MechoApedia by KREATiS

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MechoApedia is a tool dedicated to learn the scientific basis about the toxic mechanisms of action, called MechoA, which are classified within this scheme. Clik on the MechoA and their subclasses to get specific information on the class.


1.1 Non-Polar Narcosis

The non-polar narcosis (MechoA 1.1), or baseline toxicity, is assumed to be the least toxic of the known mechanisms of action for the less hydrophobic substances at least (Verhaar et al., 1992). Indeed, for similar hydrophobicity or solubility, non-polar narcosis is the mechanism which causes the lowest toxicity as shown by Dearden (2002).

Non-polar narcosis concerns chemical families like hydrocarbons, ketones, ethers, aliphatic alcohols, their halogenated derivatives etc. (Ellison et al., 2015).


1.2 Polar Narcosis

In practice, the distinction between non-polar and polar narcosis may be ambiguous. For examples, some pyridines are considered to act with MechoA 1.1 while other pyridines are associated to MechoA 1.2. 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 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 DMPC thus normalising the toxic impact between so-called polar and non-polar 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 and Hermens, 2002).

The polar narcosis (MechoA 1.2) is very similar to non-polar narcosis despite of the significant difference of toxicity observed for compounds with equivalent hydrophobicity (Escher and Hermens, 2002; Verhaar et al., 1992). Indeed effects due to carvacrol (MechoA 1.2) are comparable to 1,8-cineole (MechoA 1.1) but they are observable at smaller concentrations (de Sousa et al., 2012).

This mechanism concerns mainly simple phenols and anilines, but also amines and nitrogen heterocycles (Ellison et al., 2015).


1.3 Cationic Narcosis

The cationic narcosis (MechoA 1.3) is based on the same principle than the polar narcosis however the efficiency is higher because the bond energies involved are stronger and more favourable for interactions between cationic compounds and phospholipids of the cell membranes. The charge of a cationic compound stays at the surface of the membrane and can disrupt the charge distribution there (Wessels and Ingmer, 2013).

This mechanism concerns especially quaternary ammonium compounds.

Due to their high toxicity compared to their efficient concentration, quaternary ammoniums are often used as antimicrobials with broad spectra activity (McDonnell and Russell, 1999; Gilbert and Al-Taae, 1985; Daoud et al., 1983).


1 Narcosis

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 of this MechoA is based on a simple accumulation of substances into cell membranes thus disturbing their functions up to the loss of physical integrity. The effect may also be due to the impairment of cell proteins (e.g. ionic channel or G proteins) because their function is depending of their environment and their spatial conformation (Sikkema et al., 1995). 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. This property gives an idea of the fraction of compounds which will stay in aqueous compartment (i.e. extracellular medium or cytosol) and the fraction accumulated within hydrophobic compartments like membranes but also lipidic tissue. The potential of the narcotic effect varies according to the different affinities of non-polar and polar compounds for the membranes.

The term of narcosis used here is the same as used to describe rapid and reversible anaesthesia. Indeed, anaesthetic potential of molecules has been demonstrated to be correlated to their hydrophobicity according to the Meyer-Overton relationship (Kopp Lugli et al., 2009). That would indicate the mechanism of action leading to anaesthesia is the accumulation of molecules within the neural cell membranes. However, several current studies on the anaesthesia understanding have suggested the efficiency of narcotics have specific protein targets (like ionic channels) which have non-specific binding site (Kopp Lugli et al., 2009). After exposition to carvacrol or 1,8-cineole, some cavities are visible in cell membranes causing the cytosol to get out from the cell (cf. in opposite Figure). 1,8-cineole is a simple aliphatic ether with a MechoA 1.1 while carvacrol exerts MechoA 1.2, as a simple alkylphenol. These observations illustrate the narcotic impact of molecules. Figure: 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).


2.1 Narcotics products

Veith and Broderius (1987) have identified that simple esters lead to symptoms similar to those of narcosis. Moreover, these compounds are not very reactive in neutral aqueous medium. Since they observed an excess of toxicity compared to narcotic compounds, these authors have then described the toxicity of esters as another kind of narcosis: the esters narcosis. Russom et al. (1997) have used this category in their classification.

In fact, Jaworska et al. (1995) have shown that esters behave as non-polar narcotic compounds to protozoa (Tetrahymena pyriformis) while they were more toxic than narcotic to fish (Pimephales promelas). The authors explained this observation assuming protozoa do not, or barely, metabolise esters while fish esterase enzymes can very well hydrolyse esters (Figure).

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

This hydrolysis moves concentrations equilibrium between external medium and fish since the ester is gradually degraded. Consequently, the fish might potentially be exposed to a higher amount of the ester than if it was inert. However, the hydrolysis mechanism is in favour of the ester elimination out of the aquatic organisms since the hydrolysis products are significatively less hydrophobic than the ester parent. They are also less toxic than the ester parent. However, the accumulation of the alcohol and the acid generated after hydrolysis, combined to 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 (see MechoA 5.2).


2.2 Non-narcotic products

The hydrolysis product is not always an alcohol. For a phenol ester, for example, the degradation product will be a phenol with a more toxic MechoA like the polar narcosis (see MechoA 1.2), the uncoupling of the oxidative phosphorylation (see MechoA 5.1) for acid phenols or even the redox cycling (see MechoA 4.4) after the transformation of the p-diacetoxybenzene into p-dihydroxybenzene. Tartrates are also another example where the production of tartric acid after hydrolysis inhibits the fumarase which is an essential enzyme in the Krebs cycle (Shaw, 2002).Finally ortho-phthalates, as aromatic diesters, are a special case because the hydrolysis of one of the two ester moieties produces a monoester compound which can have endocrine disruption properties (see MechoA 6.8). This kind of compounds can induce the PPARα receptor (see MechoA 6.9) which is involved in the multiplication of the alpha peroxisomes thus leading to potential development of liver cancers (Adams et al., 1995) and antagonise FSH receptor impairing the sperm production and estrogen production.

Figure: adducts formation or acid releasing by hard electrophiles. X can be any good leaving.

Reactions a and c are related respectively to aldehydes and to epoxides and aziridines. However, these compounds often occur as intermediates in normal metabolic processes, like oxidation and reduction (Dunn et al., 2009). Indeed, both have a medium level of oxidation degree. Aldehydes can be an intermediate step in detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be easily eliminated. Epoxides are transiently used when hydrocarbons are transformed into diols to be eliminated like for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism but always transiently. They are rapidly transformed by enzymes in order to prevent their toxic MechoA.

In reaction b (see figure above), X is corresponding to a halogen. Therefore, a strong acid will be released (fluorhydric, chlorhydric acid, etc.). The rapid reaction with water prevents the same reaction with biological material instead of water (acylation) because water saturates biological media.

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 mesomere effect for instance. The leaving group has to be stabilised once separated from initial molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl group or an allyl group, such as benzyl chloride.

Basically, substances with MechoA 3.1 are mainly aldehydes, epoxides and some particular compounds with a good leaving group.


Enzymatic hydrolysis

Esters, amides, carbonates, polysaccharides and pyrophosphates are subject to hydrolysis which is mostly enzymatically driven.

The toxicity of those compounds is thus resulting from toxic effects of both parent and by-product compounds. The acidity produced together is expected to damage cells only if the amount of hydrolysed parent is high enough to exceed the buffer provided by biological media. Depending on the species, the enzymatic hydrolysis rate of compounds may differ. Generally, more complex the organisms regarding the number of cell types, better the metabolic capacity (McCarthy & Enquist, 2005). For instance, the liver is specialised in many metabolic pathways in vertebrates while it is lacking in invertebrates.


Reactive substances

This toxic mechanism is concerning substances which react spontaneously (i.e. without enzymatic catalysis) with endogenous compounds to form adducts to cell content (lipids, proteins and genetic material). Several molecular reactions can happen with different targets (e.g. thio or amino residues) depending on the kind of reactive substances.

According to HSAB theory, a hard Lewis acid (i.e. electrophile) react preferably with a hard Lewis base (i.e. nucleophile), rather hard and soft species. The concept of hardness is referring to electron distribution. Hard electrophiles and nucleophiles have a lack or an excess of electrons which is very located and barely deformable. For soft electrophiles and nucleophiles, this lack or excess can spread easily on bigger volume (Jacobs, 1997).


3.1 Hard electrophiles

Hard electrophiles have a significative and very localised lack of electron due to the presence of an electron-withdrawing group which is often a good leaving group (nucleofuge). Substances with MechoA 3.1 form adducts to amino residues of proteins and genetic material (DNA and RNA). 
The reactions which can occur along this MechoA are illustrated in Figure below.

Figure: adducts formation or acid releasing by hard electrophiles. X can be any good leaving.

Reactions a and c are related respectively to aldehydes and to epoxides and aziridines. However, these compounds often occur as intermediates in normal metabolic processes, like oxidation and reduction (Dunn et al., 2009). Indeed, both have a medium level of oxidation degree. Aldehydes can be an intermediate step in detoxication process by transforming alcohols into acids which are more hydrophilic and likely to be conjugated to be easily eliminated. Epoxides are transiently used when hydrocarbons are transformed into diols to be eliminated like for carbamazepine. Therefore, these reactive molecules can be naturally generated by the organism but always transiently. They are rapidly transformed by enzymes in order to prevent their toxic MechoA.

In reaction b (see figure above), X is corresponding to a halogen. Therefore, a strong acid will be released (fluorhydric, chlorhydric acid, etc.). The rapid reaction with water prevents the same reaction with biological material instead of water (acylation) because water saturates biological media.

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 mesomere effect for instance. The leaving group has to be stabilised once separated from initial molecule. Usually, good candidates for this reaction are the molecules having a good leaving group branched to a benzyl group or an allyl group, such as benzyl chloride.

Finally, the γ-diketones which are not disubstituted on the same carbon are another kind of hard electrophile. With very specific structural conditions, they can form pyrrole with amino residues of proteins and lead to protein cross-linking. the prototypical example of this category is hexan-2,5-dione.

Basically, substances with MechoA 3.1 are mainly aldehydes, epoxides and some particular compounds with a good leaving group.


3.2 Soft electrophiles

On the contrary of hard electrophiles, soft electrophiles have spread and deformable electron cloud. They react with soft nucleophiles like thiol residues which often occur in biological content (e.g. as lateral chain of cysteine or in glutathione molecule).

The more representative soft electrophiles have an α,β-unsaturated carbonyl group. They are typically esters, aldehydes but also quinones which are very specific α,β-unsaturated ketones. Other electron-withdrawing groups conjugated to a multiple bond, different from a carbonyl, may also have soft electrophile behaviour like, for instance, an α,β-unsaturated nitrile.

These substances and their dedicated reactivity mechanisms are shown in the figure below.

Figure: Adducts formation to proteins with soft electrophiles.


3.3 Radical-generating compounds

The radical-generating compounds have a simple and 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 one of the products). By breaking, the electrons within the bond are separated thus giving two free radical. Then, they can directly form adducts, or rather be trapped by oxygen to form superoxide anion. This chemical species is highly harmful for organisms because it reacts with endogenous molecules (lipids, proteins, genetic material) to form peroxides (Kazius et al., 2005; Munday, 1989; Wiley-VCH Verlag, 2002a).

Peroxides and disulfides are the main examples of this kind of reactivity.


Pro-active substances

This mechanism involves a metabolism step like an oxidation, a reduction or a conjugation with an endogen molecule for detoxification purpose.

The metabolisation of xenobiotic substances either aims to use them within organism processes (e.g. energy recovering by oxidising carbohydrates and lipids, production of amino acids from proteins hydrolysis, etc.), or to eliminate them, by making them more hydrophilic and less toxic, if possible. Xenobiotics are getting more hydrophilic after oxidation, then they are conjugated with hydrophilic endogen molecules, like glutathione, glucuronide or glycine. Hydrophilic compounds can be easily eliminated because they do not accumulate in membranes and adipose tissue. Instead they will preferably end up into urine. Benzylic alcohol is an example of easily metabolised compound. It is first oxidised into acid, then conjugated with glycine. The product is eliminated more easily than the parent (Nair, 2001).
However, these metabolic pathways may also generate more toxic compounds than the parent. Therefore, the MechoA related to pro-activation have been splitted up into different sub-categories of MechoA to cover different kind of metabolic products.

These mechanisms of action are often species-dependant because they involve metabolism which varies among species. For instance, mammals can metabolise 3-methoxyphenol into catechol derivative which will be toxic with RedOx cycling (Moridani et al., 2003), while fish and protozoa cannot effectively metabolise it. For those species, polar narcosis is expected for 3-methoxyphenol (Ellison et al., 2015) (see MechoA 4.3 to know more on that case).


4.1 Readily detoxified compounds

Complex organisms have yet a protective system for detoxification to oppose damaging effects, mostly from reactive compounds. Indeed, they have glutathione (GSH) and associated enzymes, aka glutathione-S-transferase (GST) and glutathione peroxidase (GPx). GSH can be oxidised into GSSG in order to reduce toxic xenobiotic. Then, GSSG can be back converted 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 and GPx can reduce peroxides. Conjugated molecules are easier to eliminate because they are not anymore reactive, and they become more hydrophilic.

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

These detoxification mechanims consum limited organism resources like GSH, glucuronic acid, and NAD(P)H. In the case of xenobitoics exposure with high or chronic doses, these resources may be drained and no more detoxification would be possible.

Readily detoxified compounds are typically hard electrophiles with a good leaving group, electron-rich aromatic aldehydes, benzylic alcohols, methacrylates and lactic acids and lactates. There can be other compounds readily detoxify not cited here.


4.2 Probably doesn’t exist

Hypothetically, this mechanism of action is related to substances which would be hydrolysed after been first metabolised (oxidation, reduction or conjugation).

Nowadays no substance has been identified to be corresponding to this definition.


4.3 Pro-reactive compounds

When epoxides or aldehydes are produced during oxidising steps, they can cause damage to cell content (proteins and DNA) if they are not rapidly transformed into less toxic compounds (see MechoA 3.1). For instance, some alcohols like α,β-unsaturated primary alcohol or 2,2,2-trifluoroethanol can generate very reactive aldehydes after oxidation before being transformed into acids (Koleva and Barzilov, 2010 ; Airaksinen et al., 1970). Generally, oxidation is the main metabolic step behind the increase of xenobiotics reactivity.

Several mechanisms for pro-reactive compounds are identified. Their related metabolism is explicated below:

  1. Aromatic hydrocarbons like Polycyclic Aromatic Hydrocarbons (PAHs) are converted into epoxides as intermediate compound (and later quinones) which cause adverse effects like DNA adducts if the elimination rate is not high enough. Note that we did not observe excess of toxicity compared to non-polar narcosis for aquatic organisms for fish, daphnids and algae for PAHs in both acute and chronic exposure. That may be due to a lack of CYP2E1 in these organisms (Ioannides, 1996).
  2. Aromatic nitros and aromatic amines (i.e. anilines) are respectively reduced and oxidised in mammals to generate nitroso compounds, which in turn are reactive (MechoA 3.1) and generate adducts with proteins and DNAs.
  3. 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.
  4. Furan and thiophen cycles which are not substituted at the same time in position 2 and 5, are oxides into epoxides, then into α,β-unsaturated dialdehyde. This very reactive compound form DNA and proteins adducts leading to cancerogenic effects (Food and Drug Administration, 2004; Smith, 2011).
  5. Vinyl alkoxy compounds (i.e. compounds with a terminal double bond branched to an ester or an ether) have been identified for having mutagenicity potency because their metabolism generates epoxides, forming DNA adducts.
  6. 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 and Bolt, 2000). All of them are metabolised into very reactive compounds in mammals. For aquatic organisms, no excess of toxicity is yet observed compared to non-polar narcotic effects.
  7. Alkynes can degrade hemes of CYP450 after being oxidised as described by Smith (2011). Few 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 ecotoxicology by Enoch et al. (2008).
  8. Hydrazines (and the compounds with a simple N-N bound) are metabolised in several ways depending on their structure, generating free radicals, so oxidative stress. Besides, diazoniums are also produced (ATSDR, 1997b). As very reactive electrophiles, diazoniums form DNA adducts (Brown and Vito, 1993).
  9. Aromatic azo compounds are likely to be metabolised by enzymes capable to break the N=N bound thus forming two anilines which will follow the mechanism of action of anilines (explained above, in paragraph ii.). They can also be metabolised into diazoniums (Brown and Vito, 1993).
  10. Benzenediols and mono-methyl ether derivatives have a specific MechoA. Mammals can demethylate a methylether group of an alkoxyphenol to generate a diol. This metabolic step is not observed in aquatic organism for which alkoxyphenols have toxicity comparable to polar narcotic compounds (MechoA 1.2) (Enoch et al., 2008; Schultz, 1987). 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 cycle (MechoA 4.4)

4.4 RedOx cycling compounds

This mechanism is dedicated to:

  • 1,2/1,4-benzenediols and quinones;
  • Paraquat and analogous;
  • Rotenoids;
  • Thiols and disulphides.

Aromatic diols are oxidised into quinones in two steps, passing through 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 into hydroquinone, semiquinone and quinone in a cyclic way, with superoxide anion generation for each oxidation step (see Figure below). Overtime, the reserve of NAD(P)H cofactors is consumed (Bolton et al., 2000; Di Francesco et al., 2004).

Figure: RedOx cycle of quinones (Bolton et al., 2000)

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 herbicide properties, but they are also toxic to animals through this same mechanism.

Figure: paraquat RedOx cycle (Blanco-Ayala et al., 2014).

Rotenoids, such as rotenone (1,2,12,12a-tetrahydro-8,9-dimethoxy-2-(1-methylethenyl-(1)-benzopyrano(2,4-b) furo(2,3-h)(1)-benzopyran-6(6H)-one), used as pesticides, are also RedOx cycling compounds. It fixes to complex I of the electron transport chain within the inner mitochondrial membrane where it is reduced of one electron. 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 stopping ATP production (Sherer et al., 2007).

Finally, thiols and disulfides can also be the cause of a RedOx cycle using gluthatione system as catalyst. Thiolates (basic form of thiols) are oxidised into thiyl radicals which can initiate a RedOx cycle with the corresponding disulphide (Munday, 1989). This mechanism is depending on stereo-electronic properties of the thiol. Indeed, tertiary thiols do not react the way described before due to steric hindrance. Furthermore, the reactivity of a thiophenol is lower because the electronic conjugation between the sulfur and the aromatic cycle stabilises the intermediate state. If the aromatic cycle has groups with electron-withdrawing effect (e.g. halogens or nitro), thiolate will be further stabilised and less reactive. On the contrary, if the cycle has electron-releasing groups (alkoxy for instance), the radical is stabilised, and oxidation is fostered.For these particular cases, gluthathione, gluthathione-S-transferase and gluthathione peroxidase are not detoxicating, but they help reactivity by catalysing the RedOx cycle (Munday, 1989).


4.5 Indirect pro-disruptors

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

A priori this MechoA could be found for many substances. To date, only ethanol is using this MechoA even if the 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, leads to liver cancer development among other deleterious effects (King, 2015a).


4.6 Docking pro-disruptors

This subcategory is related to substances which are metabolised into molecules disturbing enzymes, receptors or ion channels by directly docking in their active site. This binding can modulate activity of the protein (inhibition or activation). The final MechoA is explained in MechoA 6.

The small organo-halogenated compounds, some nitriles and diphenylamine are the main examples of MechoA 4.6.

Trichloroethylene, as other small organo-halogenated compounds, is metabolised into highly toxic compounds in mammals. The major metabolic pathway is the oxidation into chloral with cytochrome P450, then the reduction into 2,2,2-trichloroethanol (Brüning and Bolt, 2000) (see figure below). 

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

The MechoA of 2,2,2-trichloroethanol is related to ion channels modulators (MechoA 6.6). Indeed, this compound can bind to GABA receptors controlling chloride channels involved in the action potential of neurons. Also, dichloromethane is also oxidised in mammals leading to carbon monoxide production (ATSDR, 2014). The MechoA of carbon monoxide belongs to MechoA 6.9. It competes with oxygen to bind hemes and prevents its provision to cells.

Some nitriles can be hydrolysed at the alpha carbon by CYP450 to generate cyanohydrine. This reactive compound releases spontaneously cyanide anion on one hand, and an aldehyde or a ketone on the other hand (Grogan et al., 1992). Cyanide anion (MechoA 6.9) inhibits cytochrome c oxidase, which is the 4th complex within the mitochondrial electron transport chain, thus uncoupling this process from ATP production and generating oxidative stress (Way et al., 2007).

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 an explanation (Enoch et al., 2008). So far, no further substances have been identified to having similar MechoA.


Indirect enzyme disruptors

This general MechoA is dedicated to molecules which indirectly disrupt the operation of enzymes, receptors or ions channels. In that case, the xenobiotics do not directly bind to affected proteins, but rather modify their environment. Indeed, proper functioning of enzymes can be disrupted by pH, electric potential, oxidative degree of cofactors, etc.


5.1 Oxidative phosphorylation uncouplers

This mechanism is related to the disruption of a pH gradient through the inner mitochondrial membrane. This gradient is generated by the electron transport chain consisting of several membrane enzymes ultimately reducing molecular oxygen into water. The proton gradient is essential for the functioning of ATP-synthase which catalyses ATP production from ADP and inorganic phosphate while transporting one proton to internal matrix. 

Some substances are able to transport protons to internal matrix because they can go through the inner mitochondrial membrane under protonated form (acid form) in one way and under deprotonated form (basic form) in the other way. In fact, these substances decrease the proton gradient that the electron transport chain is generating, until the point where ATP-synthase cannot function anymore and no ATP is produced. This is called the oxidative phosphorylation uncoupling. (Escher et al., 1999).
Substances concerned by this mechanism are the acidic phenols.

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. In normal conditions, 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 optimal pKa for a phenol to be an oxidative phosphorylation uncoupler is between 6.6 and 8. Other authors affirm the pKa should be less than 6.5 (Schultz, 1987).
In fact, several factors are needed to get a significant uncoupling effect:

  • hydrophobicity,
  • pKa,
  • steric hindrance.

All these factors make difficult the quantification of uncoupling effect of phenols, and so their toxicity.
Some substances are known to directly interact in active site of one complex in electron transport chain thus disrupting oxidative phosphorylation. In that case, due to direct interaction of the xenobiotic and the complex, it refers to MechoA 6.


5.2 Acids and bases

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

Most of non-polar chemical species can go through cell membrane by passive diffusion, but ionic compounds cannot. For acids and bases, at the extracellular pH of around 7.4 and pH 7.2 in cytosol (Casey et al., 2010), most of the molecules are in the ionic state. Only a small portion of neutral form of acids and bases exists in both compartments. After a neutral molecule has crossed the membrane, it will release or catch electron in the cytosol to get its ionised form, driven by the acid-base equilibrium (see figure below).

Figure: example of cell acidification by acids.

On the figure, protonated acids (red) are neutral and can cross the membrane. Each release a proton taken in charge by water molecule (blue). Therefore, cytosol become more acid. Note that, after an acid molecule has passed into the cell, another acid gets protonated in extracellular medium because of acid-base equilibrium (blue). We can assume acidification can also happen in other compartments 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 in case of high exposure to acids or bases.

For instance, antimalarial effect of quinine and analogous has been recently explicated by this basification mechanism (Vallabh Minikel, 2016). The author explained that Plasmodium falciparum, the parasite responsible for the disease, does not manage to degrade hemes which accumulate in acidic compartments where they spontaneously crystallise. The basification of this compartment by quinine causes heme releasing. Furthermore, free hemes are highly toxic for the parasite because they catalyse oxidation reactions in uncontrolled and unspecific way leading to radical production. The same authors explain that bases, like quinine, can diffuse as neutral form into more acidic compartments to capture protons. We can assume the MechoA of bases is based on basification of critical cell compartment like lysosomes. In that case protease would be inhibited. Besides, cutaneous corrosivity of bases (e.g. pure bases or solutions with pH > 11) is probably also due to this basification effect.

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


5.3 Others

Others MechoAs may exist which involve an indirect disruption of the enzyme functions. The only one clearly identified for now is the MechoA related to ethanol. Indeed, ethanol is oxidised into acetaldehyde, then into acetic acid during its metabolism. The enzymes in charge of these transformations reduce NAD+ into NADH as in the Krebs’ cycle. Therefore, in case of high exposure with ethanol, glycolysis rate and fatty acids oxidation may be decreased by a lack of NAD+. Then, an excess of fatty acids in blood and in liver may be observed leading to hepatic steatosis or even to liver cirrhosis (King, 2015a). In this way, there will be indirect disruptions of a whole enzymatic system due to a lack or an excess of the common cofactor (NAD+/NADH).


Direct docking disruptors

This general MechoA is gathering the largest number of subcategories of MechoA related to any direct interaction with an enzyme, a receptor or an ion channel (called active protein here).

The most known MechoAs within direct docking disruption are: acetylcholine esterase inhibition, nicotinic or muscarinic acetylcholine receptors binding, ion channels binding (calcium, sodium, chloride, etc.), opioids receptors binding, hemes binding, hormonal receptors binding (oestrogen, androgen, thyroid), etc.

The binding of xenobiotic to an active protein can be localised in the active site of the enzyme, to an allosteric site for an enzyme, an ion channel or a receptor, or even to the main binding site of a receptor.


6.1 Acetylcholine-esterase inhibitors

This mechanism is responsible for the disruption of the acetylcholine (ACh)-dependant nervous transmission. ACh is a neurotransmitter which triggers the opening of ions channels, the receptors themselves for nicotinic receptors or separate ions channels for muscarinic receptors. The ions flux leads to the depolarisation of the postsynaptic 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 ions channels and the cell to recover its resting state. The acetylcholinesterase (AChE) hydrolyses the ACh into choline and acetate in the synaptic cleft. This enzyme is critical to ensure the nervous transmission is done properly. The choline molecules produced with AChE activity are recycled by being transported back to the first neurone which has initially secreted the ACh.

The inhibition of AChE rapidly leads to lethal effects for animals by arresting respiratory muscle functions (Elersek and Filipic, 2011; Fukuto, 1990). This mechanism is used by nerve agents for chemical weapons (e.g. VX, sarin, tabun, etc.). This is also the common mechanism of action used by various insecticides like azinphos-methyl, methomyl, aldicarb, malathion, etc.

At a molecular level, the hydrolysis of ACh counts several steps: binding of ACh to serine residue of AChE active site, elimination of choline and elimination of acetate. Inhibition of AChE is possible by imitating ACh binding to serine as presented in the figure below. As expected, the leaving group is eliminated in a second step. However, the group attached to serine is barely eliminated compared to ACh (Elersek and Filipic, 2011).

Organophosphorus compounds with structures shown in the figure are known to have strong capacities to inhibit AChE. There may be others enzymatic targets of organophosphorus compounds less known as suggested by (Elersek and Filipic, 2011). Carbamates are another chemical family with good candidates to inhibit AChE (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 to have a strong impact on the inhibition efficiency. Nevertheless, the reactivity is not the only parameter leading the inhibition. Indeed, steric hindrance plays an important role. For example, carbamates with a good leaving group like phenoxy or oxime must have an adequate three-dimensional structure corresponding to the form and polarity to the AChE active (Fukuto, 1990).

This MechoA occurs only for organism with neurons, i.e. animals with some exceptions. However, substances with MechoA 6.1 are not necessarily inert for non-animal organisms because they can inhibit other esterases, notably the organophosphorus compounds.


6.2 Acetylcholine-receptor binders

This mechanism is responsible for the disruption of the nervous transmission by binding the acetylcholine receptors (AChR). The molecule with MechoA 6.2 can be an agonist or an antagonist. The agonist produces the same effect than natural ligand, i.e. AChR activation. The antagonist blocks the receptor rather than activating it like an agonist.

Note that the ACh agonists to AChR produces a similar effect than AChE inhibitors. Indeed, they activate AChR but are not degraded by AChE, therefore the effect is not stopped, and the post-synaptic cell cannot recover its resting potential.

The most known ACh agonists are nicotine for nicotinic receptors (nAChR) and muscarine for muscarinic receptors (mAChR).

Antagonists are toxic when they are administered alone by blocking nervous transmission. However, they are antidotes to prevent adverse effects occurring with AChE inhibitors and AChR agonists.

Atropine and derivatives are typical examples of mAChR antagonists (IPCS Inchem, 2002). Besides coniine is known to block nAChRs (National Center for Biotechnology Information, 2017; Vetter, 2004). Compounds with similar structure to coniine can also cause the same effect despite of their very simple structure. The efficiency of these antagonists is depending of the affinity to the binding site (Committee on Acute Exposure Guideline Levels et al., 2012).

Agonists and antagonists to ACh share common features with ACh (see Figure below). All of them have hindered ending with a positive charge (in blue) at a few Angström distance to an electron-rich H-bond acceptor (in red).

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

Tubocucarine is also an antagonist of ACh for nAChR and it contains 2 equivalents of ACh (King, 2015b). Indeed, there are 2 binding sites for ACh in AChR which have to be occupied to open ion channel of the receptor (Droual, 2011). The reason why a molecule will have an agonist effect or antagonist effect is not yet totally understood.

Nota Bene. This mechanism is only concerning organisms with neurones whose ACh is the neurotransmitter (with AChRs). Basically, they are animals with some exceptions. 


6.3 Dopamine transport disruptors

Dopamine is a neurotransmitter which is involved in other neuronal connections than acetylcholine. Like for acetylcholine, the functioning of the nerve transmission needs dopamine to be released at synaptic cleft in order to activate a G Protein-Coupled Receptor. Then the neurotransmitter is recycled by moving back to the presynaptic neurone (Calipari and Ferris, 2013; King, 2015b).

Amphetamine and similar compounds modify these mechanisms.

Indeed, amphetamine forces dopamine release in synaptic cleft. At the same time, it inhibits the transport of dopamine back to the cell leading to overdose of dopamine in intercellular space and so, a continuous excitation of the postsynaptic neurone (Calipari and Ferris, 2013).

This mechanism is concerning only organisms with dopamine-dependant neurones, basically animals with some exceptions (Barron et al., 2010). Note that dopamine has also been identified in plants, but with a different role (Kulma and Szopa, 2007).


6.4 Metal chelators

For most of organisms, metals are needed in a great extent for enzymes functioning, inside their active site to catalyse the chemical reaction. For instance, iron ion is present in the centre of cytochrome P450 heme and metalloproteases use a magnesium or zinc ion to catalyse hydrolysis reaction. Moreover, metal ions can play a critical role in cell signaling (especially calcium ions) or they are used to create the electric action potential (sodium and potassium). Metal chelation is the formation of a stable complex between a molecule and a metal ion. Some xenobiotics have the ability to chelate metals thus competing with the enzymes. These compounds can either share the metal chelation inside the active site of the enzyme, either extract the metal ion from the enzyme to form an independent complex. In both cases, enzyme is inactivated.

In order to have good capacities to chelate metals, the molecule must have negative partial charges at the right distance corresponding to the target metal diameter. For instance, beta-dicarbonyls are efficient iron chelators and degrade cytochrome P450, provided the two carbonyls can get into a parallel position (O’Donoghue, 2001). Besides, calcium can be chelated by oxalate forming crystals which accumulate and block the blood circulation (Wiley-VCH Verlag, 2002c).

Some organisms have protecting proteins, the metallothioneins, which can chelate metal ions to control the free concentration. However genic expression of metallothioneins is finely regulated. Therefore, high exposure to xenobiotics behaving like metal chelators is deleterious for the cell (Petering and Fowler, 1986).


6.5 Photosystem II electron transport inhibitors

This mechanism is similar to RedOx cycling (MechoA 4.4). Instead of blocking mitochondrial electron transport chain, substances with a MechoA 6.5 block the electron transport chain of plants photosystem.

Several urea derivatives compounds inhibit D1 protein in the photosystem II (van Rensen, 1982) preventing electron transmission from water oxidation to other proteins of the chain which reduce NADP+ into NADPH. Besides, the electron transport chain creates a proton gradient between the stroma and the thylakoid lumen which is used by ATP synthase to produce ATP. By blocking electrons at the level of D1 protein, no more NADPH, nor ATP is produced. 

Bromacil, Diuron, Atrazine and other triazines are typical examples of compounds which inhibit D1 protein in photosystem II.


6.6 Ion channel modulators

This MechoA is related to substances which modify activity of ion channels or ionotropic receptors except for nicotinic acetylcholine receptors (nAChRs) for which a dedicated MechoA class (MechoA 6.2) exists.

Substances with MechoA 6.6 may disrupt the functioning of receptors to γ-aminobutyric acid (GABA), a neurotransmitter. There are two types of GABA receptors, GABAAR and GABABR, which are directly (GABAAR) or indirectly (GABABR) responsible of the influx of chloride ions. Activation of GABAergic neurons leads to antidepressant effects like anticonvulsants, anxiolytics, sedatives and hypnotics effects (Olsen and DeLorey, 1999).

2,2,2-trichloroethanol has a GABAergic activity by binding to GABA receptors which are then more sensitive to GABA.

Neurons have also G protein-coupled receptors to opioids which trigger the opening of channels for potassium efflux and calcium influx. The activation of the opioids receptors leads to the reduction of action potential and nerve signal propagation.

Morphine and derivatives are the most known of the opioids with MechoA 6.6 (Chahl, 1996). 

Some substances prevent the closing of sodium channels in neurons thus prolonging their excitation. 

These are basically DDT (dichlorodiphenyltrichloroethane) and analogues like methoxychlor and pyrethroids like resmethrin, cypermethrin or tefluthrin, etc. (figure below).

Figure: Structure of DDT derivatives and pyrethroids derivatives.

Besides having impact on sodium channels, type II pyrethroids also inhibit the opening of GABAergic chloride channels and Ca-Mg-ATPase thus resulting in a modification of calcium concentration in the neuron (Coats, 1990). Moreover, various substances can modulate GABAergic calcium channels which have several different binding sites with a broad range of potential modulations (see figure below).

Figure: GABA receptor and binding sites (Olsen and DeLorey, 1999).

The binding site of 2,2,2-trichloroethanol and ethanol is uncertain (Olsen and DeLorey, 1999). Pyrethroids seem to bind to the picrotoxin site (Coats, 1990). Chlorinated alicyclic insecticides are also binding to the picrotoxin site thus preventing the channel to open and the neuron to recover its resting state. These insecticides cause death by respiratory arrest (Coats, 1990). They have various structures with a non-aromatic cycle (i.e. alicyclic) and several chlorine substituents as a common point. Some of them are presented in the figure below.

Figure: chlorinated alicyclic insecticides and picrotoxinin.

Caffeine is another example of substance which have a MechoA 6.6 because it binds to GABA receptor at benzodiazepine sites. However, this mechanism is minor for caffeine (and methylxanthines, in general) which modulates several calcium channels by activating or disactivating them depending on the dose.

Furthermore, caffeine is an antagonist of adenosine receptors leading to repression of the spontaneous neuronal electric activity, the synaptic transmission inhibition and the neurotransmitter release (Nehlig et al., 1992). Caffeine would also disrupt calcium regulation in plants, but these mechanisms are not well studied (IAC Publishing Labs, n.d.).


6.7 Mitochondrial electron transport chain inhibitors

This MechoA is related to substances which inhibit one or another protein of the electron transport system in the mitochondria present in every animal cell. When one of these proteins is inhibited, the whole chain is stopped, and the proton gradient is not maintained anymore. Thus, ATP cannot be produced anymore.

Figure 20 : Couplage de l’ATP synthase et de la chaîne de transport d’électrons par le gradient de protons (from Anatomy & Physiology (OpenStax, 2014).

Most known examples are rotenone (and rotenoids in general) which inhibit complex I, cyanide ion which inhibits complex IV.


6.8 Endocrine disruptors

This MechoA concerns substances that disturb the production, transport or metabolism of hormones, or that interact with their receptors, either as agonist, antagonist, or any other mechanism that alter the number of hormone receptors or their activity.

Examples of this MechoA are ethynylestradiol (EE2), tamoxifen, kepone, bisphenol A, DDT, genistein, sodium arsenite.

The prediction of this endocrine disruptor MechoA is provided by KREATiS as a specific expert service.


6.9 Others

They are various other MechoA involving a specific interaction with an active protein which are sometimes specific to one particular molecule (e.g. amanitin). These mechanisms are listed here below but they are not detailed. There are of course other MechoAs which are not in this list, notably for pharmaceuticals. At KREATiS, we are working daily to increase the understanding of specific MechoAs and include them in the MechoA Classification Scheme.

Non-exhaustive list of other specific MechoAs:

  1. Inhibition of cyclooxygenases (COX-1 and COX-2) (e.g. salicylic acid and derivatives)
  2. Herbicides with specific MechoA (e.g. glyphosate, trifluraline)
  3. Glycine antagonist (e.g. strychnine)
  4. Specific dicofol-related endocrine disruptions
  5. Aryl hydrocarbons receptors binding (e.g. dioxines, PCBs and polychlorodibenzofuranes)
  6. Inhibition of oxygen transport by hemoglobins (e.g. carbon or sulfur monoxide, cyanide ion)
  7. Inhibition of DNA transcription by binding RNA polymerase (e.g. amanitine)
  8. Other specific MechoAs with few details (e.g. 1,1,1-trichloroethane, 3-methoxyphenol, 2,4,6-triiodophenol)
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Knowledge & research in environment and toxicology in silico