Transsulfuration Is a Significant Source of Sulfur for ...downloads.hindawi.com/journals/isrn.biochemistry/2013/637897.pdfMAT S-Adenosylmethionine Homocysteine S-Adenosylhomocysteine

Hindawi Publishing CorporationISRN BiochemistryVolume 2013 Article ID 637897 7 pageshttpdxdoiorg1011552013637897

Research ArticleTranssulfuration Is a Significant Source of Sulfur forGlutathione Production in Human Mammary Epithelial Cells

Andrea D Belalcaacutezar1 John G Ball2 Leslie M Frost1

Monica A Valentovic2 and John Wilkinson IV3

1 Department of Chemistry Marshall University One John Marshall Drive Huntington WV 25755-0003 USA2Department of Pharmacology Physiology amp Toxicology Marshall University Joan C Edwards School of MedicineOne John Marshall Drive Huntington WV 25755-0003 USA

3Department of Anatomy and Pathology Marshall University Joan C Edwards School of MedicineOne John Marshall Drive Huntington WV 25755-0003 USA

Correspondence should be addressed to John Wilkinson IV wilkinsonjmarshalledu

Received 22 January 2013 Accepted 16 February 2013

Academic Editors A-M Lambeir and B Lenarcic

Copyright copy 2013 Andrea D Belalcazar 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

The transsulfuration pathway throughwhich homocysteine from themethionine cycle provides sulfur for cystathionine formationwhich may subsequently be used for glutathione synthesis has not heretofore been identified as active in mammary cellsPrimary human mammary epithelial cells (HMECrsquos) were labeled with 35S-methionine for 24 hours following pretreatment with avehicle control the cysteine biosynthesis inhibitor propargylglycine or the gamma-glutamylcysteine synthesis inhibitor buthioninesulfoximine Cell lysates were prepared and reacted with glutathione-S-transferase and the fluorescent labeling compoundmonochlorobimane to form a fluorescent glutathione-bimane conjugate Comparison of fluorographic and autoradiographicimages indicated that glutathione had incorporated 35S-methionine demonstrating that functional transsulfuration occurs inmammary cells Pathway inhibitors reduced incorporation by roughly 80 Measurement of glutathione production in HMECrsquostreatedwith andwithout hydrogenperoxide andor pathway inhibitors indicates that the transsulfuration pathway plays a significantrole in providing cysteine for glutathione production both normally and under conditions of oxidant stress

1 Introduction

Inmammals cystathionine beta-synthase (CBS) catalyzes thefirst step in the transsulfuration pathway (see Figure 1) [1]a pyridoxal-51015840-phosphate- (PLP-) dependent condensationof serine and homocysteine to cystathionine [2 3] Thesecond step of the transsulfuration pathway is the hydrolysisof cystathionine to cysteine ammonia and 120572-ketobutyratecatalyzed by the enzyme 120574-cystathionase These reactionsform a metabolic bridge between the methionine cycle andcysteine a necessary precursor for glutathione biosynthesis

Homocystinuria is the principal disorder resulting fromimpairment of transsulfuration (TS) which leads to abnor-mally high homocysteine levels Transsulfuration impair-ment is also associated with other disorders such as autism

[4ndash8] cirrhosis [9ndash13] coronary artery disease [14ndash18] goi-ter [19] impaired coagulation [20ndash22] immune function[23] neurodegenerative disease [24ndash26] and pancreatitis[27] This variety of afflictions highlights the importance oftranssulfuration to human health Despite the widespreadnature of tissues involved in these disorders our knowledge oftranssulfuration is confined to the liver and a small number ofother tissues These include the brain [28] certain lymphoidcells [29] and the pancreas [27 30]

Mammary cells may face certain oxidative challengesfromdiets high in redmeat [31ndash36] (which delivers heme iron[37 38]) and from ethanol consumption (for which there aremany excellent reviews and meta-analyses [39ndash42]) We areinterested in whether transsulfuration occurs in breast tissueto determine its possible role in protection versus oxidant

2 ISRN Biochemistry

Methionine

MS

MATS-Adenosylmethionine

S-AdenosylhomocysteineHomocysteine

Cystathionine

CBS

Propargylglycine Gamma-cystathionase

Gamma-glutamylcysteine synthase

Cysteine

Gamma-glutamylcysteine

Glutathione synthase

Glutathione

Buthionine sulfoximine

Figure 1 The transsulfuration pathway connects methionine andglutathione biosynthesis In themethionine cyclemethionine formsS-adenosylmethionine which serves as a methyl donor generatingS-adenosyl homocysteineThis is converted to homocysteine whichis subsequently converted back into methionine Homocysteine hasan alternative fate however It can be used to produce cystathioninewhich is further converted to cysteine This latter conversion is cat-alyzed by gamma-cystathionase and inhibited by propargylglycineCysteine can then feed glutathione biosynthesis through productionof gamma-glutamylcysteine This step is catalyzed by gamma-glutamylcysteine synthase and inhibited by buthionine sulfoximine

stress for chronic diseases of mammary tissue such as breastcancer Based on our results obtained using a radioactivetracer method involving 35S-methionine incorporation intoglutathione we report that the transsulfuration pathway isactive in human mammary epithelial cells Further workemploying pathway inhibitors while measuring glutathioneproduction normally and during oxidant challenge indicatestranssulfuration provides necessary sulfur for the enhancedcysteine production that is required for these cells to respondto oxidative stress

2 Materials and Methods

21 Human Mammary Epithelial Cell Culture In order toapply the power of a radioactive tracer method to test ourhypothesis while still avoiding as many of the inherentbiases that can result from alterations in cellular biochemistryacquired by tissue culture lines as feasible we used a primaryhuman mammary epithelial cell (HMEC) model systemThese cells were obtained from Lonza (Walkersville MD)and are neither transformed nor immortalized They arecapable of undergoing a limited number of divisions andmust be maintained at less than maximum confluence toavoid triggering senescence Human mammary epithelialcells (HMEC) were grown and passaged under standardconditions in tissue culture treated plasticware (Corning

Lowell MA) at 37∘C at high humidity in water jacketed CO2

incubators (Fisher Scientific Thermo Forma) according tothe distributors instructions changing culturemedium everytwo days with standard medium prepared using their propri-etary reagents (Lonza Walkersville MD) Standard growthmedia for HMEC was mammary epithelial basal medium(MEBM) supplemented with growth factors supplied inaliquots at proprietary concentrations which include (for a500mL bottle) 2mL of bovine pituitary extract 05mL ofepidermal growth factor 05mL of insulin 05mL of hydro-cortisone and 05mL of gentamicin sulfateamphotericinB To prepare experimental cells 80 confluent T75 flaskswere split and used to seed T75 flasks each with 2E5 cellsin 15mL of standard medium Seven days after seeding(medium changed every two days) they were 50 confluentand experimental treatment began

22 Transsulfuration Assay Cells were pretreated for 24hours with standard media containing either 9mM buthio-nine sulfoximine (BSO) 25mM propargylglycine (PPG)or a PBS vehicle control Then the medium was replacedwith 10mL of radioactive labeling medium (standard mediacontaining the appropriate inhibitors and 35S methionine at25 120583CimL activity) Cells were incubated for 24 hours andthen pellets were harvested through trypsinization Pelletswere rinsed with PBS and frozen and stored briefly at minus80∘C

To fluorescently label cellular glutathione in cell lysateswe adapted the method of Kamencic et al [43] Monochloro-bimane (mCBi) is reported to specifically react with glu-tathione (a reaction catalyzed by GST) and not other cellularthiols [44] Cell pellets were removed from the freezer anequal volume of water was added and pellets were thawedand frozen an additional 3 times following each thaw pelletswere vortexed at high speed The supernatant with cytosoliccontents was isolated by spinning the cells at 4∘C and15000 rpm for 15 minutes Pellets were reextracted twice andall supernatants pooled for each sampleThe reactionmixtureto fluorescently label GSH consisted of 100120583L of cell lysate2 120583L of mCBi 20mM 20120583L of 500mM potassium phosphate(K2PO4) pH 65 and 25 120583g of GST (20120583L of 25120583g GST

dissolved in 100 120583L of PBS) in a total reaction volume of200120583L (water is used to equalize samples) Reactions wereprepared on ice then initiated by incubation at 37∘C for aperiod of 10 minutes Reactions were stopped by freezing Acontrol reaction was run by substituting the cell lysate with2 120583L of 10mM GSH in the same reaction volume of 200120583L10 120583L of each reaction mixture were spotted on 250 micronsilica gel GF uniplates (Analtech Newark DE) and analyzedby TLC using a 3 1 1 mixture of 1-butanol methanol water(solvents from Fisher Scientific Chicago IL) Two 120583L of20mM mCBi were run in a separate lane as a control toindicate the migration of nonreacted mCBi Migrations offluorescent products were compared under UV light andrecorded using a Bio-Rad universal hood digital camera(Bio-Rad laboratories Inc Segrate Milan Italy) Massspectroscopic analysis confirmed the identity of the GSH-Bimane TLC band (data not shown) Autoradiographs ofplates were prepared using Kodak BioMax light film (Sigma

ISRN Biochemistry 3

St Louis MO) and phosphorimaging using a Typhoon 9200variable mode imager (Amersham Biosciences Sweden)Note that the incorporation of methionine into glutathionerevealed through autoradiography measures only the glu-tathione produced from transsulfuration and not glutathioneproduced from cysteine derived from the media Furtherthe incorporation of the fluorescentmonochlorobimane labelinto glutathione occurs regardless of whether the glutathionederives from transsulfuration

23 Glutathione Assay As an initial step in evaluating thebiologic significance of active transsulfuration in HMECrsquoswe hypothesized that the HMECrsquos would require transsulfu-ration to produce glutathione in response to oxidant stressTo test this hypothesis cells were pretreated for 24 hourswith vehicle or 25mM PPG inhibitor in standard mediaMedia was then removed and replacedwithmedia containingpretreatments plus vehicle or 300120583M H

2O2for two hours

Cells were harvested by trypsinization after the two-hourtreatment with PBS washed pellets frozen in aliquots atminus80∘C Cell pellets were homogenized in 500 uL 05 sul-fosalicylic acid and adjusted to a 1mL volume Total glu-tathione was determined by an enzymatic method [45 46]as described previously [47ndash52] using a glutathione reduc-tase and NADPH coupled reaction with 551015840-dithiobis(2-nitrobenzoic acid) and reported as nmolg of protein Proteinwas determined using the Bradford method [53]

3 Results

31 Transsulfuration Assay To test our hypothesis that tran-ssulfuration occurs in mammary tissue we incubated pri-mary human mammary epithelial cells (HEMCrsquos) with aradioactive 35S-methionine tracer (Perkin Elmer WalthamMA) isolated cellular material following incubation anddetermined the extent to which (if any) the 35S-methionineincorporated into glutathioneThe glutathione in cell extractswas fluorescently labeled with monochlorobimane and sepa-rated fromother cell constituents using thin layer chromatog-raphy Fluorescent excitation of the TLC plate identifiedthe glutathione spots while autoradiography of those spotsindicated the extent to which 35S-methionine incorporatedinto the glutathione

In Figure 2 cell extracts containing bimane-labeled glu-tathione are analyzed by TLC and the glutathione conjugatesare indicated under the fluorescence panel Comparison ofthe images in Figure 2 (fluorescence lanes 1 and 2 withautoradiography lanes 1 and 2) indicate that the fluores-cent GSH-MCBi conjugate contains radioactivity Incor-poration of 35S-methionine into glutathione (GSH-MCBibands) demonstrates that functional transsulfuration occursin mammary cells The inhibitors propargyl glycine (PPG)or buthionine sulfoximine (BSO) (Sigma St Louis MO)were used to block the pathway at two steps critical for theevaluation of the results (see Figure 1) Cystathionine derivesfrom active transsulfuration wherein homocysteine exits themethionine cycle Conversion of cystathionine to cysteine

is blocked by PPG while conversion of cysteine to gamma-glutamylcysteine is inhibited by BSO thus PPG inhibitsincorporation of only transsulfuration derived cysteine intoglutathione while BSO inhibits incorporation of cysteinederived fromany source into glutathione [28] In Figure 2 theimpact of PPG inhibition of either total glutathione synthesis(left panel fluorescence compare PPG with Control) orthe incorporation of 35S-methionine into glutathione (rightpanel autoradiography compare PPG with Control) bothdemonstrate that active transsulfuration is taking place andconfirm the identity of the TLC spots The identity of thespots was further confirmed using mass spectroscopy (datanot shown) Pretreatment with BSO caused inhibition of totalglutathione (Figure 2 fluorescence BSO versus control) aswell as 35S-methionine incorporation (Figure 2 autoradiog-raphy BSO versus control) consistent with its action at onestep later in the pathway (Figure 1)

32 Glutathione Assay As an initial step in evaluating thebiological significance of active transsulfuration in HMECrsquoswe hypothesized that the HMECrsquos would require transsulfu-ration to produce glutathione in response to oxidant stressTo test this hypothesis cells were pretreated for 24 hourswith vehicle or 25mM PPG inhibitor in standard mediaMedia was then removed and replacedwithmedia containingpretreatments plus vehicle or 300 120583M H

2O2for two hours

and glutathione levels were determined as described undermethods

Results depicted in Figure 3 indicate that resting cellshave roughly 30 nmoles glutathione per mg of cell proteinCells respond to 2 hrs of oxidant treatment by increasing glu-tathione synthesis to achieve levels of roughly 40 nmolesmgcell protein When resting cells are subjected to a transsulfu-ration blockade using PPG treatment they contain 20 nmolesGSHmg cell protein roughly two-thirds the level of controlcells When cells are oxidant treated while simultaneouslyundergoing transsulfuration blockade they are unable torespond to the oxidant insult resulting in roughly 16 nmolesGSHmg cell protein This significantly differs (119875 lt 0002by ANOVA) from the response in oxidant treated cellswhich have an active transsulfuration pathway (see Figure 3compare H

2O2PBS versus PPG bars)

4 Discussion

Cystathionine 120573-synthase (CBS) catalyzes the first step ofthe transsulfuration pathway the conversion of homocysteineto cystathionine in effect forming a bridge between themethionine cycle and the production of cysteine a precursorfor glutathione biosynthesis The CBS enzyme is prevalentparticularly in the liver and pancreas [30] though its mRNAis also reported to be expressed to a low level in the heartbrain placenta lung skeletal muscle and kidney [54] In thissame pioneering report a dot blot did not detect expressionfor total CBS (representing all 5 known variants of theenzyme) in human mammary mRNA possibly leading to alack of further study for this tissue [54] We speculate thatthe source of this mammary mRNA must be different than

4 ISRN Biochemistry

Fluorescence Autoradiography

Free MCBi

GSH-Bi

Control BSO PPG Control BSO PPG

Figure 2 Transsulfuration is a significant source of sulfur for glutathione synthesis in human mammary cells human mammary epithelialcells (HMECrsquos) were pretreated with vehicle control pathway inhibitors buthionine sulfoximine (BSO) or propargylglycine (PPG) for24 hours then labeled with 35S-methionine for 24 hours Lysate and glutathione bimane conjugates were prepared and analyzed by thinlayer chromatography and autoradiography as described under methods Comparison of the images (fluorescence lanes 1 and 2 withautoradiography lanes 1 and 2) indicates that the fluorescent GSH-MCBi conjugate is radioactive Incorporation of 35S-methionine intoglutathione (GSH-MCBi bands) demonstrates that functional transsulfuration occurs in mammary cells PPG inhibitory impact on eitherglutathione synthesis from all cysteine sources (left panel fluorescence compare PPG with control) or the incorporation of 35S-methioninelabeled cysteine (which must be transsulfuration derived) into glutathione demonstrates both transsulfuration and the identity of the TLCspots BSO predictably inhibited production of glutathione without regard to cysteine source

Tota

l glu

tath

ione

(mea

n nm

oles

mg

prot

einplusmn

SEM

)

50

40

30

20

10

0Vehicle for 2 h 300 120583M H

2O2

for 2 h

lowast

lowast

PBSPPG

Figure 3 Impact of transsulfuration inhibition (PPG) on cellulartotal glutathione levels in humanmammary epithelial cells subjectedto oxidative challenge (H

2O2) Human mammary epithelial cells

(HMECrsquos) were grown in normal mammary epithelial growthmedium to 50 confluency and pretreated with a PBS vehicle con-trol or propargylglycine (PPG) for 24 hours followed by treatmentin the same media with vehicle or 300 uM H

2O2for two hours Cell

pellets were prepared and analyzed for total glutathione levels asdescribed under methods [45] Results are expressed as nmolmg ofcell protein (mean plusmn SEM119873 = 5-6) Asterisk indicates a significantdifference between PBSH

2O2and PPGH

2O2groups determined

by ANOVA (119875 lt 0002)

our primary human mammary epithelial cells which clearlypossesses CBS activity as 35S from methionine incorporatesinto glutathione seen in Figure 2

The limitations of our approach despite our use of non-immortalized primary cells involve the artificial nature ofan in vitro experiment We chose HMECrsquos to be able tobring the power of a radioactive tracer approach to answerthis question because generation of 35S-glutathione from

35S-methionine is the most direct and definitive proof thattranssulfuration is active in the cells Nevertheless thepossibility exists that even carefully tended primary cellswill behave differently from cells within tissues which hasthe potential to limit the significance of our findings Ourpurpose in reporting these in vitro results is to progress thefield of study in a meaningful way and perhaps provide thebasis for subsequent interest in a more involved in vivo basedwork that was beyond our means

Our results indicate that normal human mammaryepithelial cells have an active transsulfuration pathway thatsignificantly contributes to glutathione production Theincorporation of 35S-methionine into glutathione seen inFigure 2 definitively indicates that transsulfuration is activein HMECrsquos as the radioactive glutathione can only deriveif transsulfuration converts the methionine ultimately tocysteine Note that mass spectroscopy confirmed the iden-tity of the radioactive fluorescent band in the thin layerchromatography analysis The impact of the transsulfura-tion inhibitor PPG in Figure 3 where total glutathione ismeasured enzymatically also indicates that transsulfurationinhibition leads to lower levels of glutathione implying boththe existence of active transsulfuration and an estimate of itscontribution to normal glutathione levels

When cells are oxidatively challenged treatment withthe transsulfuration inhibitor PPG significantly reduces glu-tathione levels While an interesting observation in its ownright these findings imply that oxidant stress in breast tissuemay lead to changes in levels of methionine cycle inter-mediates such as the methyl donor S-adenosylmethioninewhich may result from homocysteine exiting the methioninecycle to replenish glutathione Because changes in the S-adenosylmethionine methyl donor pool have the potentialto impact on DNA methylation this discovery provides apossible tie between oxidant stress events and the epigeneticregulation of genes involved in disease states While sucha linkage has been reported for liver cells [55] and theepigenetic implications have been discussed [56] our study

ISRN Biochemistry 5

is the first to explore this area with regard to mammarycells We feel that the discovery of active transsulfuration inmammary cells is thus a highly significant finding given thatthis identifies ametabolicmechanism throughwhich oxidantstressmay be linked to the etiology of chronic diseases such asbreast cancer via the potential for changes in the epigeneticregulation of genes Additionally the method we employedto analyze transsulfuration provides a straightforward andeconomical means for such exploration into other tissues

Because transsulfuration pathwaymethionine cycle con-stituents can be impacted by nutritional interventions thatalter vitamin B6 [57] choline and cysteine [58] or methio-nine [59] our findings imply that in vivo dietary modalitiesmay ultimately be designed to attenuate the potential epige-netic impact of oxidant stress This may also contribute toour understanding of how diets rich in heme iron whichcould be a strong source of dietary oxidant stress may belinked to breast carcinogenesis Of great interest shouldthese potential relationships be demonstrated would becharacterizing the ability of individual polymorphisms ofmethionine cycletranssulfuration enzymes to influence thisprocess

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

This work was supported by pilot funding from 1 P20RR20180 from the NIHNCRR and by Grant 1R21CA133701-01A2 from the NIHNCI

References

[1] S C Lu M L Martinez-Chantar and J M Mato ldquoMethionineadenosyltransferase and S-adenosylmethionine in alcoholicliver diseaserdquo Journal of Gastroenterology and Hepatology vol21 supplement 3 pp S61ndashS64 2006

[2] S Ratnam K N Maclean R L Jacobs M E Brosnan J PKraus and J T Brosnan ldquoHormonal regulation of cystathionine120573-synthase expression in liverrdquo Journal of Biological Chemistryvol 277 no 45 pp 42912ndash42918 2002

[3] R Banerjee R Evande O Kabil S Ojha and S TaokaldquoReaction mechanism and regulation of cystathionine beta-synthaserdquo Biochimica et Biophysica Acta vol 1647 no 1-2 pp30ndash35 2003

[4] S J James S Melnyk S Jernigan et al ldquoMetabolic endopheno-type and related genotypes are associated with oxidative stressin children with autismrdquo American Journal of Medical GeneticsB vol 141 no 8 pp 947ndash956 2006

[5] S Jill James S Melnyk S Jernigan A Hubanks S Rose andD W Gaylor ldquoAbnormal transmethylationtranssulfurationmetabolism and DNA hypomethylation among parents ofchildren with autismrdquo Journal of Autism and DevelopmentalDisorders vol 38 no 10 pp 1966ndash1975 2008

[6] D A Geier J K Kern C R Garver J B Adams T Audhya andMRGeier ldquoAprospective study of transsulfuration biomarkersin autistic disordersrdquoNeurochemical Research vol 34 no 2 pp386ndash393 2009

[7] D A Geier J K Kern C R Garver et al ldquoBiomarkers ofenvironmental toxicity and susceptibility in autismrdquo Journal ofthe Neurological Sciences vol 280 no 1-2 pp 101ndash108 2009

[8] S J James S Melnyk G Fuchs et al ldquoEfficacy of methylcobal-amin and folinic acid treatment on glutathione redox status inchildren with autismrdquo American Journal of Clinical Nutritionvol 89 no 1 pp 425ndash430 2009

[9] J H Horowitz E B Rypins and J M Henderson ldquoEvidencefor impairment of transsulfuration pathway in cirrhosisrdquo Gas-troenterology vol 81 no 4 pp 668ndash675 1981

[10] C Loguercio G Nardi G Prota C Del Vecchio Blanco andMColtorti ldquoDecrease of total glutathione and cysteine SH in non-alcoholic cirrhosisrdquo Italian Journal of Gastroenterology vol 22no 1 pp 13ndash15 1990

[11] G Marchesini E Bugianesi G Bianchi et al ldquoDefectivemethionine metabolism in cirrhosis relation to severity of liverdiseaserdquo Hepatology vol 16 no 1 pp 149ndash155 1992

[12] M P Look R Riezler C Reichel et al ldquoIs the increase inserum cystathionine levels in patients with liver cirrhosis a con-sequence of impaired homocysteine transsulfuration at the levelof 120574-cystathionaserdquo Scandinavian Journal of Gastroenterologyvol 35 no 8 pp 866ndash872 2000

[13] G Bianchi M Brizi B Rossi M Ronchi G Grossi and GMarchesini ldquoSynthesis of glutathione in response to methio-nine load in control subjects and in patients with cirrhosisrdquoMetabolism Clinical and Experimental vol 49 no 11 pp 1434ndash1439 2000

[14] K Robinson EMayer andDW Jacobsen ldquoHomocysteine andcoronary artery diseaserdquo Cleveland Clinic Journal of Medicinevol 61 no 6 pp 438ndash450 1994

[15] N P B Dudman X W Guo R B Gordon P A Dawsonand D E L Wilcken ldquoHuman homocysteine catabolism threemajor pathways and their relevance to development of arterialocclusive diseaserdquo Journal of Nutrition vol 126 supplement 4pp 1295Sndash1300S 1996

[16] P Verhoef M J Stampfer J E Buring et al ldquoHomocysteinemetabolism and risk of myocardial infarction relation withvitamins B6 B12 and folaterdquoAmerican Journal of Epidemiologyvol 143 no 9 pp 845ndash859 1996

[17] S M Saw ldquoHomocysteine and atherosclerotic disease theepidemiologic evidencerdquo Annals of the Academy of MedicineSingapore vol 28 no 4 pp 565ndash568 1999

[18] P DurandM Prost N Loreau S Lussier-Cacan andD BlacheldquoImpaired homocysteine metabolism and atherothromboticdiseaserdquo Laboratory Investigation vol 81 no 5 pp 645ndash6722001

[19] Y Ingenbleek D Barclay and H Dirren ldquoNutritional signifi-cance of alterations in serum amino acid patterns in goitrouspatientsrdquo American Journal of Clinical Nutrition vol 43 no 2pp 310ndash319 1986

[20] G Palareti S Salardi and S Piazzi ldquoBlood coagulation changesin homocystinuria effects of pyridoxine and other specifictherapyrdquo Journal of Pediatrics vol 109 no 6 pp 1001ndash1006 1986

[21] G Palareti and S Coccheri ldquoLowered antithrombin III activityand other clotting changes in homocystinuria effects of apyridoxine-folate regimenrdquo Haemostasis vol 19 supplement 1pp 24ndash28 1989

[22] MM Eldibany and J A Caprini ldquoHyperhomocysteinemia andthrombosis an overviewrdquo Archives of Pathology and LaboratoryMedicine vol 131 no 6 pp 872ndash884 2007

6 ISRN Biochemistry

[23] S Garg V Vitvitsky H E Gendelman and R BanerjeeldquoMonocyte differentiation activation andmycobacterial killingare linked to transsulfuration-dependent redox metabolismrdquoJournal of Biological Chemistry vol 281 no 50 pp 38712ndash387202006

[24] K Wisniewski J A Sturman and E Devine ldquoCystathioninedisappearance with neuronal loss a possible neuronal markerrdquoNeuropediatrics vol 16 no 3 pp 126ndash130 1985

[25] F Tchantchou ldquoHomocysteine increase folate oxidative brainhomocysteine metabolism and various consequences of folatedeficiencyrdquo Journal of Alzheimerrsquos Disease vol 9 no 4 pp 421ndash427 2006

[26] N Vatanavicharn B D Pressman and W R WilcoxldquoReversible leukoencephalopathy with acute neurological dete-rioration and permanent residua in classical homocystinuria acase reportrdquo Journal of Inherited Metabolic Disease 2007

[27] SH RahmanA R Srinivasan andANicolaou ldquoTranssulfura-tion pathway defects and increased glutathione degradation insevere acute pancreatitisrdquo Digestive Diseases and Sciences vol54 no 3 pp 675ndash682 2009

[28] V Vitvitsky M Thomas A Ghorpade H E Gendelman andR Banerjee ldquoA functional transsulfuration pathway in thebrain links to glutathione homeostasisrdquo Journal of BiologicalChemistry vol 281 no 47 pp 35785ndash35793 2006

[29] J A Sturman N G Beratis L Guarini and G E GaullldquoTranssulfuration by human long term lymphoid lines Normaland cystathionase-deficient cellsrdquo Journal of Biological Chem-istry vol 255 no 10 pp 4763ndash4765 1980

[30] S H Mudd J D Finkelstein F Irreverre and L LasterldquoTranssulfuration in mammals Microassays and tissue distri-butions of three enzymes of the pathwayrdquo Journal of BiologicalChemistry vol 240 no 11 pp 4382ndash4392 1965

[31] A Ronco E De Stefani M Mendilaharsu and H Deneo-Pellegrini ldquoMeat fat and risk of breast cancer a case-controlstudy from Uruguayrdquo International Journal of Cancer vol 65no 3 pp 328ndash331 1996

[32] E F Taylor V J Burley D C Greenwood and J E Cade ldquoMeatconsumption and risk of breast cancer in the UK WomenrsquosCohort Studyrdquo British Journal of Cancer vol 96 no 7 pp 1139ndash1146 2007

[33] E Cho W Y Chen D J Hunter et al ldquoRed meat intake andrisk of breast cancer among premenopausal womenrdquo Archivesof Internal Medicine vol 166 no 20 pp 2253ndash2259 2006

[34] E Linos W C Willett E Cho G Colditz and L AFrazier ldquoRed meat consumption during adolescence amongpremenopausal women and risk of breast cancerrdquo CancerEpidemiology Biomarkers and Prevention vol 17 no 8 pp 2146ndash2151 2008

[35] S E Steck M M Gaudet S M Eng et al ldquoCooked meatand risk of breast cancermdashlifetime versus recent dietary intakerdquoEpidemiology vol 18 no 3 pp 373ndash382 2007

[36] G C Kabat and T E Rohan ldquoDoes excess iron play a rolein breast carcinogenesis An unresolved hypothesisrdquo CancerCauses and Control vol 18 no 10 pp 1047ndash1053 2007

[37] H Takkunen and R Seppanen ldquoIron deficiency and dietaryfactors in Finlandrdquo American Journal of Clinical Nutrition vol28 no 10 pp 1141ndash1147 1975

[38] E Bjorn Rasmussen L Hallberg B Isaksson and B ArvidssonldquoFood iron absorption in man Applications of the two poolextrinsic tag method to measure heme and nonheme ironabsorption from the whole dietrdquo Journal of Clinical Investiga-tion vol 53 no 1 pp 247ndash255 1974

[39] R G Dumitrescu and P G Shields ldquoThe etiology of alcohol-induced breast cancerrdquoAlcohol vol 35 no 3 pp 213ndash225 2005

[40] V Bagnardi M Blangiardo C L Vecchia and G CorraoldquoA meta-analysis of alcohol drinking and cancer riskrdquo BritishJournal of Cancer vol 85 no 11 pp 1700ndash1705 2001

[41] Collaborative Group on Hormonal Factors in Breast CancerldquoAlcohol tobacco and breast cancermdashcollaborative reanalysis ofindividual data from 53 epidemiological studies including 58515 women with breast cancer and 95 067 women without thediseaserdquo British Journal of Cancer vol 87 no 11 pp 1234ndash12452002

[42] R Suzuki N Orsini L Mignone S Saji and A Wolk ldquoAlcoholintake and risk of breast cancer defined by estrogen and pro-gesterone receptor statusmdashameta-analysis of epidemiologicalstudiesrdquo International Journal of Cancer vol 122 no 8 pp 1832ndash1841 2008

[43] H Kamencic A Lyon P G Paterson and B H J JuurlinkldquoMonochlorobimane fluorometric method to measure tissueglutathionerdquo Analytical Biochemistry vol 286 no 1 pp 35ndash372000

[44] J C Fernandez-Checa and N Kaplowitz ldquoThe use of mono-chlorobimane to determine hepatic GSH levels and synthesisrdquoAnalytical Biochemistry vol 190 no 2 pp 212ndash219 1990

[45] O W Griffith ldquoDetermination of glutathione and glutathionedisulfide using glutathione reductase and 2-vinylpyridinerdquoAnalytical Biochemistry vol 106 no 1 pp 207ndash212 1980

[46] M E Anderson ldquoDetermination of glutathione and glutathionedisulfide in biological samplesrdquoMethods in Enzymology vol 113pp 548ndash555 1985

[47] M Valentovic M K Meadows R C Harmon J G Ball S KHong and G O Rankin ldquo2-Amino-5-chlorophenol toxicity inrenal cortical slices from Fischer 344 rats effect of antioxidantsand sulfhydryl agentsrdquo Toxicology and Applied Pharmacologyvol 161 no 1 pp 1ndash9 1999

[48] M A Valentovic J G Ball H Sun and G O Rankin ldquoChara-cterization of 2-amino-45-dichlorophenol (2A45CP) in vitrotoxicity in renal cortical slices from male Fischer 344 ratsrdquoToxicology vol 172 no 2 pp 113ndash123 2002

[49] M Valentovic M Terneus R C Harmon and A B Carpen-ter ldquoS-Adenosylmethionine (SAMe) attenuates acetaminophenhepatotoxicity in C57BL6micerdquoToxicology Letters vol 154 no3 pp 165ndash174 2004

[50] R C Harmon M V Terneus K K Kiningham and MValentovic ldquoTime-dependent effect of p-Aminophenol (PAP)toxicity in renal slices and development of oxidative stressrdquoToxicology and Applied Pharmacology vol 209 no 1 pp 86ndash942005

[51] R C Harmon K K Kiningham and M A ValentovicldquoPyruvate reduces 4-aminophenol in vitro toxicityrdquo Toxicologyand Applied Pharmacology vol 213 no 2 pp 179ndash186 2006

[52] M V Terneus K K Kiningham A B Carpenter S BSullivan and M A Valentovic ldquoComparison of S-adenosyl-L-methionine and N-acetylcysteine protective effects onacetaminophen hepatic toxicityrdquo Journal of Pharmacology andExperimental Therapeutics vol 320 no 1 pp 99ndash107 2007

[53] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[54] L Bao C Vlcek V Paces and J P Kraus ldquoIdentification andtissue distribution of human cystathionine 120573-synthase mRNA

ISRN Biochemistry 7

isoformsrdquo Archives of Biochemistry and Biophysics vol 350 no1 pp 95ndash103 1998

[55] K Lertratanangkoon C J Wu N Savaraj and M L ThomasldquoAlterations of DNA methylation by glutathione depletionrdquoCancer Letters vol 120 no 2 pp 149ndash156 1997

[56] E Mosharov M R Cranford and R Banerjee ldquoThe quan-titatively important relationship between homocysteinemetabolism and glutathione synthesis by the transsulfurationpathway and its regulation by redox changesrdquo Biochemistryvol 39 no 42 pp 13005ndash13011 2000

[57] R C Bakker and D P M Brandjes ldquoHyperhomocysteinaemiaand associated diseaserdquoPharmacyWorld and Science vol 19 no3 pp 126ndash132 1997

[58] C P Lima S R Davis A DMackey J B Scheer J Williamsonand J F Gregory ldquoVitamin B-6 deficiency suppresses thehepatic transsulfuration pathway but increases glutathioneconcentration in rats fed AIN-76A or AIN-93G dietsrdquo Journalof Nutrition vol 136 no 8 pp 2141ndash2147 2006

[59] B Tang A Mustafa S Gupta S Melnyk S J Jamesand W D Kruger ldquoMethionine-deficient diet induces post-transcriptional downregulation of cystathionine 120573-synthaserdquoNutrition vol 26 no 11-12 pp 1170ndash1175 2010

Submit your manuscripts athttpwwwhindawicom

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2 ISRN Biochemistry

Methionine

MS

MATS-Adenosylmethionine

S-AdenosylhomocysteineHomocysteine

Cystathionine

CBS

Propargylglycine Gamma-cystathionase

Gamma-glutamylcysteine synthase

Cysteine

Gamma-glutamylcysteine

Glutathione synthase

Glutathione

Buthionine sulfoximine

Figure 1 The transsulfuration pathway connects methionine andglutathione biosynthesis In themethionine cyclemethionine formsS-adenosylmethionine which serves as a methyl donor generatingS-adenosyl homocysteineThis is converted to homocysteine whichis subsequently converted back into methionine Homocysteine hasan alternative fate however It can be used to produce cystathioninewhich is further converted to cysteine This latter conversion is cat-alyzed by gamma-cystathionase and inhibited by propargylglycineCysteine can then feed glutathione biosynthesis through productionof gamma-glutamylcysteine This step is catalyzed by gamma-glutamylcysteine synthase and inhibited by buthionine sulfoximine

stress for chronic diseases of mammary tissue such as breastcancer Based on our results obtained using a radioactivetracer method involving 35S-methionine incorporation intoglutathione we report that the transsulfuration pathway isactive in human mammary epithelial cells Further workemploying pathway inhibitors while measuring glutathioneproduction normally and during oxidant challenge indicatestranssulfuration provides necessary sulfur for the enhancedcysteine production that is required for these cells to respondto oxidative stress

2 Materials and Methods

21 Human Mammary Epithelial Cell Culture In order toapply the power of a radioactive tracer method to test ourhypothesis while still avoiding as many of the inherentbiases that can result from alterations in cellular biochemistryacquired by tissue culture lines as feasible we used a primaryhuman mammary epithelial cell (HMEC) model systemThese cells were obtained from Lonza (Walkersville MD)and are neither transformed nor immortalized They arecapable of undergoing a limited number of divisions andmust be maintained at less than maximum confluence toavoid triggering senescence Human mammary epithelialcells (HMEC) were grown and passaged under standardconditions in tissue culture treated plasticware (Corning

Lowell MA) at 37∘C at high humidity in water jacketed CO2

incubators (Fisher Scientific Thermo Forma) according tothe distributors instructions changing culturemedium everytwo days with standard medium prepared using their propri-etary reagents (Lonza Walkersville MD) Standard growthmedia for HMEC was mammary epithelial basal medium(MEBM) supplemented with growth factors supplied inaliquots at proprietary concentrations which include (for a500mL bottle) 2mL of bovine pituitary extract 05mL ofepidermal growth factor 05mL of insulin 05mL of hydro-cortisone and 05mL of gentamicin sulfateamphotericinB To prepare experimental cells 80 confluent T75 flaskswere split and used to seed T75 flasks each with 2E5 cellsin 15mL of standard medium Seven days after seeding(medium changed every two days) they were 50 confluentand experimental treatment began

22 Transsulfuration Assay Cells were pretreated for 24hours with standard media containing either 9mM buthio-nine sulfoximine (BSO) 25mM propargylglycine (PPG)or a PBS vehicle control Then the medium was replacedwith 10mL of radioactive labeling medium (standard mediacontaining the appropriate inhibitors and 35S methionine at25 120583CimL activity) Cells were incubated for 24 hours andthen pellets were harvested through trypsinization Pelletswere rinsed with PBS and frozen and stored briefly at minus80∘C

To fluorescently label cellular glutathione in cell lysateswe adapted the method of Kamencic et al [43] Monochloro-bimane (mCBi) is reported to specifically react with glu-tathione (a reaction catalyzed by GST) and not other cellularthiols [44] Cell pellets were removed from the freezer anequal volume of water was added and pellets were thawedand frozen an additional 3 times following each thaw pelletswere vortexed at high speed The supernatant with cytosoliccontents was isolated by spinning the cells at 4∘C and15000 rpm for 15 minutes Pellets were reextracted twice andall supernatants pooled for each sampleThe reactionmixtureto fluorescently label GSH consisted of 100120583L of cell lysate2 120583L of mCBi 20mM 20120583L of 500mM potassium phosphate(K2PO4) pH 65 and 25 120583g of GST (20120583L of 25120583g GST

dissolved in 100 120583L of PBS) in a total reaction volume of200120583L (water is used to equalize samples) Reactions wereprepared on ice then initiated by incubation at 37∘C for aperiod of 10 minutes Reactions were stopped by freezing Acontrol reaction was run by substituting the cell lysate with2 120583L of 10mM GSH in the same reaction volume of 200120583L10 120583L of each reaction mixture were spotted on 250 micronsilica gel GF uniplates (Analtech Newark DE) and analyzedby TLC using a 3 1 1 mixture of 1-butanol methanol water(solvents from Fisher Scientific Chicago IL) Two 120583L of20mM mCBi were run in a separate lane as a control toindicate the migration of nonreacted mCBi Migrations offluorescent products were compared under UV light andrecorded using a Bio-Rad universal hood digital camera(Bio-Rad laboratories Inc Segrate Milan Italy) Massspectroscopic analysis confirmed the identity of the GSH-Bimane TLC band (data not shown) Autoradiographs ofplates were prepared using Kodak BioMax light film (Sigma

ISRN Biochemistry 3



2O2for two hours


3 Results





2O2for two hours




4 Discussion


4 ISRN Biochemistry


Free MCBi

GSH-Bi



Tota

l glu

tath

ione

(mea

n nm

oles

mg

prot

einplusmn

SEM

)

50

40

30

20

10


2O2

for 2 h

lowast

lowast

PBSPPG






2O2and PPGH


by ANOVA (119875 lt 0002)






ISRN Biochemistry 5





Acknowledgments


References























6 ISRN Biochemistry

































ISRN Biochemistry 7














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biochemistry 3



2O2for two hours


3 Results





2O2for two hours




4 Discussion


4 ISRN Biochemistry


Free MCBi

GSH-Bi



Tota

l glu

tath

ione

(mea

n nm

oles

mg

prot

einplusmn

SEM

)

50

40

30

20

10


2O2

for 2 h

lowast

lowast

PBSPPG






2O2and PPGH


by ANOVA (119875 lt 0002)






ISRN Biochemistry 5





Acknowledgments


References























6 ISRN Biochemistry

































ISRN Biochemistry 7














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

4 ISRN Biochemistry


Free MCBi

GSH-Bi



Tota

l glu

tath

ione

(mea

n nm

oles

mg

prot

einplusmn

SEM

)

50

40

30

20

10


2O2

for 2 h

lowast

lowast

PBSPPG






2O2and PPGH


by ANOVA (119875 lt 0002)






ISRN Biochemistry 5





Acknowledgments


References























6 ISRN Biochemistry

































ISRN Biochemistry 7














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biochemistry 5





Acknowledgments


References























6 ISRN Biochemistry

































ISRN Biochemistry 7














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

6 ISRN Biochemistry

































ISRN Biochemistry 7














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

ISRN Biochemistry 7














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Transsulfuration Is a Significant Source of Sulfur for ...downloads.hindawi.com/journals/isrn.biochemistry/2013/637897.pdfMAT S-Adenosylmethionine Homocysteine S-Adenosylhomocysteine

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