review article the role of the keap1/nrf2 pathway in the...

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2013 Article ID 848279 8 pageshttpdxdoiorg1011552013848279

Review ArticleThe Role of the Keap1Nrf2 Pathway in the CellularResponse to Methylmercury

Yoshito Kumagai12 Hironori Kanda2 Yasuhiro Shinkai12 and Takashi Toyama2

1 Environmental Biology Section Faculty of Medicine University of Tsukuba Tsukuba Ibaraki 305-8575 Japan2Doctoral Program in Biomedical Sciences Graduate School of Comprehensive Human Sciences University of Tsukuba TsukubaIbaraki 305-8575 Japan

Correspondence should be addressed to Yoshito Kumagai yk-em-tumdtsukubaacjp

Received 28 January 2013 Revised 26 May 2013 Accepted 3 June 2013

Academic Editor Mi-Kyoung Kwak

Copyright copy 2013 Yoshito Kumagai et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Methylmercury (MeHg) is an environmental electrophile that covalently modifies cellular proteins with reactive thiols resulting inthe formation of protein adducts While such protein modifications referred to as 119878-mercuration are thought to be associated withthe enzyme dysfunction and cellular damage caused byMeHg exposure the current consensus is that (1) there is a cellular responseto MeHg through the activation of NF-E2-related factor 2 (Nrf2) coupled to 119878-mercuration of its negative regulator Kelch-likeECH-associated protein 1 (Keap1) and (2) the Keap1Nrf2 pathway protects against MeHg toxicity In this review we introduceour findings and discuss the observations of other workers concerning the S-mercuration of cellular proteins by MeHg and theimportance of the Keap1Nrf2 pathway in protection against MeHg toxicity in cultured cells and mice

1 Introduction

Methylmercury (MeHg) is strongly accumulated by fish andmarine mammals and is found at the highest concentrationsin large predatory species at the top of the aquatic foodchain MeHg readily penetrates the blood-brain barrier andaffects the central nervous system It is transferred into cellsby passive diffusion andor transport by the L-type largeneutral amino acid transporter (as its L-cysteine complex)and reacts covalently with protein thiols because of itshigh affinity for nucleophilic groups having a dissociationconstant of 10minus15 [1] This covalent modification by MeHg isreferred to as ldquoS-mercurationrdquo Some MeHg however bindsto glutathione (GSH) that is present in a variety of celltypes at relatively high concentrations (gt1mM) to form aMeHg-GSH adduct that is transported into the extracellularspace by multidrug resistance-associated proteins (MRPs)[2ndash4] (Figure 1)While theMeHg-mediated S-mercuration ofcellular proteins is believed to be involved in the toxicologicalsignificance of MeHg [5] oxidative stress from the presenceofMeHg in the body is also a critical factor associated with its

toxicity [6] Interestingly antioxidant proteins such as hemeoxygenase-1 (HO-1) glutamate-cysteine ligase (GCL a rate-limiting enzyme for GSH synthesis) and MRPs are regulatedby transcription factor NF-E2-related factor 2 (Nrf2) [7ndash12]

2 S-Mercuration of Proteins by MeHg

Although there is little doubt that MeHg is able to covalentlymodify cellular proteins and it is thought that a protein-MeHg complex is at least partly involved in MeHg toxicity(Figure 1) there have been only a limited number of studies inwhich the in vivo effects ofMeHg have been investigated withregard to S-mercuration This is probably because of the lackof appropriate methods for identifying S-mercuration Herewe describe our efforts to investigate the effects of MeHgusing chemical biology approaches (Table 1)

We have found that the subcutaneous administrationof MeHg (10mgkg) to mice resulted in a time-dependentdecrease in brain manganese-superoxide dismutase (Mn-SOD) activity whereas MeHg exposure had no effect oncuprozinc-SOD (CuZn-SOD) activity [13] Although levels

2 Oxidative Medicine and Cellular Longevity

Table 1 Molecular targets of MeHg

Target protein In vivo effect References

Mn-SOD Reduction of catalytic activity inmouse brain

Shinyashikiet al [13]

nNOS Induction of NOS in rat brain Shinyashikiet al [15]

Arginase I Reduction of activity in rat liverDecreased Mn levels in tissues

Kanda et al[18]

SDH Not defined Kanda et al[19]

ndashSminus

GSH

Nrf2GCL

MeHg

Protein

Protein

MRP

Protein

Cellular damage

pKa

ndashSH

MeHgndashSG

ndashSndashHgMe

Figure 1 S-mercuration of cellular proteins byMeHg and theMeHgdetoxification pathway

of mRNA and protein synthesis of Mn-SOD were unaffectedby MeHg administration MeHg caused a facile reduction inthe activity and a drastic decrease in the native form of Mn-SOD (but not of CuZn-SOD) with purified enzyme prepa-rations It was subsequently shown that the S-mercurationof Mn-SOD through Cys196 by MeHg results in a decreasein the enzyme activity in vivo [14] Therefore the selectiveinhibition of Mn-SOD activity caused by S-mercurationcould play a critical role in MeHg-induced neurotoxicitybecause SOD protects the cell against oxidative stress byscavenging superoxide

We also found that the subcutaneous administration ofMeHg (10mg kgminus1 dayminus1 for 8 days) to rats caused significantincreases in nitric oxide synthase (NOS) activities in thecerebrum and cerebellum with time [15] The increase inNOS activity seems to be caused by an increase in neuronalNOS (nNOS) protein levels but not an increase in inducibleNOS In contrast to the in vivo observations however theaddition of MeHg to a cerebellar enzyme preparation in vitrocaused a concentration-dependent decrease in nNOS activ-ity suggesting that MeHg modifies NOS through reactivethiol groups thereby affecting its catalytic activity in vitroExperimentswith arylmercury column chromatographyhaveconfirmed that this is possible [15] A reasonable explanationfor our observations is that the increase in nNOS proteinsobserved from MeHg exposure in vivo may be one of theinitial responses to counteract the metal-induced negativeeffects on the central nervous system

In the body MeHg accumulates to high concentrationsin the liver [16 17] but few molecular targets of MeHg havebeen identified in this tissue We hypothesized that MeHgcould interact with arginase I an abundant Mn-bindingprotein in the liver through covalent modification resultingin alterations in not only its catalytic activity but also inhepatic Mn levels through in vivo MeHg exposure Afterthe subcutaneous administration of MeHg (10mg kgminus1 dayminus1for 8 days) to rats mercury levels in the liver were greaterthan those in the cerebrum or cerebellum [18] A markedsuppression of arginase I activity was also detected underthese conditions Using purified rat arginase I we foundthat MeHg-induced S-mercuration of arginase I caused theprotein to aggregate and substantial leakage of Mn ionsfrom the arginase I active site We speculate that the MeHg-mediated suppression of hepatic arginase I activity in vivois at least partly attributable to the S-mercuration of thisprotein

In the course of the study we coincidentally detectedanother protein with a 42 kDa subunit molecule fromthe same hepatic preparation that readily underwent S-mercuration and subsequent aggregation Two-dimensionalsodium dodecyl sulfate-polyacrylamide gel electrophoresiswas used to separate the protein and it was identifiedby peptide mass fingerprinting using matrix-assisted laserdesorption and ionization time-of-flight mass spectrometry(MALDI-TOFMS) This 42 kDa protein was identified assorbitol dehydrogenase (SDH) [19] Using recombinant ratSDH possessing 10 cysteine residues in a subunit [20] wefound that MeHg was covalently bound to SDH throughCys44 Cys119 Cys129 and Cys164 resulting in the inhibitionof its catalytic activity and the release of zinc ions facilitat-ing protein aggregation [19] Subsequent mutation analysisconfirmed that Cys44 which ligates the active site zinc atom[21] and Cys129 play a crucial role in the MeHg-mediatedaggregation of SDH [19]

3 Cellular Response to MeHg viathe Keap1Nrf2 Pathway

As mentioned above MeHg reacts readily with cellularnucleophiles and also causes oxidative stress implying thatit may lead to decreased GSH levels in tissues As a resultMeHg undergoes GSH conjugation and is excreted intothe extracellular space through the action of MRPs [2ndash4]However several studies have indicated that MeHg exposurealso upregulates GCL [22ndash28] This suggests that there isan initial response to MeHg in the cells to compensate fordecreased GSH levels and to repress oxidative cell damageOn the other hand Dr Yamamoto and his associates reportedthat transcription factor Nrf2 cooperatively regulates antiox-idant proteins such as GCL and HO-1 phase-II xenobioticdetoxifying enzymes and phase-III xenobiotic transporterssuch as MRPs [10 29] They subsequently found that Nrf2is negatively regulated by Kelch-like ECH-associated protein1 (Keap1) [30] Interestingly Keap1 has 27 and 25 cysteineresidues in humans and mice respectively [31 32] and somethiols (eg Cys151 Cys273 and Cys288) that have been

Oxidative Medicine and Cellular Longevity 3

Table 2 MeHg modification sites in Keap1

Peak no Position Peptide sequence Calculated mass Observed mass

1 484ndash494 LNSAECYYPER 13455 134512 363ndash380 SGLAGCVVGGLLYAVGGR 16499 164963 151ndash169 CVLHVMNGAVMYQIDSVVR 21356 21351

P-1 484ndash494+MeHg

LNSAECYYPER+MeHg (214)

15605 15596


SGLAGCVVGGLLYAVGGR+MeHg (216)

18638 18648


CVLHVMNGAVMYQIDSVVR+MeHg (214)

23506 23495

MeHg

489257 273 288 297 613368151 22623 319

Keap1

Cysteine residuesmodified

Dexamethasone21-mesylate

Biotinylatediodoacetamide

TBQ

lowastlowastlowast

lowast lowast lowast lowast

lowastlowastlowastlowastlowast


lowastlowastlowast lowast

lowast

lowast

lowast

lowast

NTR BTB IVR DC domain

12-NQ

Figure 2 Covalent modification of Keap1 cysteine residues byelectrophiles The BTB IVR and DC domains are essential forthe formation of homodimers associated with cullin 3 and thebinding of Nrf2 12-NQ 12-naphthoquinone TBQ tert-butyl-14-benzoquinone

established as being highly reactive [31ndash34] so the ldquocysteinecoderdquo which defines the preferential target cysteine(s) anddistinct biological effects was proposed [35ndash37]These obser-vations led us to assume that MeHg could modify Keap1through S-mercuration and so activate the Nrf2-regulatedgene expression of GCL HO-1 and MRPs

Figure 2 shows a summary of the Keap1 modificationsites consisting of NTR (N-terminal region) BTB (Boardcomplex Tramtrack and Bric-a-brac) IVR (interveningregion) and DC (DGRCTR double glycine repeatC-terminal region) domains that can be modified by a varietyof electrophiles [31 33 38 39] Among them Cys151 Cys273and Cys288 are well established as being essential for regu-lating Nrf2 function [31 32 40 41] although other cysteineresidues (eg Cys257 Cys297 and Cys613) in Keap1 canalso be modified by dexamethasone 21-mesylate biotinylatediodoacetamide and 12-napthoquinone [31 33 38] OurMALDI-TOFMS analysis revealed that Keap1 undergoes S-mercuration by MeHg at three cysteine residues Cys151Cys368 and Cys489 (see Figure 3 and Table 2) suggestingthat the S-mercuration of Cys151 in Keap1 potentially causesa structural alteration activating Nrf2 while Cys368 andCys489 are also targets for 12-naphthoquinone and tert-butyl-14-benzoquinone [38 39]

1

1

2

2

3

3

P-1

P-2

P-3

Keap1 control

100

80

60

40

20

00 500 1000 1500 2000 2500 3000 3500

mz

Inte

nsity

()

Keap1 + MeHg

Figure 3 MALDI-TOFMS analysis of Keap1 reacted with MeHgRecombinant Keap1 protein (025 120583g120583L) was incubated for 30minat 37∘C with or without MeHg (10120583M) After the reaction theKeap1 proteins were trypsinized and subjected to MALDI-TOFMSanalysis Compared with the calculated mass of the unmodifiedpeptides the modified peptides P-1 to P-3 showed increased massesof 214 or 216Da corresponding to the addition of a single equivalentof MeHg

As expected MeHg activated Nrf2 in SH-SY5Y cells ina concentration- and time-dependent manner and upreg-ulated the downstream proteins such as the GCL catalyticsubunit (GCLC) and the GCL modifier subunit (GCLM)(Figure 4) [42] The same observation was seen in primarymouse hepatocytes and HepG2 cells (data not shown) Otherresearchers also reported thatMeHg led toNrf2 activation itsnuclear accumulation and upregulation of Nrf2 downstreamantioxidant genes such as HO-1 in a variety of cell types [43ndash45] Interestingly Ni et al demonstrated different responsekinetics in astrocytes and microglia upon MeHg treatment[46] They concluded that these unique sensitivities appearto be dependent on the cellular thiol status of the particularcell type This viewpoint is consistent with the evidence that


MeHg

Nrf2

GCLM

GCLC

Actin

0 1 2 4 8 (120583M)

(a)

MeHg

GCLM

025 056 612 24 12 24Control

Actin

Nrf2

(h)

GCLC

(120583M)

(b)

Figure 4 MeHg activates Nrf2 in SH-SY5Y cells (a) Cells were exposed to MeHg (1 2 4 or 8 120583M) for 6 h and the total cell lysates (20120583g)were subjected to western blotting with the antibodies indicated (b) Cells were exposed to MeHg (025 or 05 120583M) for 6 12 or 24 h andthe total cell lysates (20120583g) were subjected to western blotting with the antibodies indicated Reprinted from Toyama et al [42] with thepermission of Biochemical Biophysical Research Communications

reactive thiols of Keap1 undergo S-mercuration resulting inNrf2 activation Pretreatment with Trolox an antioxidantblocked MeHg-mediated oxidative stress determined byintracellular reactive oxygen species levels to a significantdegree but did not affect Nrf2 activation during the exposureof SH-SY5Y cells to MeHg (Toyama et al unpublisheddata) We therefore speculate that S-mercuration ratherthan oxidative stress is involved in the MeHg-dependentactivation of Nrf2

4 Protection against MeHg Toxicity throughthe Keap1Nrf2 Pathway

Several lines of evidence indicate that Nrf2-deficient miceare susceptible to the toxicity of a variety of chemicalsincluding acetaminophen 712-dimethylbenz[a]anthracenekainic acid dextran sulfate sodium and benzo[a]pyrene[47ndash52] To examine the role of Nrf2 in protecting againstMeHg we used primary hepatocytes fromNrf2++ orNrf2minusminusmice As shown in Figure 5(a) steady-state levels of Nrf2downstream proteins such as GCLC GCLM MRP1 [42]and MRP2 [42] were drastically decreased in Nrf2minusminus cellsUnder these condition the Nrf2minusminus cells were highly sensitiveto MeHg compared with the Nrf2++ cells (Figure 5(b)) [42]In agreement with these observations Ni et al demonstratedthat Nrf2 knockdown by the small hairpin RNA approachreduced the upregulation of its downstream genes such asHO-1 in primary astrocytes and microglia and decreasedviability for both cells [45 46] The accumulation of mercuryin the cerebellum cerebrum and liver after oral MeHgadministration was significantly higher in the Nrf2minusminus micethan in the Nrf2++ mice (Figure 5(c)) [53] because detoxify-ing enzymes associated with MeHg excretion were relativelylow in the Nrf2minusminus mice Consistent with this result theNrf2minusminus mice were also highly susceptible to MeHg in vivo(Figure 5(d)) MeHg-induced neuropathological changes inthe cerebellum were evaluated in the Nrf2++ and Nrf2minusminusmice (Figure 6) and the degeneration of Purkinje cells

(Figure 6(a)) and vacuolar degeneration of the medulla(Figure 6(b)) were observed in the cerebellum of MeHg-treated mice Nrf2 deletion clearly increased these types ofMeHg-induced degeneration These results suggest that Nrf2is a crucial transcription factor for protecting against MeHgtoxicity in vitro and in vivo

5 Conclusions

We found that MeHg S-mercuration reactions on Mn-SODand arginase I cause dysfunction in the activities of these cat-alysts and therefore possibly cause oxidative stress in mousebrain and the release of Mn from the active site leading toa decrease in hepatic Mn levels (seen in rats) The evidenceshowed that MeHg also S-mercurates Keap1 at its reactivethiols including Cys151 resulting in Nrf2 activation in avariety of cell types and thereby upregulation of the down-stream gene products such as antioxidant proteins phase IIxenobiotic-metabolizing enzymes and phase III transporters(as shown in Figure 7) Because these Nrf2-regulated geneproducts contribute to the blocking of oxidative stress and thefacilitation of the detoxification and excretion of MeHg thefindings indicate that the Keap1Nrf2 system plays a criticalrole not only in the cellular response toMeHg but also in thesuppression of MeHg toxicity in vitro and in vivo

Conflicts of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors thank Dr Masayuki Yamamoto Department ofMedical Biochemistry TohokuUniversityGraduate School ofMedicine for his contributions to the studies This work wassupported by a grant-in-aid (no 23117703 to Yoshito Kuma-gai) for scientific research from the Ministry of EducationCulture Sports Science and Technology of Japan


Actin

GCLC

GCLM

Nrf2

Nrf2

MRP1

MRP2

Nrf2

5998400-NT

++ minusminus

++ minusminus

(a)

Cel

l via

bilit

y (

of c

ontro

l)

120

0

20

40

60

80

100

0 1 5 10 20MeHg (120583M)

lowastlowast

lowastlowast

Nrf2++

Nrf2minusminus

(b)

0

04

08

12

16

2

Cerebellum Cerebrum Liver

lowastlowast

Nrf2++

Nrf2minusminus

Tota

l Hg

(120583g

g of

tiss

ue)

lowast lowast

(c)

0

20

40

60

80

100

120

1 5 9 13 17 21 25 29

Surv

ival

()

Day

Nrf2++

Nrf2minusminus

(d)

Figure 5 Nrf2 confers protection against MeHg toxicity (a) Total cell lysates (upper) or crude membrane fractions (lower) from primaryhepatocytes from Nrf2++ or Nrf2minusminus mice were subjected to western blotting with the antibodies indicated (b) Primary hepatocytes fromNrf2++ or Nrf2minusminus mice were exposed to MeHg (1 5 10 and 20 120583M) for 24 h and then the MTT assay was performed Each value representsthe mean plusmn SE of three independent experiments (c) Nrf2++ or Nrf2minusminus mice were orally administratedMeHg (1mgkg) After 48 h the totalmercury contents of the cerebellum cerebrum and liver were determined by atomic absorption mercury detection Each value representsthe mean plusmn SE of five independent experiments (d) Nrf2++ or Nrf2minusminus mice were orally administered MeHg (5mg kgminus1 dayminus1) for 12 days(119899 = 4) and mortality was recorded lowast119875 lt 005 and lowastlowast119875 lt 001 for the Nrf2minusminus mice compared to the Nrf2++ mice Partially reprinted fromToyama et al [42 53] with the permission of Biochemical Biophysical Research Communications and Environmental Health Perspectives


Control MeHg (12 days)

Nrf2

++

Nrf2

minusminus

(a)


Nrf2

++

Nrf2

minusminus

(b)

Figure 6 Pathological observations of brains from Nrf2++ or Nrf2minusminus mice given MeHg Nrf2++ or Nrf2minusminus mice were orally administratedMeHg (5mg kgminus1 dayminus1) for 12 days and pathophysiological changes in the cerebellum were observed Bar = 50 120583m (a) Photographs of thecerebellar granule cell layer (HE stain) Arrows indicate theMeHg-induced degeneration of Purkinje cells (b) Photographs of the cerebellummedulla (KB stain) Arrows indicate the MeHg-induced vacuolar degeneration

RGCNNNCGTCA

Nrf2

Nrf2

Nrf2

Keap1

SH

Nrf2

SmallMaf

SmallMaf

Cul3

Ub

UbUb

Keap1

S

Cul3Ub

MeHg MeHg

Cytoplasm

Normal condition

Nucleus

GCLMRPs

HO-1

EpREARE

UbiquitinProteasome

system

S-mercuration

Figure 7 Cellular protective response to MeHg through the Keap1-Nrf2 system

References

[1] R B Simpson ldquoAssociation constants of methylmercury withsulfhydryl and other basesrdquo Journal of the American ChemicalSociety vol 83 no 23 pp 4711ndash4717 1961

[2] N Ballatori ldquoTransport of toxic metals by molecular mimicryrdquoEnvironmental Health Perspectives vol 110 supplement 5 pp689ndash694 2002

[3] M S Madejczyk D A Aremu T A Simmons-Willis TW Clarkson and N Ballatori ldquoAccelerated urinary excretionof methylmercury following administration of its antidote119873-acetylcysteine requires Mrp2Abcc2 the apical multidrugresistance-associated proteinrdquoThe Journal of Pharmacology andExperimental Therapeutics vol 322 no 1 pp 378ndash384 2007

[4] C C Bridges L Joshee and R K Zalups ldquoMRP2 and thehandling of mercuric ions in rats exposed acutely to inorganicand organic species of mercuryrdquo Toxicology and Applied Phar-macology vol 251 no 1 pp 50ndash58 2011

[5] T W Clarkson ldquoThe pharmacology of mercury compoundsrdquoAnnual Review of Pharmacology vol 12 pp 375ndash406 1972

[6] M Farina M Aschner and J B Rocha ldquoOxidative stress inMeHg-induced neurotoxicityrdquo Toxicology and Applied Pharma-cology vol 256 no 3 pp 405ndash417 2011

[7] J Alam D Stewart C Touchard S Boinapally A M Choi andJ L Cook ldquoNrf2 a CaprsquonrsquoCollar transcription factor regulatesinduction of the heme oxygenase-1 generdquo Journal of BiologicalChemistry vol 274 no 37 pp 26071ndash26078 1999

[8] A C Wild H R Moinova and R T Mulcahy ldquoRegulation of120574-glutamylcysteine synthetase subunit gene expression by thetranscription factor Nrf2rdquo The Journal of Biological Chemistryvol 274 no 47 pp 33627ndash33636 1999

[9] J Y Chan and M Kwong ldquoImpaired expression of glutathionesynthetic enzyme genes in mice with targeted deletion of theNrf2 basic-leucine zipper proteinrdquo Biochimica et BiophysicaActa vol 1517 no 1 pp 19ndash26 2000

[10] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[11] V Vollrath A M Wielandt M Iruretagoyena and J ChianaleldquoRole of Nrf2 in the regulation of the Mrp2 (ABCC2) generdquoBiochemical Journal vol 395 no 3 pp 599ndash609 2006

[12] J M Maher M Z Dieter L M Aleksunes et al ldquoOxidativeand electrophilic stress inducesmultidrug resistance-associatedprotein transporters via the nuclear factor-E2-related factor-2transcriptional pathwayrdquo Hepatology vol 46 no 5 pp 1597ndash1610 2007

[13] M Shinyashiki Y Kumagai S Homma-Takeda et al ldquoSelectiveinhibition of the mouse brain Mn-SOD by methylmercuryrdquoEnvironmental Toxicology and Pharmacology vol 2 no 4 pp359ndash366 1996

[14] Y Kumagai S Homma-Takeda M Shinyashiki and NShimojo ldquoAlterations in superoxide dismutase isozymes bymethylmercuryrdquo Applied Organometallic Chemistry vol 11 no8 pp 635ndash643 1997


[15] M Shinyashiki Y Kumagai H Nakajima et al ldquoDifferentialchanges in rat brain nitric oxide synthase in vivo and in vitro bymethylmercuryrdquo Brain Research vol 798 no 1-2 pp 147ndash1551998

[16] A Yasutake A Nakano K Miyamoto and K Eto ldquoChroniceffects of methylmercury in rats I biochemical aspectsrdquo TheTohoku Journal of Experimental Medicine vol 182 no 3 pp185ndash196 1997

[17] J L Rodrigues J M Serpeloni B L Batista S S Souzaand F Barbosa Jr ldquoIdentification and distribution of mercuryspecies in rat tissues following administration of thimerosal ormethylmercuryrdquo Archives of Toxicology vol 84 no 11 pp 891ndash896 2010

[18] H Kanda D Sumi A Endo et al ldquoReduction of arginaseI activity and manganese levels in the liver during exposureof rats to methylmercury a possible mechanismrdquo Archives ofToxicology vol 82 no 11 pp 803ndash808 2008

[19] H Kanda T Toyama A Shinohara-Kanda et al ldquo119878-mercuration of rat sorbitol dehydrogenase by methylmercurycauses its aggregation and the release of the zinc ion from theactive siterdquo Archives of Toxicology vol 86 no 11 pp 1693ndash17022012

[20] C Karlsson H Jornvall and J O Hoog ldquoSorbitol dehydro-genase cDNA coding for the rat enzyme variations withinthe alcohol dehydrogenase family independent of quaternarystructure and metal contentrdquo European Journal of Biochemistryvol 198 no 3 pp 761ndash765 1991

[21] K Johansson M El-Ahmad C Kaiser et al ldquoCrystal structureof sorbitol dehydrogenaserdquoChemico-Biological Interactions vol130ndash132 no 1ndash3 pp 351ndash358 2001

[22] J S Woods H A Davis and R P Baer ldquoEnhancement of 120574-glutamylcysteine synthetase mRNA in rat kidney by methylmercuryrdquo Archives of Biochemistry and Biophysics vol 296 no1 pp 350ndash353 1992

[23] A Yasutake and K Hirayama ldquoAcute effects of methylmercuryon hepatic and renal glutathionemetabolisms inmicerdquoArchivesof Toxicology vol 68 no 8 pp 512ndash516 1994

[24] S Li S A Thompson and J S Woods ldquoLocalization of 120574-lutamylcysteine synthetase mRNA expression in mouse brainfollowingmethylmercury treatment using reverse transcriptionin situ PCR amplificationrdquo Toxicology and Applied Pharmacol-ogy vol 140 no 1 pp 180ndash187 1996

[25] S A Thompson C C White C M Krejsa et al ldquoInductionof glutamate-cysteine ligase (120574-glutamylcysteine synthetase) inthe brains of adult female mice subchronically exposed tomethylmercuryrdquo Toxicology Letters vol 110 no 1-2 pp 1ndash91999

[26] S A Thompson C C White C M Krejsa D L Eaton andT J Kavanagh ldquoModulation of glutathione and glutamate-L-cysteine ligase by methylmercury during mouse developmentrdquoToxicological Sciences vol 57 no 1 pp 141ndash146 2000

[27] D Dıaz C M Krejsa C C White C L Keener F M Farinand T J Kavanagh ldquoTissue specific changes in the expres-sion of glutamate-cysteine ligase mRNAs in mice exposed tomethylmercuryrdquo Toxicology Letters vol 122 no 2 pp 119ndash1292001

[28] D Dıaz C M Krejsa C C White J S Charleston and TJ Kavanagh ldquoEffect of methylmercury on glutamate-cysteineligase expression in the placenta and yolk sac during mousedevelopmentrdquoReproductive Toxicology vol 19 no 1 pp 117ndash1292004

[29] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[30] K Itoh N Wakabayashi Y Katoh et al ldquoKeap1 repressesnuclear activation of antioxidant responsive elements by Nrf2through binding to the amino-terminal Neh2 domainrdquo Genesamp Development vol 13 no 1 pp 76ndash86 1999

[31] A T Dinkova-Kostova W D Holtzclaw R N Cole et alldquoDirect evidence that sulfhydryl groups of keap1 are the sensorsregulating induction of phase 2 enzymes that protect againstcarcinogens and oxidantsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 99 no 18 pp11908ndash11913 2002

[32] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of Nrf2and for stabilization of Nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[33] A L Eggler G Liu J M Pezzuto R B Van Breemen and AD Mesecar ldquoModifying specific cysteines of the electrophile-sensing human keap1 protein is insufficient to disrupt bindingto the Nrf2 domain Neh2rdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 29 pp10070ndash10075 2005

[34] FHongK R SekharM L Freeman andDC Liebler ldquoSpecificpatterns of electrophile adduction trigger keap1 ubiquitinationand Nrf2 activationrdquo Journal of Biological Chemistry vol 280no 36 pp 31768ndash31775 2005

[35] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[36] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystem keap1-Nrf2 comprises a multiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[37] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the keap1-Nrf2 pathway in stress response andcancer evolutionrdquoGenes to Cells vol 16 no 2 pp 123ndash140 2011

[38] Y Abiko T Miura B H Phuc Y Shinkai and Y Kuma-gai ldquoParticipation of covalent modification of keap1 in theactivation of Nrf2 by tert-butylbenzoquinone an electrophilicmetabolite of butylated hydroxyanisolerdquoToxicology andAppliedPharmacology vol 255 no 1 pp 32ndash39 2011

[39] TMiura Y ShinkaiH Y Jiang et al ldquoInitial response and cellu-lar protection through the keap1Nrf2 system during the expo-sure of primary mouse hepatocytes to 12-naphthoquinonerdquoChemical Research in Toxicology vol 24 no 4 pp 559ndash567 2011

[40] A L Levonen A Landar A Ramachandran et al ldquoCellularmechanisms of redox cell signalling role of cysteine mod-ification in controlling antioxidant defences in response toelectrophilic lipid oxidation productsrdquo Biochemical Journal vol378 no 2 pp 373ndash382 2004

[41] N Wakabayashi A T Dinkova-Kostova W D Holtzclaw et alldquoProtection against electrophile and oxidant stress by inductionof the phase 2 response fate of cysteines of the keap1 sensormodified by inducersrdquo Proceedings of the National Academy ofSciences of the United States of America vol 101 no 7 pp 2040ndash2045 2004


[42] T Toyama D Sumi Y Shinkai et al ldquoCytoprotective role ofNrf2Keap1 system inmethylmercury toxicityrdquo Biochemical andBiophysical Research Communications vol 363 no 3 pp 645ndash650 2007

[43] L Wang H Jiang Z Yin M Aschner and J Cai ldquoMethylmer-cury toxicity and Nrf2-dependent detoxification in astrocytesrdquoToxicological Sciences vol 107 no 1 pp 135ndash143 2009

[44] V Lamsa A L Levonen H Leinonen S Yla-Herttuala MYamamoto and J Hakkola ldquoCytochrome P450 2A5 constitutiveexpression and induction by heavy metals is dependent onredox-sensitive transcription factor nrf2 in liverrdquo ChemicalResearch in Toxicology vol 23 no 5 pp 977ndash985 2010

[45] M Ni X Li Z Yin et al ldquoMethylmercury induces acute oxida-tive stress altering Nrf2 protein level in primary microglialcellsrdquo Toxicological Sciences vol 116 no 2 pp 590ndash603 2010

[46] M Ni X Li Z Yin et al ldquoComparative study on the responseof rat primary astrocytes and microglia to methylmercurytoxicityrdquo Glia vol 59 no 5 pp 810ndash820 2011

[47] A Enomoto K Itoh E Nagayoshi et al ldquoHigh sensitiv-ity of Nrf2 knockout mice to acetaminophen hepatotoxicityassociated with decreased expression of ARE-regulated drugmetabolizing enzymes and antioxidant genesrdquo ToxicologicalSciences vol 59 no 1 pp 169ndash177 2001

[48] Y Aoki H Sato N Nishimura S Takahashi K Itoh and MYamamoto ldquoAccelerated DNA adduct formation in the lung ofthe Nrf2 knockoutmouse exposed to diesel exhaustrdquoToxicologyand Applied Pharmacology vol 173 no 3 pp 154ndash160 2001

[49] C Xu M T Huang G Shen et al ldquoInhibition of 712-dimethylbenz(119886)anthracene-induced skin tumorigenesis inC57BL6 mice by sulforaphane is mediated by nuclear factorE2-related factor 2rdquo Cancer Research vol 66 no 16 pp8293ndash8296 2006

[50] A D Kraft J M Lee D A Johnson Y W Kan and J AJohnson ldquoNeuronal sensitivity to kainic acid is dependent ontheNrf2-mediated actions of the antioxidant response elementrdquoJournal of Neurochemistry vol 98 no 6 pp 1852ndash1865 2006

[51] T O Khor M T Huang K H Kwon J Y Chan B SReddy and A N Kong ldquoNrf2-deficient mice have an increasedsusceptibility to dextran sulfate sodium-induced colitisrdquoCancerResearch vol 66 no 24 pp 11580ndash11584 2006

[52] Y Aoki A H Hashimoto K Amanuma et al ldquoEnhancedspontaneous and benzo(119886)pyrene-induced mutations in thelung of Nrf2-deficient gpt delta micerdquo Cancer Research vol 67no 12 pp 5643ndash5648 2007

[53] T Toyama Y Shinkai A Yasutake K Uchida M Yamamotoand Y Kumagai ldquoIsothiocyanates reduce mercury accumula-tion via an Nrf2-dependent mechanism during exposure ofmice tomethylmercuryrdquoEnvironmentalHealth Perspectives vol119 no 8 pp 1117ndash1122 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Molecular targets of MeHg

Target protein In vivo effect References

Mn-SOD Reduction of catalytic activity inmouse brain

Shinyashikiet al [13]

nNOS Induction of NOS in rat brain Shinyashikiet al [15]

Arginase I Reduction of activity in rat liverDecreased Mn levels in tissues

Kanda et al[18]

SDH Not defined Kanda et al[19]

ndashSminus

GSH

Nrf2GCL

MeHg

Protein

Protein

MRP

Protein

Cellular damage

pKa

ndashSH

MeHgndashSG

ndashSndashHgMe

Figure 1 S-mercuration of cellular proteins byMeHg and theMeHgdetoxification pathway

of mRNA and protein synthesis of Mn-SOD were unaffectedby MeHg administration MeHg caused a facile reduction inthe activity and a drastic decrease in the native form of Mn-SOD (but not of CuZn-SOD) with purified enzyme prepa-rations It was subsequently shown that the S-mercurationof Mn-SOD through Cys196 by MeHg results in a decreasein the enzyme activity in vivo [14] Therefore the selectiveinhibition of Mn-SOD activity caused by S-mercurationcould play a critical role in MeHg-induced neurotoxicitybecause SOD protects the cell against oxidative stress byscavenging superoxide

We also found that the subcutaneous administration ofMeHg (10mg kgminus1 dayminus1 for 8 days) to rats caused significantincreases in nitric oxide synthase (NOS) activities in thecerebrum and cerebellum with time [15] The increase inNOS activity seems to be caused by an increase in neuronalNOS (nNOS) protein levels but not an increase in inducibleNOS In contrast to the in vivo observations however theaddition of MeHg to a cerebellar enzyme preparation in vitrocaused a concentration-dependent decrease in nNOS activ-ity suggesting that MeHg modifies NOS through reactivethiol groups thereby affecting its catalytic activity in vitroExperimentswith arylmercury column chromatographyhaveconfirmed that this is possible [15] A reasonable explanationfor our observations is that the increase in nNOS proteinsobserved from MeHg exposure in vivo may be one of theinitial responses to counteract the metal-induced negativeeffects on the central nervous system

In the body MeHg accumulates to high concentrationsin the liver [16 17] but few molecular targets of MeHg havebeen identified in this tissue We hypothesized that MeHgcould interact with arginase I an abundant Mn-bindingprotein in the liver through covalent modification resultingin alterations in not only its catalytic activity but also inhepatic Mn levels through in vivo MeHg exposure Afterthe subcutaneous administration of MeHg (10mg kgminus1 dayminus1for 8 days) to rats mercury levels in the liver were greaterthan those in the cerebrum or cerebellum [18] A markedsuppression of arginase I activity was also detected underthese conditions Using purified rat arginase I we foundthat MeHg-induced S-mercuration of arginase I caused theprotein to aggregate and substantial leakage of Mn ionsfrom the arginase I active site We speculate that the MeHg-mediated suppression of hepatic arginase I activity in vivois at least partly attributable to the S-mercuration of thisprotein

In the course of the study we coincidentally detectedanother protein with a 42 kDa subunit molecule fromthe same hepatic preparation that readily underwent S-mercuration and subsequent aggregation Two-dimensionalsodium dodecyl sulfate-polyacrylamide gel electrophoresiswas used to separate the protein and it was identifiedby peptide mass fingerprinting using matrix-assisted laserdesorption and ionization time-of-flight mass spectrometry(MALDI-TOFMS) This 42 kDa protein was identified assorbitol dehydrogenase (SDH) [19] Using recombinant ratSDH possessing 10 cysteine residues in a subunit [20] wefound that MeHg was covalently bound to SDH throughCys44 Cys119 Cys129 and Cys164 resulting in the inhibitionof its catalytic activity and the release of zinc ions facilitat-ing protein aggregation [19] Subsequent mutation analysisconfirmed that Cys44 which ligates the active site zinc atom[21] and Cys129 play a crucial role in the MeHg-mediatedaggregation of SDH [19]

3 Cellular Response to MeHg viathe Keap1Nrf2 Pathway

As mentioned above MeHg reacts readily with cellularnucleophiles and also causes oxidative stress implying thatit may lead to decreased GSH levels in tissues As a resultMeHg undergoes GSH conjugation and is excreted intothe extracellular space through the action of MRPs [2ndash4]However several studies have indicated that MeHg exposurealso upregulates GCL [22ndash28] This suggests that there isan initial response to MeHg in the cells to compensate fordecreased GSH levels and to repress oxidative cell damageOn the other hand Dr Yamamoto and his associates reportedthat transcription factor Nrf2 cooperatively regulates antiox-idant proteins such as GCL and HO-1 phase-II xenobioticdetoxifying enzymes and phase-III xenobiotic transporterssuch as MRPs [10 29] They subsequently found that Nrf2is negatively regulated by Kelch-like ECH-associated protein1 (Keap1) [30] Interestingly Keap1 has 27 and 25 cysteineresidues in humans and mice respectively [31 32] and somethiols (eg Cys151 Cys273 and Cys288) that have been







15605 15596



18638 18648



23506 23495

MeHg

489257 273 288 297 613368151 22623 319

Keap1




TBQ

lowastlowastlowast





lowast

lowast

lowast

lowast


12-NQ




1

1

2

2

3

3

P-1

P-2

P-3

Keap1 control

100

80

60

40

20

00 500 1000 1500 2000 2500 3000 3500

mz

Inte

nsity

()

Keap1 + MeHg




MeHg

Nrf2

GCLM

GCLC

Actin

0 1 2 4 8 (120583M)

(a)

MeHg

GCLM

025 056 612 24 12 24Control

Actin

Nrf2

(h)

GCLC

(120583M)

(b)






5 Conclusions




Acknowledgments



Actin

GCLC

GCLM

Nrf2

Nrf2

MRP1

MRP2

Nrf2

5998400-NT

++ minusminus

++ minusminus

(a)

Cel

l via

bilit

y (

of c

ontro

l)

120

0

20

40

60

80

100

0 1 5 10 20MeHg (120583M)

lowastlowast

lowastlowast

Nrf2++

Nrf2minusminus

(b)

0

04

08

12

16

2


lowastlowast

Nrf2++

Nrf2minusminus

Tota

l Hg

(120583g

g of

tiss

ue)

lowast lowast

(c)

0

20

40

60

80

100

120

1 5 9 13 17 21 25 29

Surv

ival

()

Day

Nrf2++

Nrf2minusminus

(d)




Nrf2

++

Nrf2

minusminus

(a)


Nrf2

++

Nrf2

minusminus

(b)


RGCNNNCGTCA

Nrf2

Nrf2

Nrf2

Keap1

SH

Nrf2

SmallMaf

SmallMaf

Cul3

Ub

UbUb

Keap1

S

Cul3Ub

MeHg MeHg

Cytoplasm

Normal condition

Nucleus

GCLMRPs

HO-1

EpREARE

UbiquitinProteasome

system

S-mercuration


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















15605 15596



18638 18648



23506 23495

MeHg

489257 273 288 297 613368151 22623 319

Keap1




TBQ

lowastlowastlowast





lowast

lowast

lowast

lowast


12-NQ




1

1

2

2

3

3

P-1

P-2

P-3

Keap1 control

100

80

60

40

20

00 500 1000 1500 2000 2500 3000 3500

mz

Inte

nsity

()

Keap1 + MeHg




MeHg

Nrf2

GCLM

GCLC

Actin

0 1 2 4 8 (120583M)

(a)

MeHg

GCLM

025 056 612 24 12 24Control

Actin

Nrf2

(h)

GCLC

(120583M)

(b)






5 Conclusions




Acknowledgments



Actin

GCLC

GCLM

Nrf2

Nrf2

MRP1

MRP2

Nrf2

5998400-NT

++ minusminus

++ minusminus

(a)

Cel

l via

bilit

y (

of c

ontro

l)

120

0

20

40

60

80

100

0 1 5 10 20MeHg (120583M)

lowastlowast

lowastlowast

Nrf2++

Nrf2minusminus

(b)

0

04

08

12

16

2


lowastlowast

Nrf2++

Nrf2minusminus

Tota

l Hg

(120583g

g of

tiss

ue)

lowast lowast

(c)

0

20

40

60

80

100

120

1 5 9 13 17 21 25 29

Surv

ival

()

Day

Nrf2++

Nrf2minusminus

(d)




Nrf2

++

Nrf2

minusminus

(a)


Nrf2

++

Nrf2

minusminus

(b)


RGCNNNCGTCA

Nrf2

Nrf2

Nrf2

Keap1

SH

Nrf2

SmallMaf

SmallMaf

Cul3

Ub

UbUb

Keap1

S

Cul3Ub

MeHg MeHg

Cytoplasm

Normal condition

Nucleus

GCLMRPs

HO-1

EpREARE

UbiquitinProteasome

system

S-mercuration


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MeHg

Nrf2

GCLM

GCLC

Actin

0 1 2 4 8 (120583M)

(a)

MeHg

GCLM

025 056 612 24 12 24Control

Actin

Nrf2

(h)

GCLC

(120583M)

(b)






5 Conclusions




Acknowledgments



Actin

GCLC

GCLM

Nrf2

Nrf2

MRP1

MRP2

Nrf2

5998400-NT

++ minusminus

++ minusminus

(a)

Cel

l via

bilit

y (

of c

ontro

l)

120

0

20

40

60

80

100

0 1 5 10 20MeHg (120583M)

lowastlowast

lowastlowast

Nrf2++

Nrf2minusminus

(b)

0

04

08

12

16

2


lowastlowast

Nrf2++

Nrf2minusminus

Tota

l Hg

(120583g

g of

tiss

ue)

lowast lowast

(c)

0

20

40

60

80

100

120

1 5 9 13 17 21 25 29

Surv

ival

()

Day

Nrf2++

Nrf2minusminus

(d)




Nrf2

++

Nrf2

minusminus

(a)


Nrf2

++

Nrf2

minusminus

(b)


RGCNNNCGTCA

Nrf2

Nrf2

Nrf2

Keap1

SH

Nrf2

SmallMaf

SmallMaf

Cul3

Ub

UbUb

Keap1

S

Cul3Ub

MeHg MeHg

Cytoplasm

Normal condition

Nucleus

GCLMRPs

HO-1

EpREARE

UbiquitinProteasome

system

S-mercuration


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Actin

GCLC

GCLM

Nrf2

Nrf2

MRP1

MRP2

Nrf2

5998400-NT

++ minusminus

++ minusminus

(a)

Cel

l via

bilit

y (

of c

ontro

l)

120

0

20

40

60

80

100

0 1 5 10 20MeHg (120583M)

lowastlowast

lowastlowast

Nrf2++

Nrf2minusminus

(b)

0

04

08

12

16

2


lowastlowast

Nrf2++

Nrf2minusminus

Tota

l Hg

(120583g

g of

tiss

ue)

lowast lowast

(c)

0

20

40

60

80

100

120

1 5 9 13 17 21 25 29

Surv

ival

()

Day

Nrf2++

Nrf2minusminus

(d)




Nrf2

++

Nrf2

minusminus

(a)


Nrf2

++

Nrf2

minusminus

(b)


RGCNNNCGTCA

Nrf2

Nrf2

Nrf2

Keap1

SH

Nrf2

SmallMaf

SmallMaf

Cul3

Ub

UbUb

Keap1

S

Cul3Ub

MeHg MeHg

Cytoplasm

Normal condition

Nucleus

GCLMRPs

HO-1

EpREARE

UbiquitinProteasome

system

S-mercuration


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Nrf2

++

Nrf2

minusminus

(a)


Nrf2

++

Nrf2

minusminus

(b)


RGCNNNCGTCA

Nrf2

Nrf2

Nrf2

Keap1

SH

Nrf2

SmallMaf

SmallMaf

Cul3

Ub

UbUb

Keap1

S

Cul3Ub

MeHg MeHg

Cytoplasm

Normal condition

Nucleus

GCLMRPs

HO-1

EpREARE

UbiquitinProteasome

system

S-mercuration


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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