Event: 1487

Key Event Title


Binding, SH/seleno proteins

Short name


Binding, SH/seleno proteins

Biological Context


Level of Biological Organization

Cell term


Organ term


Key Event Components


Process Object Action

Key Event Overview

AOPs Including This Key Event


AOP Name Role of event in AOP
Oxidative stress and Developmental Neurotoxicity MolecularInitiatingEvent



Taxonomic Applicability


Term Scientific Term Evidence Link
rat Rattus norvegicus NCBI
mouse Mus musculus NCBI
zebra fish Danio rerio NCBI
human Homo sapiens NCBI
Gallus gallus Gallus gallus NCBI

Life Stages


Life stage Evidence
During brain development, adulthood and aging High

Sex Applicability


Term Evidence
Unspecific High

Key Event Description


Thiol (SH)- and seleno-containing proteins are located in different organelles and in the cytoplasm of the different neural cell types (Comini, 2016; Hoppe et al. 2008; Barbosa et al. 2017; Zhu et al. 2017). Binding of chemicals to these proteins induces either their inactivation or favor their degradation and/or inhibition of their synthesis (Farina et al. 2009; Zemolin et al. 2012). Therefore, we will directly include in the description of this MIE and of the protein of interest the main chemicals (mercury, acrylamide and acrolein) able to bind and to interfere with these proteins. (See Evidences for perturbations of this MIE by stressors)

How It Is Measured or Detected


The interference of MeHg, acrylamide and acrolein  with the normal catalytic function of thiol- or selenol-containing enzymes, transporters, channels, etc can be determined by different analytical methodologies. The activity of enzymes are typically determined by spectrophotometric, spectrofluorometric  or radiometrical methodologies that quantify the rate of product appearance or the disappearance of  substrate. The examples of HMM-SH or HMM-SeH enzymes that are altered by MeHg, acrylamide and acrolein presented in Table 3 (Table 3 and 4) are normally determined by spectrophotometric methodologies. Below we give a brief description on how to measure the enzymes listed in Table(s) 3 and 4.

Creatine Kinase (CK). CK activity can be measured using phosphocreatine and ADP as substrates. The formed creatine is estimated colorimetrically  at 520 nm as described by Hughes (Hughes, 1962).

Glutathione (GSH): Total glutathione level was determined using the Glutathione Assay Kit (Sigma-Aldrich, CS0260) according to the manufacturer’s instructions. Quantity of GSH was assessed by measuring the continuous reduction of 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) to 5-thio-2-nitrobenzoic acid (TNB) by spectrophotometry (Synergy MX) at 412 nm. Measures were taken at 1 min intervals for 5 minutes. The production rate of TNB is proportional to the concentration of glutathione up to 2 µM and values for GSH concentration were calculated as the difference between TNB absorbance values measured at time 0 versus 5 min with reduced glutathione as standard.

Glutathione peroxidase (GPx) is usually determined spectroscopically at 340 nm using a coupled assay with glutathione reductase (GR). Another methodologies can be found in Flohé, L., Günzler, W.A. (1984). The reaction mixture usually contains (in mmol/L or mM) 50 phosphate buffer (pH 7.0), 10-100 µl sample, 0.24-1.0 U of glutathione reductase (usually from yeast), 1-4  GSH, 0.6-4.3  EDTA and 0.15-0.34  NADPH . The reaction is started by adding 10-100 µl peroxide (hydrogen peroxide, cumene hydroperoxide or tert-butylperoxide) to a final concentration of 0.1-2.0 mM . For quantification in crude extracts, the addition of azide is required to inhibit the catalase reaction, when H2O2 is used as substrate. The decrease in absorbance is followed at 340 nm from 1 to 10 min. The blank is made by substituting the sample by the same buffer in which the sample is prepared.

Thioredoxin Reductase (TrxR). TrxR activity is normally measured by the method of Holmgren and Bjornstedt (1995). The reaction mixture consisted of the following (in mM): 0.24 NADPH, 10 EDTA, 100 potassium phosphate buffer (pH 7.0), 5 5,5’-dithiobis-2-nitrobenzoic acid (DTNB), and 0.2mg/mL of BSA. The partially purified TrxR was added (to final concentration of 6–10 microg/ml of protein) to the cuvette containing the reaction mixture, and the absorbance is followed at 412 nm for 4 min.

Type 2 5’-deiodinase (DIO 2). Deiodinase is usually determined by measuring [125]I released from [125I]reverse T3 (rT3) in in a gamma counter after separation of [125]I by ion exchange chromatography (Dowex 50W-X2 resin) as described by Mori et al., 1996. The reaction medium contains (in mM):  100 potassium phosphate buffer (pH 7.0), 1 EDTA, 20 dithiothreitol (DTT), 1 6-proryl-2-thiouracil (PTU), and 2 nM rT3.

Glutamine synthetase (GS). GS can be measured by different methods: a) the of formation of inorganic phosphate (Pi), b) ADP at 340 nm (using the enzymes pyruvate kinase and lactate dehydrogenase as coupled reactions), c) glutamine (e.g., determining the transformation of 14C-glutamate to 14C-glutamine) or d) the colorimetric formation of glutamylhydroxamate assay method. The glutamylhydroxamate assay method usually is determined in the presence of (in mM) 0.1 ml of enzyme solution (0.1 ml) plus 0.9 ml of the reaction solution with 50 imidazole-HCl buffer, 20 MgCI2, 25 mercaptoethanol, 50 sodium L-glutamate, 100 hydroxylamine, and 10 ATP. After incubation, 1.5 ml of FeCl3 (370 mM FeCl3, 670 mM HCl, and 200 mM trichloroacetic acid) is added. The mixture is centrifuged and the supernatant is used to determine the absorbance at 535 nm (Patel et al., 1982; Pishak and Phillips, 1979).

Ca2+-ATPase. Ca2+-ATPase can be determined directly by the quantification of inorganic phosphate released from ATP or indirectly by determining the 45Ca2+ uptake by brain microsomes (Freitas et al. 1996).  The assay mixture for Ca2+ uptake determination has (in mM) 50 MOPS-Tris (pH 7.4), 5 MgCl2, , 1 ATP, 20 Pi (inorganic phosphate) and 0.04 CaCl2 (0.5 µCi/ml 45CaCl2).  The microsome is then filtrated through Millipore filters (0.45 µm) and flushed with La(NO3) 3 and the radioactivity in the filters is counted on a scintillation counter.

Complex I. Complex I activity was measured by the rate of NADH-dependent ferricyanide reduction as described in (Cassina and Radi,1996). In short, the NADH dehydrogenase can be determined in by the reduction of ferricyanide  at 420 nm in the presence of (in mM) 0.2 NADH and 0.5 ferrycianide. The activity is determine in the presence of 5 µM rotenone.

Complex II and Complex II-III. The complex II activity or succinate -2,6-dichloroindophenol (DCIPI) reductase activity  and the complex III (succinate: cytochrome c oxidoreductase or complex II-CoQ-complex III activity) can be determined by the method of Fischer in the presence of (in mM): 50 potassium phosphate buffer (pH 7.4), 20 succinate, 2 KCN and 0.05 DCIPI at 600 nm or 0.05 of oxidized cytochrome c at  550 nm.

Complex IV. Cytochrome c oxidase (complex IV) activity can be determined spectrophotometrically by the method of Rustin et al. 1994 at 550 nm.

Domain of Applicability


Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event


Interferences of the chemical initiators with SH-/seleno-containing proteins


MeHg can interact with different functional groups found abundantly in biomolecules (e.g., carboxylate, primary and secondary amine groups, etc; Rabestein 1978a); however, its affinity for thiol and selenol groups are 6 to 12 orders of magnitude superior to that for hard nucleophile centers found in biomolecules (Table 1). The constants described in Table 1 indicate that MeHg behaves as a strong soft electrophile, i.e., it has much higher affinity for the soft nucleophiles centers of thiol- and selenol-containing molecules (Pearson, 1963; Rabestein 1978; Arnold et al. 1986; Sugiura et al., 1976; 1978), than for hard nucleophiles centers found in the functional groups of proteins, RNA, DNA, carbohydrates and lipids (Rabeinstein, 1978a).  Furthermore, the rate constant for the reaction of MeHg with thiol/thiolate (R-SH/R-S-) has been estimated to be about 6 x 108 M-1.sec-1, which indicates a very fast reaction (Rabenstein and Fairhurst, 1975).  As corollary, the occurrence of free MeHg or bound to other ligands such as carboxylates, amines, chloride or hydroxyl anions in the physiological media of living cells is insignificant or nonexistent (George et al. 2008). The binding of MeHg to abundant low molecular mass thiols or LMM-SH (e.g., cysteine and reduced glutathione-GSH) and high molecular thiol-containing proteins or HMM-SH (e.g., albumin, hemoglobin, etc) is critical for the MeHg distribution from non-target to target organs and cells (Farina et al. 2017).

Table 1 - Affinity constants of methylmercury for important chemical groups found in biomolecules (adapted from a Rabestein, 1978, Rabestein and Bravob using different thiol-containing molecules with the arylmercurial para-mercurybenzenosulfonate,  and cIsab 1991; and from dArnold et al. 1986 taking into consideration that the calculated formation constant of –Se-MeHg conjugates was 0.1 to 1.2 order greater than that of –S-MeHg). The values represent the Log of the constants.


Functional Group



Formation constant

Carboxyl/Carboxylate (-COOH/-COO-)

Amino acids, proteins, fatty acid


Amino or primary amine (-NH2/-NH3+)

Amino acids, proteins, nitrogenous bases, nucleosides, nucleotides

≈7.0-8.0 a

Secundary amine


Amino acids, proteins, nitrogenous bases, nucleosides, nucleotides

≈7.0-9.0 a

Thioester (-S-)


≈2.0 a

Thiol/thiolate (-SH/-S-)

Cysteine, glutathione, proteins

≈14-18 a,b

Thiol/thiolate (-SH/-S-)



Selenol/selenolate  (-SeH/Se-)

Selenocysteinyl residues in selenoproteins

≈ 16-18d



Here we will not discuss factors that can modify MeHg distribution, specifically, we will assume that MeHg-S conjugates reach the mitochondria, where MeHg will bind to thiol- and selenol-containing proteins via the exchange reactions of MeHg from one –SH to another –SH or –SeH groups  (Figure 1; Rabenstein 1978b; Rabenstein and Fairhurst, 1975; Rabenstein et al., 1974; 1982; Rabenstein and Reid, 1984; Farina et al. 2011, 2017; Dórea et al. 2013). But we have to emphasize that what we call of binding to –SH or –SeH groups is, in fact, an exchange reaction of MeHg from MeHg-S conjugates (e.g., MeHg-cysteine or MeHg-Cys and MeHg-glutathione or MeHg-SG conjugates; Figure 1) to  a free thiol- or selenol-group from non-target or target proteins (Figure 1). Thus, the interaction of MeHg with its target proteins in the brain usually involves the exchange of MeHg from LMM-S-conjugate to a thiol or selenol group in different types of proteins. The Molecular Initiating Event  (MIE) of targeting thiol- or selenol-groups in mitochondrial brain proteins is expected to start a cascade of related events, which will culminate in mitochondrial failure, oxidative stress, thiol depletion, glutamate dyshomeostasis, inflammation, cell death and learning disabilities (Wormser et al. 2012; Roos et al. 2012; Ciccatelli et al. 2010; Montgomery et al. 2008, Stringari et al. 208)

Figure 1 – Binding of MeHg (CH3Hg+) to target thiol- (HMM-SH) or selenol-containing proteins (HMM-SeH). Note that, in fact, the binding of MeHg to their high molecular mass target proteins is mediated by exchange reactions of MeHg from low molecular mass thiol (LMM-SH) molecules to HMM-SH (represented by Prot-SH) or HMM-SeH (represented by Prot-SeH). The scheme also demonstrated that MeHg conjugated with one LMM-SH (here represented by either Cys1-SHgCH3 or G1SHgCH3) can exchange with others LMM-SH (here represented by Cys2-SH or G2SH). After one exchange reaction, the conjugated Cys1-SHgCH3 and G1SHgCH3 release the free LMM-SH molecules Cys1-SH or G1SH. This type of exchange reaction can also occur in the extracellular space.

In view of the strong affinity of MeHg for thiol-groups and the relative high abundance of LMM-SH molecules over HMM-SH and high molecular mass selenol containing proteins (HMM-SeH) (Table 2), the probability of finding MeHg molecules bound to LMM-SH molecules is high. In fact, at physiological pH, the affinity (constant formation) of MeHg with GSH or hemoglobin was higher for GSH than hemoglobin  (about 1 order of magnitude, Reid and Rabestein, 1982). However, the studies performed by professor Dallas Rabestein have clearly demonstrated that MeHg can migrate rapidly/easily from one LMM-SH to either other LMM-SH or HMM-SH groups and vice and verse (Rabenstein 1978b; Rabenstein and Fairhurst, 1975; Rabenstein et al., 1974; 1982; Rabenstein and Reid, 1984; Arnold et al. 1986; Farina et al. 2011, 2017; Dórea et al. 2013).  The studies of Rabenstein and others have also pointed out that the affinity of MeHg for –SeH groups is higher than for analog –SH groups (Sugira et al. 1976; 1978; Arnold et al. 1986). Thus, one would guess that –SeH-containing molecules (i.e., selenoproteins) should be the preferential targets for MeHg (Farina et al. 2011). Although this can be the case, the great abundance of –SH-containing molecules over the very limited occurrence of selenoproteins (-SeH groups) and the potential change in the reactivity of specific –SH groups at the microenvironment of thiol-containing proteins, made the picture a little more complicate. Despite of this, several studies have demonstrate that the selenoenzymes glutathione peroxidase (GPx), thioredoxin reductase (TrxR) and 5′-deiodinase (DIO) can be inhibited after in vitro and in vivo exposure to MeHg (Li et al. 2008; Carvalho et al., 2008; 2011, Farina et al.,  2009; Franco et al., 2009; Wagner et al., 2010; Branco et al., 2011; 2012; 2014, 2017; Dalla Corte et al., 2013; Meinerz et al., 2017)

Table 2 – Occurrence of Soft Nucleophilic Centers (SNC) that can bind the Soft Electrophile Methylmercury (MeHg) with high affinity. The thiol (-SH) groups can be found in thousands of proteins and in a few low molecular mass molecules. In constrast, the selenol (-SeH) group is found only in a few number of selenoproteins.


Thiol-containing proteins - High molecular mass thiol molecules


–Cysteinyl residues (Cys)



in thousands of proteins



Selenoproteins - High molecular mass selenol molecules


–Selenocysteinyl residues (Sec)



in few dozens of proteins



Low molecular mass thiol molecules (-SH)




glutathione (GSH)





Low molecular mass selenol molecules (-SeH)





aThe exact concentration of thiol-containing proteins is not well characterized (except for hemoglobin and albumin, which have reactive cysteinyl residues in the mmol/L range.) The pmol/L is an estimation. bThe actual concentrations of selenoproteins have not been well characterized and the presented range is an estimation.

The binding of MeHg to the –SH or –SeH groups of proteins can directly inactivate their function or can indirectly facilitate the denaturation of the proteins even after the exchanging or transference of MeHg to another LMM-SH, HMM-SH or HMM-SeH  molecules (Farina et al. 2011; Farina et al. 2017). The hypothetical types of interactions between LMM-S-MeHg conjugates with thiol- and selenol-containing proteins (HMM-SH or HMM-SeH molecules) is depicted in Figure 2.   As commented above, the binding of MeHg to redox sensitive thiol- or selenol-groups can disrupt the activity of enzymes or the biochemical role of non-enzymatic brain proteins. Some examples of thiol- and selenol-containing brain enzymes that have been reported to be disrupted after MeHg exposure are presented in Tables 3 and 4Table 5 shows some of the mitochondrial processes that can be disturbed by MeHg.

Figure 2 – Hypothetical Binding of MeHg to different types of target proteins. The binding of MeHg to proteins can cause either a transitory inhibition of the protein fucntion (first line, the yellow protein was reactivated by interacting with LMM-SH or R-SH). The pink protein is an example of protein that after the binding of MeHg suffered a change in the structure in a such way that it cannot be reactivated by LMM-SH or R-SH.  The third protein (blue) is an example of protein that was permanently denaturated after MeHg binding and even after the removal of MeHg the activity was not recovered. The same type of interactions can be applied to the selenol-containing proteins (i.e., the selenoproteins). Here we have not included the non-targets proteins or thiol-containing proteins that can bind MeHg without interfering in the protein function.


Table 3 – Examples of thiol- and selenol-containing proteins that are inhibited by MeHg

Protein (complex) activity inhibited by MeHg



Functional group





Creatine Kinase (CK)


in vitro



Adult mice cerebral cortex

C6 glioma cells


50-1500 µM -IC50=87 µM

1-50 µM -IC50≈50 µM


Glasser et al. 2010b


Total GPx






in vitro






SH-SY5Y cells


0.5-2.0 µM Max. Inh.≈40%


Franco et al. 2009

Mouse  neuroblastoma

2.5 - 5.0  µM (24h)   Max. Inh.≈15-40%

Kromidas et al. 1990

PC12 cells

1.0-7.5 µM (24h) –  Max. Inh.≈7%

Li et al. 2008

Rat Fetal Telenchepalic cells

Aggregating immature and mature cells  (Cu2+ +ascorbate) + 1-100 nM MeHg

Sorg et al. 1998


Cytoplasmic TrxR






Nuclear TrxR








Cytoplasmic Gpx



in vivo



mice (gestacional and lactacional)


22 days-old C57BL/6J mice – 5 mg/L

cerebrum- male ¯

cerebrum- female ­

cerebellum- male ¯

cerebrum- female =


cerebrum- female =

cerebrum- male ¯


cerebellum- female =

cerebellum male  ¯




cerebrum- male ¯

cerebrum- female ­

cerebellum- male =

cerebellum- female =



Ruszkiewicz et al. 2016













in vivo




Adult Swiss male  mice-



21 days - 40 mg/L water


Cerebellum (immunocontent and activity) ¯

Cortex (activity) ¯


Cerebellum (immunocontent and activity) ¯

Cortex (immunocontent and activity) ¯



Cerebellum (activity)¯

Cortex (activity)¯



Zemolin et al. 2012

Total GPx


in vivo





Adult Swiss mice-cerebellum

21 days - 40 mg/L water

male ¯


Malagutti et al. 2009

1-, 11-, 21-day old mice (brain)

gestational exposure (1,3 or 10 mg/L water)

≈29, 84 or280 µg MeHg/day/dam


Stringari et al. 2008

Adult rat brain

5 or 10 mg/kg MeHg – 7 days

Cheng et al. 2005

Thioredoxin Reductase (TrxR)


in vivo

-SeH  and –SH



Adult rat brain


21 days - 5 mg/kg


Dalla Corte et al. 2012

in vitro

-SeH  and –SH

Adult mice brain

50-1.000 nM-IC50≈100 nM

Wagner et al. 2010







Type 2 5′-deiodinase (DIO2)


in vitro


NB41A3 neuroblastoma cells

10-100 nM -IC50≈30 nM

Mori et al. 2006

in vitro


Pituitary tumors GH3 cells

0.3-3 µM -IC50≈0.3-1.0  µM

Mori et al. 2007

Glutamine synthetase

in vitro





in vivo


Hypocampus  6-wk-old male ICR mice

Adult male Sprague/Dawley rats

Frontal cortex (0.1-100 µM -IC50≈50 µM)

Hippocampus  (0.1-100 µM -IC50≈50 µM)

Cerebellum  (0.1-100 µM -IC50≈20 µM)


6-wk-old ICR mice (2,4 and 10 mg/kg, i.p., once)

Hippocampus -  inhibition (12,17 and 21%)




Kwon and Park 2003[FT1] 




in vitro




Adult rat brain microsomes


0.5-10 µM-IC50≈4 µM (Ca2+-uptake and ATP hydrolysis)


Freitas et al. 1996


Table 4 – Some mitochondrial thiol- or selenol-containing proteins that are inhibited by MeHg

Mitochondrial creatine kinase (mtCK)

in vivo


Adult Swiss male mice

21 days - 40 mg/L water

Glasser et al. 2010a; 2014

Complex I      

in vivo


Adult Swiss male mice, cerebral cortex

21 days - 40 mg/L water

Glasser et al. 2010a; 2013

Complex II

in vivo


Adult Swiss male mice, cerebral cortex


Adult male rats

21 days - 40 mg/L water


5 days, 10 mg/kg, p.o., cerebellum

Glasser et al. 2010a; 2013

Mori et al. 2011

Succinate dehydratase

in vivo


Adult Swiss male mice

Brain and spinal cord, 7 days, 1 mg/kg, s.c.

Bapu et al. 2003

Complex III

in vivo


Adult Swiss male mice, cerebral cortex  

21 days - 40 mg/L water

Glasser et al. 2010a; 2013

Complex IV

in vivo


Adult Swiss male mice, cerebral cortex  

21 days - 40 mg/L water

Glasser et al. 2010a; 2013

Mitochondrial total GPx

in vivo


Adult rats

5 days – 10 mg/kg, p.o., cerebellum and cerebrum

Mori et al. 2007

Mitochondrial total GPx

in vivo


Adult Swiss male mice brain

21 days - 40 mg/L water

Franco et al. 2009



















Table 5– Mitochondrial processes that are disrupted by MeHg exposure and can be associated with over-production of reactive oxygen species (ROS) and oxidative stress (OS).





Functional group





MTT reduction


in vitro


Stratiatal synaptosomes male rats


7 day-old (0.5-10 µM -IC50≈5 µM)

14 day-old (0.5-10 µM -IC50≈5 µM)

21 day-old (0.5-10 µM -IC50≈5 µM)

2-3 month-old (0.5-10 µM -IC50≈8 µM)


Dreiem et al. 2005



2-3 month old (1-10 µM -IC50≈7.5 µM)


Dreiem & Seegal, 2007


C6 glioma cells



IC50 between 1-10 µM (3-24 h exposure)


Belletti et al. 2002


in vivo

Adult male rat (brain)


21 days, 5 mg/kg; i.p.


Dalla Corte et al. 2013


DYm (mitochondrial membrane potential)

in vitro


Stratiatal synaptosomes male rats

7 day-old (0.5-2.5 µM -IC50≈0.3 µM)

14 day-old (0.5-2.5 µM -IC50≈0.4 µM)

21 day-old (0.5-2.5 µM -IC50≈0.6 µM)

2-3 month-old (0.5-2.5 µM -IC50≈0.6 µM)



Dreiem et al. 2005


Cerebellar granule cells  (Marty and Atchison, 1997).


7-day-old Sprague–Dawley rats (0.5 µM – total collapse of DYm in 25 min


Limke and Atchison, 2002




1,5 and 10  µM – 15-40% collapse of DYm (1-6h)


Yin et al. 2007


P19 murine embryonal carcinoma (EC) cells


1.5  µM –50% DYm collapse after 50 min


Polunas et al. 2011

Day 5 P19-derived neurons

1.5  µM –50% DYm collapse after 20 min

Ultrastructural changes consistent with an inhibition of

mitochondrial respiration

in vivo



in vivo



-SH and –SeH



-SH and     –SeH


Sprague-Dawley rats cerebral cortex

1.5 mg/kg day 2 to 50 (each 48h)

O’Kusky (1983)

Number of Mitochondria

and ultrastructure

Adult Swiss male mice

21 days-40 mg/L water         

Glasser et al. 2014

Oxygen consumption

Adult rats

5 days – 10 mg/kg, p.o., cerebellum

Mori et al. 2007


In short, the stable or transitory interaction (binding) of MeHg with critical thiol and selenol groups in target enzymes can disrupt the biological function of different types of enzymes (Table 3). In addition to enzymes, MeHg can disrupt the physiological activity of transporters and receptors.  As indicated in Table 3, mitochondrial and non-mitochondrial  oxidoreductases containing thiol and selenol redox centers have been reported to be disrupted by MeHg.  The dysregulation of cerebral glutathione (GSH and GSSG) and thioredoxin [Trx(SH)2]  systems by MeHg (Farina et al. 2011; Branco et al. 2017) can impair the fine cellular redox balance via disruption of sensitive cysteinyl- or thiol-containing proteins (Go and Jones, 2013; Go et al. 2015; Jones 2015).


Acrylamide and Acrolein

         Acrylamide and acrolein are a,β-unsaturated (conjugated) reactive molecule, which can react with thiol (-SH) and amino (-NH2) groups in proteins proteins (LoPachin, 2004; LoPachin et al. 2007; 2009; 2011;  Friedman, 2003; Bent et al. 2016; Martyniuk et al.2011; LoPachin and Gavin, 2014 ). However, the rate constant for the reaction between acrylamide with thiol/thiolate groups are much lower than that for MeHg (Table x).  The rate of reaction of these compounds with HMM-SH and LMM-SH is slow but can occur under physiological conditions (Tong et al. 2004; LoPachin, 2004). The inhibition of brain enzyme by acrylamide have been studied and the inhibition caused by acrylamide in some HMM-SH can be reversible  (Howland et al. 1980 ). Despite of this, we can infer that some targets of MeHg and acrylamide and acrolein can overlap.  Accordingly, some targets reported in Table 3 for MeHg have also been shown to be inhibited after exposure to acrylamide (Yousef and Demerdash, 2006; Lapadula et al. 1989; Kopańska et al. 2015). Of particular toxicological significance, both MeHg, acrylamide and acrolein have been reported to change the normal dynamic of synaptic function via interaction with specific HMM-SH (LoPachin et al. 2004 ; Farina et al. 2017).  Acrylamide can also be metabolized to an epoxide intermediate (glycidamide), which can also form adducts with cysteinyl residues in HMM-SH target proteins (Bergmark et al. 1991).

Table 6-Second order rate constants for the reaction of MeHg Acrylamide and acrolein with thiol/thiolate groups of biomolecules


Thiol/thiolate source

Rate constant




≈6.0 x 108 M-1.sec-1



Human serum albumin

≈5.4 x 10-3 M-1.sec-1




≈0.15-2.1 x 10-2 M-1.sec-1




≈0.2-3.2 x 10-3 M-1.sec-1



GADPH (Cys152)

≈5.3 x 10-2M-1.sec-1



GADPH (Cys152)

≈3.0 x 102 M-1.sec-1




≈2.15 M-1.sec-1







GADPH-glyceraldehyde 3-phosphate dehydrogenase



Arnold, A.P., Tan, K.-S., Rabenstein, D.L. Nuclear Magnetic Resonance Studies of the Solution Chemistry of Metal Complexes. 23. Complexation of Methylmercury by Selenohydryl-Containing Amino Acids and Related Molecules (1986) Inorganic Chemistry, 25 (14), pp. 2433-2437.

Bapu, C., Sood, P.P., Nivsarkar, M. Organelle specific enzyme markers as indicators of methylmercury neurotoxicity and antidotal efficacy in mice (2003) BioMetals, 16 (2), pp. 279-284.

Barbosa, N.V., Nogueira, C.W., Nogara, P.A., De Bem, A.F., Aschner, M., Rocha, J.B.T. Organoselenium compounds as mimics of selenoproteins and thiol modifier agents (2017) Metallomics, 9 (12), pp. 1703-1734.

Belletti S, Orlandini G, Vettori MV, Mutti A, Uggeri J, Scandroglio R, Alinovi R, Gatti R. Time course assessment of methylmercury effects on C6 glioma cells: submicromolar concentrations induce oxidative DNA damage and apoptosis. Journal of neuroscience research. 2002 Dec 1;70(5):703-11.

Bent GA, Fairman RA, Grierson L. Towards a Deeper Understanding of the Mechanisms of Interaction between Acrylamide and Key Body-Fluid Thiols. J Clin Toxicol. 2016;6(304):2161-0495.

Bergmark E, Calleman CJ, Costa LG. Formation of hemoglobin adducts of acrylamide and its epoxide metabolite glycidamide in the rat. Toxicology and applied pharmacology. 1991; 111(2):352-63.

Cambier S, Benard G, Mesmer-Dudons N, Gonzalez P, Rossignol R, Brethes D, Bourdineaud JP. At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio). The international journal of biochemistry & cell biology. 2009 Apr 30;41(4):791-9.

Carvalho, C.M.L., Lu, J., Zhang, X., Arnér, E.S.J., Holmgren, A. Effects of selenite and chelating agents on mammalian thioredoxin reductase inhibited by mercury: Implications for treatment of mercury poisoning (2011) FASEB Journal, 25 (1), pp. 370-381.

Cassina A, Radi R. Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport. Archives of biochemistry and biophysics. 1996 Apr 15;328(2):309-16.

Ceccatelli, S., Daré, E., Moors, M.Methylmercury-induced neurotoxicity and apoptosis (2010) Chemico-Biological Interactions, 188 (2), pp. 301-308.

Cheng JP, Yang YC, Hu WX, Yang L, Wang WH, Jia JP, Lin XY(2005) Effect of methylmercury on some neurotransmitters and oxidative damage of rats. J Environ  Sci (China) 17:469-473.

Comini MA. Measurement and meaning of cellular thiol: disufhide redox status. Free radical research. 2016 Feb 1;50(2):246-71.

Eriksson, S., Svenson, A. Catalytic effects by thioltransferase on the transfer of methylmercury and p-mercuribenzoate from macromolecules to low molecular weight thiol compounds (1978) Toxicology, 10 (C), pp. 115-122.

Dreiem A, Gertz CC, Seegal RF. The effects of methylmercury on mitochondrial function and reactive oxygen species formation in rat striatal synaptosomes are age-dependent. Toxicol Sci. 2005 Sep;87(1):156-62.

Dreiem A, Seegal RF. Methylmercury-induced changes in mitochondrial function in striatal synaptosomes are calcium-dependent and ROS-independent. Neurotoxicology. 2007 Jul;28(4):720-6.

Fischer JC, Ruitenbeek W, Berden JA, Trijbels JF, Veerkamp JH, Stadhouders AM, Sengers RC, Janssen AJ. Differential investigation of the capacity of succinate oxidation in human skeletal muscle. Clinica chimica acta. 1985 Nov 29;153(1):23-36.

Flohé, L., Günzler, W.A. (1984)   Assays of Glutathione Peroxidase (1984) Methods in Enzymology, 105 (C), pp. 114-120.

Fonnum, F., Lock, E.A. The contributions of excitotoxicity, glutathione depletion and DNA repair in chemically induced injury to neurones: Exemplified with toxic effects on cerebellar granule cells (2004) Journal of Neurochemistry, 88 (3), pp. 513-531.

Fonfría, E., Vilaró, M.T., Babot, Z., Rodríguez-Farré, E., Suñol, C. Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cells (2005) Journal of Neuroscience Research, 79 (4), pp. 545-553.

Franco, J.L., Posser, T., Dunkley, P.R., Dickson, P.W., Mattos, J.J., Martins, R., Bainy, A.C.D., Marques, M.R., Dafre, A.L., Farina, M. Methylmercury neurotoxicity is associated with inhibition of the antioxidant enzyme glutathione peroxidase (2009) Free Radical Biology and Medicine, 47 (4), pp. 449-457.

Freitas, A. J., Rocha, J. B. T., Wolosker, H., & Souza, D. O. G. (1996). Effects of Hg2+ and CH3Hg+ on Ca2+ fluxes in rat brain microsomes. Brain research, 738(2), 257-264.

Friedman M. Chemistry, biochemistry, and safety of acrylamide. A review. Journal of agricultural and food chemistry. 2003 Jul 30;51(16):4504-26.

George, G.N., Singh, S.P., Prince, R.C., Pickering, I.J. Chemical forms of mercury and selenium in fish following digestion with simulated gastric fluid (2008) Chemical Research in Toxicology, 21 (11), pp. 2106-2110.

Glaser, V., Nazari, E.M., Müller, Y.M.R., Feksa, L., Wannmacher, C.M.D., Rocha, J.B.T., Bem, A.F.D., Farina, M., Latini, A. Effects of inorganic selenium administration in methylmercury-induced neurotoxicity in mouse cerebral cortex (2010a) International Journal of Developmental Neuroscience, 28 (7), pp. 631-637.

Glaser, V., Leipnitz, G., Straliotto, M.R., Oliveira, J., dos Santos, V.V., Wannmacher, C.M.D., de Bem, A.F., Rocha, J.B.T., Farina, M., Latini, A. Oxidative stress-mediated inhibition of brain creatine kinase activity by methylmercury  (2010b) NeuroToxicology, 31 (5), pp. 454-460.

Holmgren and Bjornstedt (1995) Thioredoxin and thioredoxin reductase. Methods Enzymol. 252: 199-208.

Hoppe, B., Bräuer, A.U., Kühbacher, M., Savaskan, N.E., Behne, D., Kyriakopoulos, A. Biochemical analysis of selenoprotein expression in brain cell lines and in distinct brain regions (2008) Cell and Tissue Research, 332 (3), pp. 403-414.

Howland RD, Vyas IL, Lowndes HE, Argentieri TM. The etiology of toxic peripheral neuropathies: in vitro effects of acrylamide and 2, 5-hexanedione on brain enolase and other glycolytic enzymes. Brain Research. 1980 Nov 24;202(1):131-42.

Hughes, B.P. (1962) A method for the estimation of serum creatine kinase and its use in comparing creatine kinase and aldolase activity in normal and pathological sera. Clinica Chimica Acta, 7, 597-603.

Isab, A.A. Estimated formation constants for the complexation of methylmercury(II) by captopril {1-[(2S)-3-mercapto-2-methyl-1-oxopropyl]-L-proline}: Evidence of stronger binding to the cis isomer of the drug (1991) Journal of the Chemical Society, Dalton Transactions, (3), pp. 449-452.

Johansson, C., Castoldi, A.F., Onishchenko, N., Manzo, L., Vahter, M., Ceccatelli, S. Neurobehavioural and molecular changes induced by methylmercury exposure during development (2007) Neurotoxicity Research, 11 (3-4), pp. 241-260.

Kopańska M, Lukáč N, Kapusta E, Formicki G. Acrylamide influence on activity of acetylcholinesterase, thiol groups, and malondialdehyde content in the brain of Swiss mice. Journal of Biochemical and Molecular Toxicology. 2015 Oct 1;29(10):472-8.

Kwon, O.-S., Park, Y.-J. In vitro and in vivo dose-dependent inhibition of methylmercury on glutamine synthetase in the brain of different species (2003) Environmental Toxicology and Pharmacology, 14 (1-2), pp. 17-24.

Lapadula DM, Bowe M, Carrington CD, Dulak L, Friedman M, Abou-Donia MB. In vitro binding of [14C] acrylamide to neurofilament and microtubule proteins of rats. Brain research. 1989 Feb 27;481(1):157-61.

Li, Y., Shi, W., Li, Y., Zhou, Y., Hu, X., Song, C., Ma, H., Wang, C., Li, Y. Neuroprotective effects of chlorogenic acid against apoptosis of PC12 cells induced by methylmercury (2008) Environmental Toxicology and Pharmacology, 26 (1), pp. 13-21.

Limke TL, Atchison WD. Acute exposure to methylmercury opens the mitochondrial permeability transition pore in rat cerebellar granule cells. Toxicology and applied pharmacology. 2002 Jan 1;178(1):52-61.

Liu, W., Xu, Z., Deng, Y., Xu, B., Wei, Y., Yang, T.  Protective effects of memantine against methylmercury-induced glutamate dyshomeostasis and oxidative stress in rat cerebral cortex (2013) Neurotoxicity Research, 24 (3), pp. 320-337.

LoPachin RM. The changing view of acrylamide neurotoxicity. Neurotoxicology. 2004 Jun 1;25(4):617-30.

LoPachin RM, Gavin T. Molecular mechanisms of aldehyde toxicity: a chemical perspective. Chemical research in toxicology. 2014 Jun 17;27(7):1081-91.

LoPachin RM, Schwarcz AI, Gaughan CL, Mansukhani S, Das S. In vivo and in vitro effects of acrylamide on synaptosomal neurotransmitter uptake and release. Neurotoxicology. 2004 Mar 1;25(3):349-63.

LoPachin RM, Gavin T, Geohagen BC, Das S. Neurotoxic mechanisms of electrophilic type-2 alkenes: soft–soft interactions described by quantum mechanical parameters. Toxicological sciences. 2007 May 22;98(2):561-70.

LoPachin RM, Gavin T, Petersen DR, Barber DS. Molecular mechanisms of 4-hydroxy-2-nonenal and acrolein toxicity: nucleophilic targets and adduct formation. Chemical research in toxicology. 2009 Jul 17;22(9):1499-508.

LoPachin RM, Gavin T, DeCaprio A, Barber DS. Application of the hard and soft, acids and bases (HSAB) theory to toxicant–target interactions. Chemical research in toxicology. 2011 Nov 16;25(2):239-51.

Malagutti, K.S., da Silva, A.P., Braga, H.C., Mitozo, P.A., Soares dos Santos, A.R., Dafre, A.L., de Bem, A.F., Farina, M. 17β-estradiol decreases methylmercury-induced neurotoxicity in male mice (2009) Environmental Toxicology and Pharmacology, 27 (2), pp. 293-297.

Martyniuk CJ, Fang B, Koomen JM, Gavin T, Zhang L, Barber DS, LoPachin RM. Molecular mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by α, β-unsaturated carbonyl derivatives. Chemical research in toxicology. 2011 Nov 29;24(12):2302-11.

Montgomery, K.S., Mackey, J., Thuett, K., Ginestra, S., Bizon, J.L., Abbott, L.C. Chronic, low-dose prenatal exposure to methylmercury impairs motor and mnemonic function in adult C57/B6 mice (2008) Behavioural Brain Research, 191 (1), pp. 55-61.

Mori KO, Stone SC, Braverman LE, Devito WJ. Involvement of tyrosine phosphorylation in the regulation of 5'-deiodinases in FRTL-5 rat thyroid cells and rat astrocytes. Endocrinology. 1996 Apr 1;137(4):1313-8.

Mori, K., Yoshida, K., Nakagawa, Y., Hoshikawa, S., Ozaki, H., Ito, S., Watanabe, C. Methylmercury inhibition of type II 5′-deiodinase activity resulting in a decrease in growth hormone production in GH3 cells (2007) Toxicology, 237 (1-3), pp. 203-209.

Mori, K., Yoshida, K., Tani, J.-I., Hoshikawa, S., Ito, S., Watanabe, C. Methylmercury inhibits type II 5′-deiodinase activity in NB41A3 neuroblastoma cells (2006) Toxicology Letters, 161 (2), pp. 96-101.

Mori N, Yasutake A, Hirayama K. Comparative study of activities in reactive oxygen species production/defense system in mitochondria of rat brain and liver, and their susceptibility to methylmercury toxicity. Archives of toxicology. 2007 Nov 1;81(11):769-76.

Mori, N., Yasutake, A., Marumoto, M. and Hirayama, K., 2011. Methylmercury inhibits electron transport chain activity and induces cytochrome c release in cerebellum mitochondria. The Journal of toxicological sciences, 36(3), pp.253-259.

Ni, M., Li, X., Yin, Z., Sidoryk-Weogonekgrzynowicz, M., Jiang, H., Farina, M., Rocha, J.B.T., Syversen, T., Aschner, M. Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity (2011) GLIA, 59 (5), pp. 810-820.

Ni, M., Li, X., Yin, Z., Jiang, H., Sidoryk-Wegrzynowicz, M., Milatovic, D., Cai, J., Aschner, M. Methylmercury induces acute oxidative stress, altering Nrf2 protein level in primary microglial cells (2010) Toxicological Sciences, 116 (2), pp. 590-603.

O'Kusky, J., 1983. Methylmercury poisoning of the developing nervous system: morphological changes in neuronal mitochondria. Acta neuropathologica, 61(2), pp.116-122.

Patel AJ, Hunt A, Gordon RD, Balazs R: The activities in different neural cell types of certain enzymes associated with the metabolic compartmentation glutamate. Brain research 1982, 256:3-11.

Pishak MR, Phillips AT: A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts. Analytical biochemistry 1979, 94:82-88.

Polunas, M., Halladay, A., Tjalkens, R.B., Philbert, M.A., Lowndes, H. and Reuhl, K., 2011. Role of oxidative stress and the mitochondrial permeability transition in methylmercury cytotoxicity. Neurotoxicology, 32(5), pp.526-534.

Rabenstein, D.L. The Aqueous Solution Chemistry of Methylmercury and Its Complexes (1978b) Accounts of Chemical Research, 11 (3), pp. 100-107.

Rabenstein, D.L., Arnold, A.P., Guy, R.D. 1H-NMR study of the removal of methylmercury from intact erythrocytes by sulfhydryl compounds (1986) Journal of Inorganic Biochemistry, 28 (2-3), pp. 279-287.

Rabenstein, D.L., Bravo, J. Nuclear Magnetic Resonance Studies of the Solution Chemistry of Metal Complexes: 24: Arylmercury(II) Complexes of Sulfhydryl-Containing Ligands (1987) Inorganic Chemistry, 26 (17), pp. 2784-2787.

Rabenstein, D.L., Evans, C.A. The mobility of methylmercury in biological systems (1978) Bioinorganic Chemistry, 8 (2), pp. 107-114.

Rabenstein, D.L., Fairhurst, M.T. Nuclear Magnetic Resonance Studies of the Solution Chemistry of Metal Complexes. XI. Binding of Methylmercury by Sulfhydryl-Containing Amino Acids and by Glutathione (1975) Journal of the American Chemical Society, 97 (8), pp. 2086-2092.

Rabenstein, D.L., Reid, R.S. Nuclear Magnetic Resonance Studies of the Solution Chemistry of Metal Complexes. 20. Ligand-Exchange Kinetics of Methylmercury(II)-Thiol Complexes (1984) Inorganic Chemistry, 23 (9), pp. 1246-1250.

Rabenstein, D.L., Reid, R.S., Isab, A.A. 1H nmr study of the effectiveness of various thiols for removal of methylmercury from hemolyzed erythrocytes (1983) Journal of Inorganic Biochemistry, 18 (3), pp. 241-251.

Reid, R.S., Rabenstein, D.L. Nuclear Magnetic Resonance Studies of the Solution Chemistry of Metal Complexes. 19. Formation Constants for the Complexation of Methylmercury by Glutathione, Ergothioneine, and Hemoglobin (1982) Journal of the American Chemical Society, 104 (24), pp. 6733-6737.

Roos, D., Seeger, R., Puntel, R., Vargas Barbosa, N. Role of calcium and mitochondria in MeHg-mediated cytotoxicity (2012) Journal of Biomedicine and Biotechnology, 2012, art. no. 248764,

Rowe, W..B., Ronzio, R.A., Wellner, V.P., Meister, A. (1970) Glutamine synthetase (sheep brain).Meth ods in Enzymology,17, Part A, 900-910.

Rustin, P., Chretien, D., Bourgeron, T., Gerard, B., Rötig, A., Saudubray, J. M., & Munnich, A. (1994). Biochemical and molecular investigations in respiratory chain deficiencies. Clinica chimica acta, 228(1), 35-51.

Shenker BJ, Guo TL, Shapiro IM. Low-level methylmercury exposure causes human T-cells to undergo apoptosis: evidence of mitochondrial dysfunction. Environmental research. 1998 May 31;77(2):149-59.   

Shenker BJ, Guo TL, Insug O, Shapiro IM. Induction of apoptosis in human T-cells by methyl mercury: temporal relationship between mitochondrial dysfunction and loss of reductive reserve. Toxicology and applied pharmacology. 1999 May 15;157(1):23-35.

Sorg, O., Schilter, B., Honegger, P., Monnet-Tschudi, F.Increased vulnerability of neurones and glial cells to low concentrations of methylmercury in a prooxidant situation (1998) Acta Neuropathologica, 96 (6), pp. 621-627.

Stringari, J., Nunes, A.K.C., Franco, J.L., Bohrer, D., Garcia, S.C., Dafre, A.L., Milatovic, D., Souza, D.O., Rocha, J.B.T., Aschner, M., Farina, M. Prenatal methylmercury exposure hampers glutathione antioxidant system ontogenesis and causes long-lasting oxidative stress in the mouse brain (2008) Toxicology and Applied Pharmacology, 227 (1), pp. 147-154.

Tong GC, Cornwell WK, Means GE. Reactions of acrylamide with glutathione and serum albumin. Toxicology letters. 2004 Mar 1;147(2):127-31.

Wagner, C., Sudati, J.H., Nogueira, C.W., Rocha, J.B.T. In vivo and in vitro inhibition of mice thioredoxin reductase by methylmercury (2010) BioMetals, 23 (6), pp. 1171-1177.

Wormser, U., Brodsky, B., Milatovic, D., Finkelstein, Y., Farina, M., Rocha, J.B., Aschner, M. Protective effect of a novel peptide against methylmercury-induced toxicity in rat primary astrocytes (2012) NeuroToxicology, 33 (4), pp. 763-768.

Yin, Z., Milatovic, D., Aschner, J.L., Syversen, T., Rocha, J.B.T., Souza, D.O., Sidoryk, M., Albrecht, J., Aschner, M.   Methylmercury induces oxidative injury, alterations in permeability and glutamine transport in cultured astrocytes (2007) Brain Research, 1131 (1), pp. 1-10.

Yousef MI, El-Demerdash FM. Acrylamide-induced oxidative stress and biochemical perturbations in rats. Toxicology. 2006 Feb 15;219(1-3):133-41.

Zemolin, A.P.P., Meinerz, D.F., de Paula, M.T., Mariano, D.O.C., Rocha, J.B.T., Pereira, A.B., Posser, T., Franco, J.L.  Evidences for a role of glutathione peroxidase 4 (GPx4) in Methylmercury induced neurotoxicity in vivo (2012) Toxicology, 302 (1), pp. 60-67.

Zhu, S.-Y., Li, X.-N., Sun, X.-C., Lin, J., Li, W., Zhang, C., Li, J.-L. Biochemical characterization of the selenoproteome in Gallus gallus via bioinformatics analysis: structure-function relationships and interactions of binding molecules (2017) Metallomics, 9 (2), pp. 124-131.