mechanism of action used by various insecticides like azinphos-methyl, methomyl, aldicarb, malathion, etc.

Carbamates are another chemical family that can act as AChE inhibitors (Fukuto, 1990).

Some structure-activity relationships for these compounds have been established. Ester reactivity (for organophosphorus) and the strength of the leaving group (for carbamates) have been identified as having a strong impact on the inhibition efficiency. Nevertheless, reactivity is not the only parameter leading to inhibition. Steric hindrance also plays a significant role. For example, carbamates with a good leaving group such as a phenoxy or an oxime must have an adequate three-dimensional structure corresponding to the form and polarity of AChE active site (Fukuto, 1990).

This MechoA occurs only for organisms with neurons, i.e. most animals with a few exceptions. However, substances with MechoA 6.1 (notably organophosphorus compounds) are not necessarily inert for non-animal organisms because they can inhibit other esterases, especially in the family of serine hydrolases (which includes AChE) present in various organisms (Mangas et al., 2017).

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

T. Elersek, M. Filipic, T. Elersek, M. Filipic, Organophosphorous Pesticides - Mechanisms of Their Toxicity, in: Pesticides - The Impacts of Pesticides Exposure, IntechOpen, 2011. https://doi.org/10.5772/14020.

A.J. Franjesevic, S.B. Sillart, J.M. Beck, S. Vyas, C.S. Callam, C.M. Hadad, Resurrection and Reactivation of Acetylcholinesterase and Butyrylcholinesterase, Chemistry 25 (2019) 5337–5371. https://doi.org/10.1002/chem.201805075.

T.R. Fukuto, Mechanism of action of organophosphorus and carbamate insecticides., Environ Health Perspect 87 (1990) 245–254.

I. Hreljac, M. Filipič, Organophosphorus pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 671 (2009) 84–92. https://doi.org/10.1016/j.mrfmmm.2009.09.011.

G. Levet, Fonctionnalisation et utilisation de dérivés de cyclodextrine dans la lutte contre les agents neurotoxiques organophosphorés, thesis, Normandie, 2021. https://theses.fr/2021NORMR030 (accessed September 24, 2025).

D.M. Quinn, J. Topczewski, N. Yasapala, A. Lodge, Why is Aged Acetylcholinesterase So Difficult to Reactivate?, Molecules 22 (2017) 1464. https://doi.org/10.3390/molecules22091464.

P.C. Von Der Ohe, R. Kühne, R.-U. Ebert, R. Altenburger, M. Liess, G. Schüürmann, Structural Alerts-A New Classification Model to Discriminate Excess Toxicity from Narcotic Effect Levels of Organic Compounds in the Acute Daphnid Assay, Chem. Res. Toxicol. 18 (2005) 536–555. https://doi.org/10.1021/tx0497954.

This mechanism is responsible for the disruption of the acetylcholine (ACh)-dependant nervous transmission. ACh is a neurotransmitter which is released at the end of one neuron to the space between that neuron and the next cell (another neuron or a muscle cell) to transmit the signal. This inter-cell space is called the synapse, and the two neurons the presynaptic neuron and post-synaptic cell (e.i. neuron or muscle cell). ACh then binds to ACh receptors (two types: nicotinic or muscarinic) at the surface of the post-synaptic neuron, which triggers the opening of ions channels, which are either the receptors themselves for nicotinic receptors or separate ions channels for muscarinic receptors. The ion flux leads to depolarisation of the post-synaptic cell thus initiating the electric signal through it. Once the transmission of the signal is achieved, the activity due to ACh must be stopped in order to close ion channels and the cell to recover its resting state. This is the role of the acetylcholinesterase (AChE) which hydrolyses the ACh into choline and acetate in the synaptic cleft. AChE is therefore critical to ensure that the nervous transmission is executed. The choline molecules produced with AChE activity are recycled by transporting them back to the neuron which initially secreted the ACh (Campbell, 1993). 

Figure 6.1.1: acetylcholine-mediated nerve signal transmission

The inhibition of AChE rapidly leads to lethal effects for animals by arresting respiratory muscle functions (Elersek & Filipic, 2011; Fukuto, 1990). Nerve agents used in chemical weapons are typical examples of this mechanism (e.g. VX, sarin, tabun, etc.). They usually have a phosphorous atom attached to a double bond linked to a sulfur or oxygen atom, to a good leaving group (e.g. fluoride, cyano, alkyl-thioether, etc.) and to alkyl or alkyl-ether side chains.

At a molecular level, the hydrolysis of ACh includes several steps: binding of ACh to the serine residue at the AChE active site, followed by the formation of choline and acetate with action of water.

Inhibition of AChE is possible by imitating ACh binding to serine as presented in the figure below for neurotoxic organophosphates. The phosphate atom is attacked by the serine leading to the elimination of the leaving group in a second step. However, the elimination with the action of water of the phosphate now attached to the serine (Elersek & Filipic, 2011; Von Der Ohe et al., 2005) is in competition with another path: the formation of irreversible phosphorylated acetylcholine esterase by further dealkylation, thus blocking the AChE binding site (Elersek & Filipic, 2011; Hreljac & Filipič, 2009)). This induces muscular or neurologic issues and associated neuropathies (paralysis, heart attacks eventually leading to death).

Figure 6.1.2: Scheme of organophosphate binding regarding acetylcholine-esterase inhibition (adapted from Levet, 2021, otherwise coming from adapted scheme of Quinn, 2017 and Franjesevic, 2019)

Organophosphorus described above are known to have strong capacities to inhibit AChE. This would be the case for most species, from mammals to insects. There may be other less well-known enzymatic targets of organophosphorus compounds as suggested by Elersek & Filipic (2011) but because of the severity of this mechanism, priority was given to MechoA6.1 in the decision tree of MechoA scheme.

This is also a common mechanism of action used by various insecticides like azinphos-methyl, methomyl, aldicarb, malathion, etc.

Carbamates are another chemical family that can act as AChE inhibitors (Fukuto, 1990).

Some structure-activity relationships for these compounds have been established. Ester reactivity (for organophosphorus) and the strength of the leaving group (for carbamates) have been identified as having a strong impact on the inhibition efficiency. Nevertheless, reactivity is not the only parameter leading to inhibition. Steric hindrance also plays a significant role. For example, carbamates with a good leaving group such as a phenoxy or an oxime must have an adequate three-dimensional structure corresponding to the form and polarity of AChE active site (Fukuto, 1990).

This MechoA occurs only for organisms with neurons, i.e. most animals with a few exceptions. However, substances with MechoA 6.1 (notably organophosphorus compounds) are not necessarily inert for non-animal organisms because they can inhibit other esterases, especially in the family of serine hydrolases (which includes AChE) present in various organisms (Mangas et al., 2017).

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

T. Elersek, M. Filipic, T. Elersek, M. Filipic, Organophosphorous Pesticides - Mechanisms of Their Toxicity, in: Pesticides - The Impacts of Pesticides Exposure, IntechOpen, 2011. https://doi.org/10.5772/14020.

A.J. Franjesevic, S.B. Sillart, J.M. Beck, S. Vyas, C.S. Callam, C.M. Hadad, Resurrection and Reactivation of Acetylcholinesterase and Butyrylcholinesterase, Chemistry 25 (2019) 5337–5371. https://doi.org/10.1002/chem.201805075.

T.R. Fukuto, Mechanism of action of organophosphorus and carbamate insecticides., Environ Health Perspect 87 (1990) 245–254.

I. Hreljac, M. Filipič, Organophosphorus pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 671 (2009) 84–92. https://doi.org/10.1016/j.mrfmmm.2009.09.011.

G. Levet, Fonctionnalisation et utilisation de dérivés de cyclodextrine dans la lutte contre les agents neurotoxiques organophosphorés, thesis, Normandie, 2021. https://theses.fr/2021NORMR030 (accessed September 24, 2025).

D.M. Quinn, J. Topczewski, N. Yasapala, A. Lodge, Why is Aged Acetylcholinesterase So Difficult to Reactivate?, Molecules 22 (2017) 1464. https://doi.org/10.3390/molecules22091464.

P.C. Von Der Ohe, R. Kühne, R.-U. Ebert, R. Altenburger, M. Liess, G. Schüürmann, Structural Alerts-A New Classification Model to Discriminate Excess Toxicity from Narcotic Effect Levels of Organic Compounds in the Acute Daphnid Assay, Chem. Res. Toxicol. 18 (2005) 536–555. https://doi.org/10.1021/tx0497954.

This mechanism is responsible for the disruption of the acetylcholine (ACh)-dependant nervous transmission. ACh is a neurotransmitter which is released at the end of one neuron to the space between that neuron and the next cell (another neuron or a muscle cell) to transmit the signal. This inter-cell space is called the synapse, and the two neurons the presynaptic neuron and post-synaptic cell (e.i. neuron or muscle cell). ACh then binds to ACh receptors (two types: nicotinic or muscarinic) at the surface of the post-synaptic neuron, which triggers the opening of ions channels, which are either the receptors themselves for nicotinic receptors or separate ions channels for muscarinic receptors. The ion flux leads to depolarisation of the post-synaptic cell thus initiating the electric signal through it. Once the transmission of the signal is achieved, the activity due to ACh must be stopped in order to close ion channels and the cell to recover its resting state. This is the role of the acetylcholinesterase (AChE) which hydrolyses the ACh into choline and acetate in the synaptic cleft. AChE is therefore critical to ensure that the nervous transmission is executed. The choline molecules produced with AChE activity are recycled by transporting them back to the neuron which initially secreted the ACh (Campbell, 1993). 

Figure 6.1.1: acetylcholine-mediated nerve signal transmission

The inhibition of AChE rapidly leads to lethal effects for animals by arresting respiratory muscle functions (Elersek & Filipic, 2011; Fukuto, 1990). Nerve agents used in chemical weapons are typical examples of this mechanism (e.g. VX, sarin, tabun, etc.). They usually have a phosphorous atom attached to a double bond linked to a sulfur or oxygen atom, to a good leaving group (e.g. fluoride, cyano, alkyl-thioether, etc.) and to alkyl or alkyl-ether side chains.

At a molecular level, the hydrolysis of ACh includes several steps: binding of ACh to the serine residue at the AChE active site, followed by the formation of choline and acetate with action of water.

Inhibition of AChE is possible by imitating ACh binding to serine as presented in the figure below for neurotoxic organophosphates. The phosphate atom is attacked by the serine leading to the elimination of the leaving group in a second step. However, the elimination with the action of water of the phosphate now attached to the serine (Elersek & Filipic, 2011; Von Der Ohe et al., 2005) is in competition with another path: the formation of irreversible phosphorylated acetylcholine esterase by further dealkylation, thus blocking the AChE binding site (Elersek & Filipic, 2011; Hreljac & Filipič, 2009)). This induces muscular or neurologic issues and associated neuropathies (paralysis, heart attacks eventually leading to death).

Figure 6.1.2: Scheme of organophosphate binding regarding acetylcholine-esterase inhibition (adapted from Levet, 2021, otherwise coming from adapted scheme of Quinn, 2017 and Franjesevic, 2019)

Organophosphorus described above are known to have strong capacities to inhibit AChE. This would be the case for most species, from mammals to insects. There may be other less well-known enzymatic targets of organophosphorus compounds as suggested by Elersek & Filipic (2011) but because of the severity of this mechanism, priority was given to MechoA6.1 in the decision tree of MechoA scheme.

This is also a common mechanism of action used by various insecticides like azinphos-methyl, methomyl, aldicarb, malathion, etc.

Carbamates are another chemical family that can act as AChE inhibitors (Fukuto, 1990).

Some structure-activity relationships for these compounds have been established. Ester reactivity (for organophosphorus) and the strength of the leaving group (for carbamates) have been identified as having a strong impact on the inhibition efficiency. Nevertheless, reactivity is not the only parameter leading to inhibition. Steric hindrance also plays a significant role. For example, carbamates with a good leaving group such as a phenoxy or an oxime must have an adequate three-dimensional structure corresponding to the form and polarity of AChE active site (Fukuto, 1990).

This MechoA occurs only for organisms with neurons, i.e. most animals with a few exceptions. However, substances with MechoA 6.1 (notably organophosphorus compounds) are not necessarily inert for non-animal organisms because they can inhibit other esterases, especially in the family of serine hydrolases (which includes AChE) present in various organisms (Mangas et al., 2017).

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

T. Elersek, M. Filipic, T. Elersek, M. Filipic, Organophosphorous Pesticides - Mechanisms of Their Toxicity, in: Pesticides - The Impacts of Pesticides Exposure, IntechOpen, 2011. https://doi.org/10.5772/14020.

A.J. Franjesevic, S.B. Sillart, J.M. Beck, S. Vyas, C.S. Callam, C.M. Hadad, Resurrection and Reactivation of Acetylcholinesterase and Butyrylcholinesterase, Chemistry 25 (2019) 5337–5371. https://doi.org/10.1002/chem.201805075.

T.R. Fukuto, Mechanism of action of organophosphorus and carbamate insecticides., Environ Health Perspect 87 (1990) 245–254.

I. Hreljac, M. Filipič, Organophosphorus pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 671 (2009) 84–92. https://doi.org/10.1016/j.mrfmmm.2009.09.011.

G. Levet, Fonctionnalisation et utilisation de dérivés de cyclodextrine dans la lutte contre les agents neurotoxiques organophosphorés, thesis, Normandie, 2021. https://theses.fr/2021NORMR030 (accessed September 24, 2025).

D.M. Quinn, J. Topczewski, N. Yasapala, A. Lodge, Why is Aged Acetylcholinesterase So Difficult to Reactivate?, Molecules 22 (2017) 1464. https://doi.org/10.3390/molecules22091464.

P.C. Von Der Ohe, R. Kühne, R.-U. Ebert, R. Altenburger, M. Liess, G. Schüürmann, Structural Alerts-A New Classification Model to Discriminate Excess Toxicity from Narcotic Effect Levels of Organic Compounds in the Acute Daphnid Assay, Chem. Res. Toxicol. 18 (2005) 536–555. https://doi.org/10.1021/tx0497954.

This mechanism is responsible for the disruption of the nervous transmission by binding to the acetylcholine receptors (AChRs). A molecule acting through this MechoA 6.1 can be an agonist or an antagonist. Agonists produce the same effect as natural ligands, i.e. AChR activation. Antagonists block the AChR receptor rather than activating it like an agonist.

Note that agonists to AChR produce a similar effect as AChE inhibitors as they activate AChR but are not degraded by AChE. When degradation and elimination by the organism is too slow and insufficient,  these AChR binders are maintained at the receptor for too long. This leads to toxicity effects, particularly known as “cholinergic toxicity” (Hudson & Jones, 2025). The best known ACh agonists are nicotine for nicotinic receptors (nAChR) and muscarine for muscarinic receptors (mAChR). Antagonists when they are administered alone are toxic by blocking nervous transmission. However, they can be used as antidotes to prevent adverse effects occurring due to exposure to AChE inhibitors and AChR agonists (Campbell, 1993; Goodman et al., 2008).

Atropine and derivatives are typical examples of mAChR antagonists (IPCS Inchem, 2002). Besides, coniine is known to block nAChRs (NCBI, 2017; Vetter, 2004). Compounds similar to coniine can also cause the same effect despite their quite simple structure. The efficiency of these antagonists depends on their affinity to the binding site (Committee on Acute Exposure Guideline Levels et al., 2012). Tubocurarine is also a potent antagonist of ACh for nAChR as a single molecule of tubocurarine is equivalent to 2 molecules of ACh (King, 2015b). Indeed, there are 2 binding sites for ACh in AChR which must be occupied to open ion channel of the receptor (Droual, 2011) and tubocurarine occupies both of them as a single molecule. The reason why a ligand will have an agonist or antagonist effect is not yet totally understood.

Agonists and antagonists to ACh share common features with ACh (see Figure below). All of them have an amine or ammonium (or a nitrogen atom with similar properties) often accompanied by an aromatic ring and/or other nitrogen or oxygen atoms, all these molecules having thus a region that would be positively charged (deficient in electrons) at physiological pH, and another region that is rich in electrons. More specific criteria are coded in MechoA scheme alerts but are not detailed here given the variety of structures hitting this MechoA, as can be seen with just a few examples below.

Figure 6.1.3: Molecular structures of acetylcholine and some agonists or antagonists.

NB. This mechanism only concerns organisms with neurons which use ACh as their neurotransmitter (with AChRs). Basically, these are animals with some exceptions.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

C. Gotti, Michael.J. Marks, N.S. Millar, S. Wonnacott, Nicotinic acetylcholine receptors (nACh) in GtoPdb v.2023.3, IUPHAR/BPS Guide to Pharmacology CITE 2023 (2023). https://doi.org/10.2218/gtopdb/F76/2023.3.

B. Droual, Chapter 8 - Synaptic Transmission and Neural Integration, Courses Taught by Dr. Droual at Modesto Junior College (2011). 

http://droualb.faculty.mjc.edu/Course%20Materials/Physiology%20101/Chapter%20Notes/Fall%202011/chapter_8%20Fall%202011.htm (accessed July 18, 2017).

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

E.L. Hudson, E.B. Jones, Cholinergic Toxicity, in: StatPearls, StatPearls Publishing, Treasure Island (FL), 2025. http://www.ncbi.nlm.nih.gov/books/NBK539783/ (accessed June 6, 2025).

Committee on Acute Exposure Guideline Levels, Committee on Toxicology, Board on Environmental Studies and Toxicology, Division on Earth and Life Studies, National Research Council, Piperidine, in: Acute Exposure Guideline Levels for Selected Airbone Chemicals, National Academies Press (US), 2012. http://www.ncbi.nlm.nih.gov/books/NBK241478/ (accessed September 16, 2016).

IPCS Inchem, Monograph on Atropine - Antidotes for poisoning by organophosphorus pesticides, (2002). http://www.inchem.org/documents/antidote/antidote/atropine.htm#7.1 (accessed April 28, 2017).

M.W. King, Biochemistry of Neurotransmitters and Nerve Transmission, (2015). http://themedicalbiochemistrypage.org/nerves.php (accessed September 25, 2015).

National Center for Biotechnology Information, CONIINE | C8H17N, PubChem Compound Database (2017). https://pubchem.ncbi.nlm.nih.gov/compound/441072 (accessed December 21, 2016).

J. Vetter, Poison hemlock (Conium maculatum L.), Food and Chemical Toxicology 42 (2004) 1373–1382. https://doi.org/10.1016/j.fct.2004.04.009.

This mechanism is responsible for the disruption of the nervous transmission by binding to the acetylcholine receptors (AChRs). A molecule acting through this MechoA 6.1 can be an agonist or an antagonist. Agonists produce the same effect as natural ligands, i.e. AChR activation. Antagonists block the AChR receptor rather than activating it like an agonist.

Note that agonists to AChR produce a similar effect as AChE inhibitors as they activate AChR but are not degraded by AChE. When degradation and elimination by the organism is too slow and insufficient,  these AChR binders are maintained at the receptor for too long. This leads to toxicity effects, particularly known as “cholinergic toxicity” (Hudson & Jones, 2025). The best known ACh agonists are nicotine for nicotinic receptors (nAChR) and muscarine for muscarinic receptors (mAChR). Antagonists when they are administered alone are toxic by blocking nervous transmission. However, they can be used as antidotes to prevent adverse effects occurring due to exposure to AChE inhibitors and AChR agonists (Campbell, 1993; Goodman et al., 2008).

Atropine and derivatives are typical examples of mAChR antagonists (IPCS Inchem, 2002). Besides, coniine is known to block nAChRs (NCBI, 2017; Vetter, 2004). Compounds similar to coniine can also cause the same effect despite their quite simple structure. The efficiency of these antagonists depends on their affinity to the binding site (Committee on Acute Exposure Guideline Levels et al., 2012). Tubocurarine is also a potent antagonist of ACh for nAChR as a single molecule of tubocurarine is equivalent to 2 molecules of ACh (King, 2015b). Indeed, there are 2 binding sites for ACh in AChR which must be occupied to open ion channel of the receptor (Droual, 2011) and tubocurarine occupies both of them as a single molecule. The reason why a ligand will have an agonist or antagonist effect is not yet totally understood.

Agonists and antagonists to ACh share common features with ACh (see Figure below). All of them have an amine or ammonium (or a nitrogen atom with similar properties) often accompanied by an aromatic ring and/or other nitrogen or oxygen atoms, all these molecules having thus a region that would be positively charged (deficient in electrons) at physiological pH, and another region that is rich in electrons. More specific criteria are coded in MechoA scheme alerts but are not detailed here given the variety of structures hitting this MechoA, as can be seen with just a few examples below.

Figure 6.1.3: Molecular structures of acetylcholine and some agonists or antagonists.

NB. This mechanism only concerns organisms with neurons which use ACh as their neurotransmitter (with AChRs). Basically, these are animals with some exceptions.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

C. Gotti, Michael.J. Marks, N.S. Millar, S. Wonnacott, Nicotinic acetylcholine receptors (nACh) in GtoPdb v.2023.3, IUPHAR/BPS Guide to Pharmacology CITE 2023 (2023). https://doi.org/10.2218/gtopdb/F76/2023.3.

B. Droual, Chapter 8 - Synaptic Transmission and Neural Integration, Courses Taught by Dr. Droual at Modesto Junior College (2011). 

http://droualb.faculty.mjc.edu/Course%20Materials/Physiology%20101/Chapter%20Notes/Fall%202011/chapter_8%20Fall%202011.htm (accessed July 18, 2017).

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

E.L. Hudson, E.B. Jones, Cholinergic Toxicity, in: StatPearls, StatPearls Publishing, Treasure Island (FL), 2025. http://www.ncbi.nlm.nih.gov/books/NBK539783/ (accessed June 6, 2025).

Committee on Acute Exposure Guideline Levels, Committee on Toxicology, Board on Environmental Studies and Toxicology, Division on Earth and Life Studies, National Research Council, Piperidine, in: Acute Exposure Guideline Levels for Selected Airbone Chemicals, National Academies Press (US), 2012. http://www.ncbi.nlm.nih.gov/books/NBK241478/ (accessed September 16, 2016).

IPCS Inchem, Monograph on Atropine - Antidotes for poisoning by organophosphorus pesticides, (2002). http://www.inchem.org/documents/antidote/antidote/atropine.htm#7.1 (accessed April 28, 2017).

M.W. King, Biochemistry of Neurotransmitters and Nerve Transmission, (2015). http://themedicalbiochemistrypage.org/nerves.php (accessed September 25, 2015).

National Center for Biotechnology Information, CONIINE | C8H17N, PubChem Compound Database (2017). https://pubchem.ncbi.nlm.nih.gov/compound/441072 (accessed December 21, 2016).

J. Vetter, Poison hemlock (Conium maculatum L.), Food and Chemical Toxicology 42 (2004) 1373–1382. https://doi.org/10.1016/j.fct.2004.04.009.

This mechanism is responsible for the disruption of the nervous transmission by binding to the acetylcholine receptors (AChRs). A molecule acting through this MechoA 6.1 can be an agonist or an antagonist. Agonists produce the same effect as natural ligands, i.e. AChR activation. Antagonists block the AChR receptor rather than activating it like an agonist.

Note that agonists to AChR produce a similar effect as AChE inhibitors as they activate AChR but are not degraded by AChE. When degradation and elimination by the organism is too slow and insufficient,  these AChR binders are maintained at the receptor for too long. This leads to toxicity effects, particularly known as “cholinergic toxicity” (Hudson & Jones, 2025). The best known ACh agonists are nicotine for nicotinic receptors (nAChR) and muscarine for muscarinic receptors (mAChR). Antagonists when they are administered alone are toxic by blocking nervous transmission. However, they can be used as antidotes to prevent adverse effects occurring due to exposure to AChE inhibitors and AChR agonists (Campbell, 1993; Goodman et al., 2008).

Atropine and derivatives are typical examples of mAChR antagonists (IPCS Inchem, 2002). Besides, coniine is known to block nAChRs (NCBI, 2017; Vetter, 2004). Compounds similar to coniine can also cause the same effect despite their quite simple structure. The efficiency of these antagonists depends on their affinity to the binding site (Committee on Acute Exposure Guideline Levels et al., 2012). Tubocurarine is also a potent antagonist of ACh for nAChR as a single molecule of tubocurarine is equivalent to 2 molecules of ACh (King, 2015b). Indeed, there are 2 binding sites for ACh in AChR which must be occupied to open ion channel of the receptor (Droual, 2011) and tubocurarine occupies both of them as a single molecule. The reason why a ligand will have an agonist or antagonist effect is not yet totally understood.

Agonists and antagonists to ACh share common features with ACh (see Figure below). All of them have an amine or ammonium (or a nitrogen atom with similar properties) often accompanied by an aromatic ring and/or other nitrogen or oxygen atoms, all these molecules having thus a region that would be positively charged (deficient in electrons) at physiological pH, and another region that is rich in electrons. More specific criteria are coded in MechoA scheme alerts but are not detailed here given the variety of structures hitting this MechoA, as can be seen with just a few examples below.

Figure 6.1.3: Molecular structures of acetylcholine and some agonists or antagonists.

NB. This mechanism only concerns organisms with neurons which use ACh as their neurotransmitter (with AChRs). Basically, these are animals with some exceptions.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

C. Gotti, Michael.J. Marks, N.S. Millar, S. Wonnacott, Nicotinic acetylcholine receptors (nACh) in GtoPdb v.2023.3, IUPHAR/BPS Guide to Pharmacology CITE 2023 (2023). https://doi.org/10.2218/gtopdb/F76/2023.3.

B. Droual, Chapter 8 - Synaptic Transmission and Neural Integration, Courses Taught by Dr. Droual at Modesto Junior College (2011). 

http://droualb.faculty.mjc.edu/Course%20Materials/Physiology%20101/Chapter%20Notes/Fall%202011/chapter_8%20Fall%202011.htm (accessed July 18, 2017).

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

E.L. Hudson, E.B. Jones, Cholinergic Toxicity, in: StatPearls, StatPearls Publishing, Treasure Island (FL), 2025. http://www.ncbi.nlm.nih.gov/books/NBK539783/ (accessed June 6, 2025).

Committee on Acute Exposure Guideline Levels, Committee on Toxicology, Board on Environmental Studies and Toxicology, Division on Earth and Life Studies, National Research Council, Piperidine, in: Acute Exposure Guideline Levels for Selected Airbone Chemicals, National Academies Press (US), 2012. http://www.ncbi.nlm.nih.gov/books/NBK241478/ (accessed September 16, 2016).

IPCS Inchem, Monograph on Atropine - Antidotes for poisoning by organophosphorus pesticides, (2002). http://www.inchem.org/documents/antidote/antidote/atropine.htm#7.1 (accessed April 28, 2017).

M.W. King, Biochemistry of Neurotransmitters and Nerve Transmission, (2015). http://themedicalbiochemistrypage.org/nerves.php (accessed September 25, 2015).

National Center for Biotechnology Information, CONIINE | C8H17N, PubChem Compound Database (2017). https://pubchem.ncbi.nlm.nih.gov/compound/441072 (accessed December 21, 2016).

J. Vetter, Poison hemlock (Conium maculatum L.), Food and Chemical Toxicology 42 (2004) 1373–1382. https://doi.org/10.1016/j.fct.2004.04.009.

This mechanism is responsible for the disruption of the nervous transmission by binding to the acetylcholine receptors (AChRs). A molecule acting through this MechoA 6.1 can be an agonist or an antagonist. Agonists produce the same effect as natural ligands, i.e. AChR activation. Antagonists block the AChR receptor rather than activating it like an agonist.

Note that agonists to AChR produce a similar effect as AChE inhibitors as they activate AChR but are not degraded by AChE. When degradation and elimination by the organism is too slow and insufficient,  these AChR binders are maintained at the receptor for too long. This leads to toxicity effects, particularly known as “cholinergic toxicity” (Hudson & Jones, 2025). The best known ACh agonists are nicotine for nicotinic receptors (nAChR) and muscarine for muscarinic receptors (mAChR). Antagonists when they are administered alone are toxic by blocking nervous transmission. However, they can be used as antidotes to prevent adverse effects occurring due to exposure to AChE inhibitors and AChR agonists (Campbell, 1993; Goodman et al., 2008).

Atropine and derivatives are typical examples of mAChR antagonists (IPCS Inchem, 2002). Besides, coniine is known to block nAChRs (NCBI, 2017; Vetter, 2004). Compounds similar to coniine can also cause the same effect despite their quite simple structure. The efficiency of these antagonists depends on their affinity to the binding site (Committee on Acute Exposure Guideline Levels et al., 2012). Tubocurarine is also a potent antagonist of ACh for nAChR as a single molecule of tubocurarine is equivalent to 2 molecules of ACh (King, 2015b). Indeed, there are 2 binding sites for ACh in AChR which must be occupied to open ion channel of the receptor (Droual, 2011) and tubocurarine occupies both of them as a single molecule. The reason why a ligand will have an agonist or antagonist effect is not yet totally understood.

Agonists and antagonists to ACh share common features with ACh (see Figure below). All of them have an amine or ammonium (or a nitrogen atom with similar properties) often accompanied by an aromatic ring and/or other nitrogen or oxygen atoms, all these molecules having thus a region that would be positively charged (deficient in electrons) at physiological pH, and another region that is rich in electrons. More specific criteria are coded in MechoA scheme alerts but are not detailed here given the variety of structures hitting this MechoA, as can be seen with just a few examples below.

Figure 6.1.3: Molecular structures of acetylcholine and some agonists or antagonists.

NB. This mechanism only concerns organisms with neurons which use ACh as their neurotransmitter (with AChRs). Basically, these are animals with some exceptions.

N.A. Campbell, Biology, 3rd ed, Benjamin/Cummings, Redwood City, Calif, 1993.

C. Gotti, Michael.J. Marks, N.S. Millar, S. Wonnacott, Nicotinic acetylcholine receptors (nACh) in GtoPdb v.2023.3, IUPHAR/BPS Guide to Pharmacology CITE 2023 (2023). https://doi.org/10.2218/gtopdb/F76/2023.3.

B. Droual, Chapter 8 - Synaptic Transmission and Neural Integration, Courses Taught by Dr. Droual at Modesto Junior College (2011). 

http://droualb.faculty.mjc.edu/Course%20Materials/Physiology%20101/Chapter%20Notes/Fall%202011/chapter_8%20Fall%202011.htm (accessed July 18, 2017).

L.S. Goodman, A. Gilman, L.L. Brunton, eds., Goodman & Gilman’s manual of pharmacology and therapeutics, McGraw-Hill Medical, New York, 2008.

E.L. Hudson, E.B. Jones, Cholinergic Toxicity, in: StatPearls, StatPearls Publishing, Treasure Island (FL), 2025. http://www.ncbi.nlm.nih.gov/books/NBK539783/ (accessed June 6, 2025).

Committee on Acute Exposure Guideline Levels, Committee on Toxicology, Board on Environmental Studies and Toxicology, Division on Earth and Life Studies, National Research Council, Piperidine, in: Acute Exposure Guideline Levels for Selected Airbone Chemicals, National Academies Press (US), 2012. http://www.ncbi.nlm.nih.gov/books/NBK241478/ (accessed September 16, 2016).

IPCS Inchem, Monograph on Atropine - Antidotes for poisoning by organophosphorus pesticides, (2002). http://www.inchem.org/documents/antidote/antidote/atropine.htm#7.1 (accessed April 28, 2017).

M.W. King, Biochemistry of Neurotransmitters and Nerve Transmission, (2015). http://themedicalbiochemistrypage.org/nerves.php (accessed September 25, 2015).

National Center for Biotechnology Information, CONIINE | C8H17N, PubChem Compound Database (2017). https://pubchem.ncbi.nlm.nih.gov/compound/441072 (accessed December 21, 2016).

J. Vetter, Poison hemlock (Conium maculatum L.), Food and Chemical Toxicology 42 (2004) 1373–1382. https://doi.org/10.1016/j.fct.2004.04.009.

This mechanism is responsible for the disruption of the nervous transmission by binding to the acetylcholine receptors (AChRs). A molecule acting through this MechoA 6.1 can be an agonist or an antagonist. Agonists produce the same effect as natural ligands, i.e. AChR activation. Antagonists block the AChR receptor rather than activating it like an agonist.

Note that agonists to AChR produce a similar effect as AChE inhibitors as they activate AChR but are not degraded by AChE. When degradation and elimination by the organism is too slow and insufficient,  these AChR binders are maintained at the receptor for too long. This leads to toxicity effects, particularly known as “cholinergic toxicity” (Hudson & Jones, 2025). The best known ACh agonists are nicotine for nicotinic receptors (nAChR) and muscarine for muscarinic receptors (mAChR). Antagonists when they are administered alone are toxic by blocking nervous transmission. However, they can be used as antidotes to prevent adverse effects occurring due to exposure to AChE inhibitors and AChR agonists (Campbell, 1993; Goodman et al., 2008).

Atropine and derivatives are typical examples of mAChR antagonists (IPCS Inchem, 2002). Besides, coniine is known to block nAChRs (NCBI, 2017; Vetter, 2004). Compounds similar to coniine can also cause the same effect despite their quite simple structure. The efficiency of these antagonists depends on their affinity to the binding site (Committee on Acute Exposure Guideline Levels et al., 2012). Tubocurarine is also a potent antagonist of ACh for nAChR as a single molecule of tubocurarine is equivalent to 2 molecules of ACh (King, 2015b). Indeed, there are 2 binding sites for ACh in AChR which must be occupied to open ion channel of the receptor (Droual, 2011) and tubocurarine occupies both of them as a single molecule. The reason why a ligand will have an agonist or antagonist effect is not yet totally understood.

Agonists and antagonists to ACh share common features with ACh (see Figure below). All of them have an amine or ammonium (or a nitrogen atom with similar properties) often accompanied by an aromatic ring and/or other nitrogen or oxygen atoms, all these molecules having thus a region that would be positively charged (deficient in electrons) at physiological pH, and another region that is rich in electrons. More specific criteria are coded in MechoA scheme alerts but are not detailed here given the variety of structures hitting this MechoA, as can be seen with just a few examples below.