API

Event: 1487

Key Event Title

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Binding, Thiol/seleno-proteins involved in protection against oxidative stress

Short name

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Binding, SH/SeH proteins involved in protection against oxidative stress

Biological Context

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Level of Biological Organization
Molecular

Cell term

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Organ term

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Organ term
brain


Key Event Components

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Process Object Action

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
Oxidative stress and Developmental impairment in learning and memory MolecularInitiatingEvent

Stressors

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Taxonomic Applicability

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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

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Life stage Evidence
During brain development High

Sex Applicability

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Term Evidence
Female High
Male High

Key Event Description

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Thiol (SH)- and seleno-containing proteins involved in protection against oxidative stress are mainly located in mitochondria and in the cytoplasm of the different neural cell types (Comini, 2016; Hoppe et al. 2008; Barbosa et al. 2017; Zhu et al. 2017). The main SH-containing peptide involved in protection against oxidative stress is Glutathione (GSH), a tripeptide acting as a cofactor for the enzyme peroxidase and thus serving as an indirect antioxidant donating the electrons necessary for its decomposition of H2O2. The seleno-containing proteins of interest are: (i) the Glutathione Peroxidase (GPx) family, involved in detoxification of hydroperoxides; (ii) the Thioredoxin Reductase (TrxR) family, involved in the regeneration of reduced thioredoxin (Pillai et al., 2014; ), and the less studied SelH, K, S, R, W, and P selenoproteins (Pisoschi and Pop, 2015, Reeves and Hoffmann, 2009).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). Of particular importance, the GSH/GPx and thioredoxin (Trx)/TrxR systems are the two main redox regulators of mammalian cells and the disruption of their activities can compromise cell viability (Ren et al. 2016).


How It Is Measured or Detected

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 The interference of MeHg, Hg2+ and acrylamide with the normal catalytic function of thiol- or selenol-containing enzymes (i.e., GPx and TrxR) can be determined by different analytical methodologies. The activity of enzymes are typically determined by spectrophotometric, methodologies that quantify the rate of product appearance or the disappearance of  substrate. Commercial kits are also available to quantify GPx and TrxR.  The quantification of GSH and reduced Trx1 or Trx2 can be made by commercial kits.

Glutathione Peroxidase

Glutathione peroxidase 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 ul 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 ul 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 where the sample is prepared.

Reduction of GPx activity. The activity of GPx can be measured by a colorimetric assay, using a commercially available kit (e.g., Abcam ab102530)-

 

Thioredoxin Reductase

TrxR Activity. TrxR activity is normally measured by the method of Holmgren and Bjornstedt. The reaction mixture consisted of the following (in mmol/l): 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 is added (to final concentration of 6–10  ug/ml of protein) to the cuvette containing the reaction mixture, and the absorbance is followed at 412 nm for 4 min.

 

-   Reduction of TrxR activity. The activity of TrxR can be measured by a colorimetric assay, using a commercially available kit (e.g., Abcam ab83463)

 

Glutathione (GSH)

Glutathione (GSH) depletion. GSH can be measured by assaying the ratio of reduced to oxidized glutathione (GSH:GSSG) using a commercially available kit (e.g., http://www.abcam.com/gshgssg-ratio-detection-assay-kit-fluorometric-green-ab138881.html)

 

Thioredoxin (Trx)

Reduction of Trx levels. The levels of Trx can be measured by a colorimetric assay, using a commercially available kit (https://www.caymanchem.com/pdfs/11527.pdf; Ruszkiewicz et al. 2016 ).


Domain of Applicability

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Due to the ubiquitous distribution of the SH-/ and seleno-proteins involved in protection against oxidative stress and inview of the strong affinity of MeHg and Hg2+ for thiolate and selenolate groupsthe binding of MeHg and Hg2+ to thiol and selenol groups is expected to occur in the living cells of all taxonomic groups found in the biosphere.The conservation of these effects across different vertebrate species indicates that thiol- and selenol-containing proteins (particularly, TrxR and GPx) can also be important targets of electrophilic forms of Hg(EpHg+ or MeHg and Hg2+) toxicity in fish and birds (Heinz, 1979; Carvalho et al. 2008b; Heinz et al. 2009). The disruption of the Trx and GSH systems by MeHg and Hg2+have been demonstrated in zebra-seabreams  (Branco et al. 2011; 2012a,b) and salmon (Salmo salar, Bernstssen et al. 2003).  MeHg can also interfere with the Trx and GSH systems in zebrafish (Yang et al. 2007; Cambier et al. 2012).


Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

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Mercury (Methylmercury, mercury chloride)

 The binding of Methylmercury (MeHg) to redox sensitive thiol- or selenol-groups can disrupt the activity of enzymes or the biochemical role of non-enzymatic brain proteins. 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, particularly of the antioxidant selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase isoforms. The dysregulation of cerebral glutathione (GSH and GSSG) and thioredoxin [Trx or 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 etal., 2013; Go et al. 2014; Jones 2015).

 

 

Figure 1 – 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 such a 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).

 

The affinity of Mercury chloride (Hg2+) for thiol and selenol groups is higher than that of MeHg (compare Table 2 with Table 1). The constants described in Table 1 and 2 indicate that MeHg and Hg2+ behave as  strong soft electrophiles, i.e., theyhave much higher affinity for the soft nucleophiles centers of thiol- and selenol-containing molecules (Rabenstein 1978a; Arnold et al. 1986; Sugiura et al., 1976).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,  indicating that the reactions of electrophilic forms of Hg (EpHg; here MeHg and Hg2+) with thiolate and selenolate groups are diffusion controlled reactions (Rabenstein  and Fairhurst, 1975). The constant indicates that the binding of EpHg+ to thiolate (-S-) or selenolate (-Se-) groups will occurr almost instaneously, when an EpHg+ collides with –S- or -Se- groups.

 The studies of Rabenstein and others have also pointed out that the affinity of MeHg for –SeH groups is higher than for  –SH groups (Sugira et al. 1976; Arnold et al. 1986). Consequently, –SeH-containing molecules (i.e., selenoproteins) should be the preferential targets for MeHg (Farina et al. 2011). Accordingly, several studies have demonstrate that the selenoenzymes glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) were inhibited after in vitro and in vivo exposure to MeHg  or Hg2+ (Carvalho et al., 2008a; 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).

As corollary, the occurrence of free MeHg and Hg2+ 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 mass 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). The coordination of MeHg with one –S- group of a LMM-SH will determine MeHg distribution to its targets organs, including the brain. The coordination of Hg2+ with two –S- of LMM-SH molecules (particularly, cysteine or Cys) will determine the distribution of Hg2+ to kidney (which is its main target) and to non-classical targets organs, such as the brain (Oliveira et al. 2017). The entrance of Hg2+ into the brain is proportionally small, but recent literature data have indicated the neurotoxicity of very low and environmentally relevant doses of Hg2+ in rodents (Mello-Carpes et al. 2013 ), which confirms data obtained with toxic doses in rodents (Peixoto et al. 2007 ;  Franciscato et al. 2009 ; Chehimi et al. 2012).

Table 1 - Affinity constants of methylmercury for important chemical groups found in biomolecules (adapted from aRabestein, 1978a, bRabestein and Bravo using different thiol-containing molecules with the arylmercurialpara-mercurybenzenosulfonate,  and from cArnold 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 Occurrence Formation constant
Thiol/thiolate (-SH/-S-) Cysteine, glutathione, proteins ≈14-18 a,b

Selenol/selenolate

(-SeH/Se-)
Selenocysteinyl residues in selenoproteins ≈ 16-18c

 

Table 2. Formation constants of Hg2+ with some representative nucleophilic centers from biomolecules.

Functional group Hg2+
R-S-R ≈ 6-12
R-SH ≈ 40-50
R-SeH ≈ 50-60

The approximate (≈) Log of the constants. The values were adapted  from Stricks and Kolthoff 1953; Mousavi 2011 and Liem-Nguyem et al. 2017.

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) to  a free thiol/thiolate- or selenol/selenolate-group from non-target or target proteins. Thus, the interaction of MeHg with its target proteins in the brain usually involves the exchange of MeHg from low-molecular mass conjugates (LMM-S-conjugates) to a thiol or selenol group in different types of proteins (Rabenstein 1978b; Rabenstein and Fairhurst, 1975; Reid and Rabenstein et al.; 1982; Rabenstein and Reid, 1984; Arnold et al. 1986; Farina et al. 2011, 2017; Dórea et al. 2013).

Figure 2 – 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.

 

Table 3: References for the inhibition by MeHg and Hg2+ of SH-/seleno-proteins involved in protection against oxidative stress

Protein activity inhibited by MeHg

 

exposure

Functional group likely involved in the inhibition

 

organism-preparation

 

glutathione peroxidase (total GPx)

in vivo

-SeH

Adult mice

Glasser et al. 2013

Total GPx

in vivo

-SeH

Adult mice

Glasser et al. 2010a

Mitochondrial total GPx

in vivo

-SeH

Adult mice

Franco et al. 2009

Total GPx

in vitro

-SeH

SH-SY5Y cells

Franco et al. 2009

GPx1 and GPx4

in vivo

-SeH

Adult mice

Zemolin et al. 2012

Total GPx

in vivo

-SeH

Adult male mice

Malagutti et al. 2009

Total GPx

in vitro

-SeH

PC12 cells

Li et al. 2008

Total GPx

in vivo

-SeH

Mice gestational exposure

Stringari et al. 2008

Total GPx

in vivo

-SeH

Adult rats

Cheng et al. 2005

Total GPx

in vitro

-SeH

Fetal Telencepalic cells from rats

Sorg et al. 1998

Total GPx

in vitro

-SeH

Mice neuroblastoma cells

Kromidas et al. 1990

Thioredoxin Reductase (TrxR)

in vivo

-SeH  and –SH

Adult mice

Zemolin et al. 2012

TrxR

in vitro

-SeH  and –SH

Adult mice

Wagner et al. 2010

TrxR

in vivo

-SeH-  and –SH

Adult rats

Dalla Corte et al. 2012

Mitochondrial total Gpx

In vivo

-SeH

Adult rat

Mori et al., 2007

Mitochondrial total Gpx

In vivo

-SeH

Adult Swiss male mice brain

Franco et al., 2009

 

 

 

 

 

Total brain TrxR

In vivo

-SeH and -SH

Juvenile fish (zebra-seabreams)

Branco et al. 2011

Branco et al. 2012

 

 

 

 

 

Protein activity inhibited by Hg2+

 

exposure

Functional group likely involved in the inhibition

 

organism-preparation

 

Total brain TrxR

In vivo

-SeH and -SH

Juvenile fish (zebra-seabreams)

Branco et al. 2012

 

Acrylamide

            Acrylamide is ana,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 is much lower than that for MeHg (Table x).  The rate of reaction of this compound with HMM-SH and LMM-SH is slow but can occur under physiological conditions (Tong et al. 2004; LoPachin, 2004). The inhibition of brain enzymes 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 can overlap, in particular GSH,where the rate constant for MeHg and acrylamide are ≈6.0 x 108 M-1.sec-1 and ≈0.15-2.1 x 10-2 M-1.sec-1, respectively (Yousef and Demerdash, 2006; Lapadula et al. 1989; Kopańska et al. 2015). 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).

 



References

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