Evaluation of 8-Hydroxy-2'- Deoxyguanosine Concentration and Antioxidant Enzyme Activities in Bladder Cancer Patients

Transcription

1 ORİJİNAL ARAŞTIRMA Evaluation of 8-Hydroxy-2'- Deoxyguanosine Concentration and Antioxidant Enzyme Activities in Bladder Cancer Patients Yıldız DİNÇER, MD, a Tülay AKÇAY, a Ali Rıza KURAL, MD, b Süleyman ATAUS, MD, b Eylem E. KOÇ, MD, a Sinharib ÇİTGEZ, MD, b Mustafa Bilal TUNA, MD b Departments of a Biochemistry, b Urology, İstanbul University Cerrahpaşa Faculty of Medicine, İstanbul Ge liş Ta ri hi/re ce i ved: Ka bul Ta ri hi/ac cep ted: Ya zış ma Ad re si/cor res pon den ce: Yıldız DİNÇER, MD İstanbul University Cerrahpaşa Faculty of Medicine, Department of Biochemistry, İstanbul, TÜRKİYE/TURKEY stare63@yahoo.com ABS TRACT Ob jec ti ve: Oxi dant/an ti o xi dant ba lan ce has be en sug ges ted as an im por tant fac tor for ini ti a ti on and prog res si on of can cer. The pur po se of this study was to exa mi ne 8-hydroxy-2 -de - oxy gu a no si ne (8-OHdG) le vel which is a mar ker of oxi da ti ve DNA da ma ge, su pe ro xi de dis mu ta se (SOD), glu tat hi o ne pe ro xi da se (GPx) and glu tat hi o ne S-trans fe ra se (GST) ac ti vi ti es as an ti o xi dant enz ymes, in se rum of uri nary blad der can cer, and to de ter mi ne re la ti ons bet we en me a su red pa ra - me ters and tu mor cha rac te ris tics such as his to lo gi cal gra de, lo cal in va si on and tu mor si ze. Ma te ri - al and Met hods: Forty pa ti ents with uri nary blad der can cer we re inc lu ded in the study. Blo od samp les we re col lec ted just be fo re the re sec ti on. Se rum le vels of 8-OHdG we re me a su red with a com pe ti ti ve ELI SA kit, SOD and GPx ac ti vi ti es we re me a su red by spec trop ho to met ric kits, GST ac ti vity was de ter mi ned by spec trop ho to met ric as say. Re sults: The re was no sig ni fi cant dif fe ren ce bet we en pa ti ent and con trol gro ups for se rum 8-OHdG le vel and GPx ac ti vity. Ho we ver se rum SOD ac ti vity was sig ni fi cantly lo wer (P< 0.001) and GST ac ti vity was sig ni fi cantly hig her (P< 0.001) in the pa ti ent gro up as com pa red to con trol gro up. Tu mor si ze fo und to be ne ga ti vely cor re la ted with GST ac ti vity (r: -0.43, P< 0.01). Conc lu si on: Da ta show that se rum le vel of 8-OHdG does not have a prog nos tic po ten ti al for uri nary blad der can cer. An ti o xi dant ba lan ce is dis tur bed in the se pati ents however chan ges in an ti o xi dant enz yme ac ti vi ti es does not have a prog nos tic va lu e. The most pro mi sing an ti o xi dant enz yme is GST be ca u se of its ne ga ti ve as so ci a ti on with tu mor si ze. Key Words: Glu tat hi o ne pe ro xi da se; su pe ro xi de dis mu ta se; glu tat hi o ne trans fe ra se; 8-hydroxy-2 -de oxy gu a no si ne; uri nary blad der ne op lasms ÖZET Amaç: Ok si dan/an ti ok si dan den ge nin kan ser olu şu mu ve iler le me sin de önem li bir fak tör ol du ğu ile ri sü rül mek te dir. Bu ça lış ma nın ama cı me sa ne kan ser li has ta lar da se rum da DNA ok si - das yo nu be lir te ci olan 8-hid rok si-2 -de ok si gu a no zin (8-OHdG) dü ze yi ile an ti ok si dan ak ti vi te ola rak sü pe rok sit dis mu taz (SOD), glu tat yon pe rok si daz (GPx) ve glu tat yon S-trans fe raz (GST) ak ti vi te le ri ni ölç mek ve bu pa ra met re le rin his to lo jik gra de, lo kal in vaz yon, tü mör bü yük lü ğü gi - bi tü mör ka rak te ris tik le ri ile iliş ki le ri ni be lir le mek tir. Ge reç ve Yön tem ler: Me sa ne kan ser li 40 has ta ça lış ma kap sa mı na alın dı. Kan ör nek le ri ame li ya tın he men ön ce sin de top lan dı. Se rum 8- OHdG dü zey le ri ya rış ma lı ELI SA ki ti, SOD ve GPx ak ti vi te le ri spek tro fo to met rik kit ler le, GST ak - ti vi te si spek tro fo to met rik yön tem le be lir len di. Bul gu lar: Has ta ve kon trol grup la rı ara sın da se rum 8-OHdG dü ze yi ve GPx ak ti vi te si ba kı mın dan an lam lı fark yok tu. An cak, kon trol gru bu ile kar - şı laş tı rıl dı ğın da has ta gru bun da SOD ak ti vi te si an lam lı ola rak dü şük (P< 0.001), GST ak ti vi te si an lam lı ola rak yük sek ti (P< 0.001). Tü mör bü yük lü ğü GST ak ti vi te si ile ne ga tif ko re le ola rak bu - lun du (r: -0.43, P< 0.01). So nuç: Bul gu lar me sa ne kan ser li has ta lar da se rum 8-OHdG dü ze yi nin prog nos tik bir öne mi ol ma dı ğı nı gös ter mek te dir. Bu has ta lar da an ti ok si dan den ge bo zul muş tur fa - kat an ti ok si dan en zim ak ti vi te le rin de ki de ği şik lik le rin prog nos tik bir de ğe ri yok tur. Tü mör bü - yük lü ğü ile ne ga tif ko re las yon gös ter me si ne de niy le bu ko nu da en umut ve ri ci an ti ok si dan en zim GST dır. Anah tar Ke li me ler: Glu tat yon pe rok si daz; sü pe rok sid dis mu taz; glu tat yon trans fe raz; 8-hid rok si-2 -de ok si gu a no zin; me sa ne tü mör le ri doi: /medsci Cop yright 2011 by Tür ki ye Kli nik le ri Turkiye Klinikleri J Med Sci 2011;31(3):553-8 Turkiye Klinikleri J Med Sci 2011;31(3) 553

2 Dinçer ve ark. e cent stu di es ha ve de mons tra ted the ro le of oxy gen fre e ra di cals (OFR) in car ci no ge ne - sis. By in te rac ting with ge no mic DNA, OFR have be en sug ges ted to da ma ge spe ci fic ge nes which con trol cell growth and dif fe ren ti a ti on, 1 incre a se the ac ti vity of car ci no ge nic xe no bi o tics, 2 and sti mu la te fas ter growth of ma lig nant cells. 3 OFR are highly re ac ti ve mo le cu les be ca u se of un pa i red electrons on the ex te ri or or bi tals. As a re sult of at tacks by OFR to DNA many types of oxi di zed nuc le o si - de ha ve be en de ter mi ned. 8-hydroxy-2 -de oxy gu - a no si ne (8-OHdG) is the most fre qu ently de tec ted and stu di ed oxi da ti ve DNA le si on. 8-OHdG has a pro-mu ta ge nic po ten ti al since it mispairs with A re si du es and le a ds to an in cre a sed fre qu ency of spon ta ne o us G:C T:A trans ver si on. This mu ta ti - on is ge ne rally ob ser ved in mu ta ted pro to on co ge - nes and tu mor sup pres sor ge nes. 4 Upon DNA re pa ir, 8-OHdG is ex cre ted in the uri ne. Se rum or uri nary 8-OHdG le vel is con si de red as a bi o mar ker of ge ne ra li zed cel lu lar oxi da ti ve stress and is lin - ked to de ge ne ra ti ve di se a ses inc lu ding can cer. 5 De fen se aga inst OFR is pro vi ded by a system of enz ymes and an ti o xi dant com po unds ca pab le of pre ven ting ex cess OFR pro duc ti on and ne ut ra li sing OFR. Su pe ro xi de dis mu ta se (SOD) and glu tat hi o ne pe ro xi da se (GPx) are enz yma tic an ti o xi dants which ca taly ze the de to xi fi ca ti on of su pe ro xi de ani on (O 2 ) and hydro gen pe ro xi de (H 2 O 2 ), res pec ti vely. 6 Glu tat hi o ne S-trans fe ra ses (GST) con ju ga te glu tat - hi o ne to va ri o us po ten ti ally car ci no ge nic com po - unds, fa ci li ta ting the ir eli mi na ti on from the body. Alt ho ugh GPx, SOD and GST are cel lu lar enz ymes that are ava i lab le in the plas ma at a de tec tab le le - vel. 7-9 in cre a sed oxi da ti ve stress un der pat ho lo gi cal cir cums tan ces may ex ha ust the an ti o xi dant ca pa - bi lity of cells and as a re sult, the sus cep ti bi lity of tar get mo le cu les to oxi da ti ve stress in cre a ses. Oxi - dant/an ti o xi dant ba lan ce has be en sug ges ted as an im por tant fac tor for ini ti a ti on and prog res si on of can cer. 10 We ai med to exa mi ne 8-OHdG le vel, SOD, GPx and GST ac ti vi ti es in se rum of uri nary blad der can cer, and to de ter mi ne re la ti ons bet we - en me a su red pa ra me ters and tu mor cha rac te ris tics such as his to lo gi cal gra de, lo cal in va si on and tu mor si ze. We hypot he si zed that se rum le vel of 8-OHdG and ac ti vi tes of the se enz ymes may be prog nos tic mar kers which can be ea sily ac ces sib le for uri nary blad der can cer. MA TE RI AL AND MET HODS Tıbbi Biyokimya SUB JECTS A to tal of 40 pa ti ent who ad mit ted to Is tan bul Univer sity, Cer rah pa sa Me di cal Fa culty, De part ment of Uro logy we re inc lu ded in the study (Tab le 1). Cerrah pa sa Me di cal Fa culty Et hi cal Com mit te e ap pro - val was ta ken in ac cor dan ce with the prin cip les of Dec la ra ti on of Hel sin ki and in for med con sent was ob ta i ned from the ca ses. Uri nary blad der can cer was di ag no sed ac cor ding to pat ho logy re ports. No ne of the pa ti ents had un der go ne any pre vi o us tre at ments such as che mo te rapy or sur gery. The con trol gro up was cons ti tu ted by 21 he althy vo lun te ers. Both pati ents and con trols we re euth yro id and had nor mal li ver and re nal func ti ons. Sub jects with chro nic inf lam ma tory di se a ses we re exc lu ded. Sub jects who had any inf lam ma tory con di ti on or in fec ti o us di se - a se, and who we re re ce i ving drugs ca pab le of in ter - fe ring with oxi dant/an ti o xi dant system in pre vi o us six months we re exc lu ded from the study. LA BO RA TORY ME A SU RE MENTS Eight ml of ve no us blo od samp le was col lec ted befo re the ope ra ti on. Fol lo wing cen tri fu ga ti on at 2000 X g for 10 mi nu tes, se rum was re mo ved and kept at the-80 0 C until the ti me of analy sis. Se rum le vel of TABLE 1: Patient and control characteristics. Urinary bladder cancer Control group group (n= 40) (n= 21) Mean age (years) 65 ± ± 9 Gender Male Female 10 7 Smoking (%) Tumor Grade High (%) 52 Low (%) 48 Local invasion (%) 52 Metastasis (%) 5 Tumor size (cm) 2.5 ± Turkiye Klinikleri J Med Sci 2011;31(3)

3 Medical Biochemistry 8-OHdG was me a su red with a com pe ti ti ve ELI SA kit ob ta i ned from Oxis (Port land, OR; USA). 11 Ac ti - vity of SOD and GPx in se rum we re me a su red by spec trop ho to met ric kits from Ran dox (Crum lin, UK). 12,13 GST ac ti vity was de ter mi ned ac cor ding to the met hod of Ha big et al. 14 using 1-chlo ro, 2.4 di - nit ro ben ze ne as subs tra te. For ma ti on of the S-con - ju ga te was fol lo wed by its ab sor ban ce at 340 nm. STA TIS TI CAL ANALY SIS The re sults of 8-OHdG le vel, SOD, GPx and GST ac ti vi ti es are ex pres sed as me an ± SD (stan dard devi a ti on of the me an). Sin ce ma jo rity of da ta we re not nor mally dis tri bu ted, sta tis ti cal analy sis of me - a su red pa ra me ters we re per for med by non pa ra - met ric Mann Whit ney U test. Dif fe ren ces bet we en gro ups we re con si de red sig ni fi cant at P< Spe - ar man cor re la ti on co ef fi ci ent was used for cor re la - ti on analy sis. RE SULTS The re was no sig ni fi cant dif fe ren ce bet we en pa ti - ent and con trol gro ups for se rum 8-OHdG le vel and GPx ac ti vity. Alt ho ugh 8-OHdG le vel was hig - her in the pa ti ent gro up, it did not re ach a sig ni fi - cant le vel. Ho we ver, se rum SOD ac ti vity was sig ni fi cantly lo wer (P< 0.001) and GST ac ti vity was sig ni fi cantly hig her (P< 0.001) in the pa ti ent gro up as com pa red to the con trol gro up (Tab le 2). When the pa ti ent gro up was di vi ded in to sub gro ups with res pect to lo cal in va si on, GST ac ti vity was fo und to be lo wer in the pre sen ce of lo cal in va si on (Tab le 3). With res pect to the his to lo gi cal gra de of tu mor, no sig ni fi cant dif fe ren ce was fo und bet we en low gra de tu mor gro up and high gra de tu mor gro up for any pa ra me ter (Tab le 4). Sin ce a to tal of two pa ti - TABLE 2: Measured parameters in the study groups. Control group Urinary bladder cancer (n= 21) group (n= 40) 8-OHdG (ng/ml) 3.08 ± ± 1.99 SOD (U/ml) 0.53 ± ± 0.19 a G-Px (U/L) 2.44 ± ± 0.62 GST (mu/l) 50 ± ± 52a a P< versus control. Dinçer et al TABLE 3: Measured parameters in the patient group with respect to the local invasion. Local invasion (+) Local invasion (-) group (n= 21) group (n= 19) 8-OHdG (ng/ml) 3.09 ± ± 0.24 SOD (U/ml) 0.38 ± ± 0.16 G-Px (U/L) 2.19 ± ± 0.41 GST (mu/l) 78 ± ± 67 a a P< 0.01 versus control. TABLE 4: Measured parameters in the patient group with respect to the tumor grade. High grade Low grade (n= 21) group (n= 19) 8-OHdG (ng/ml) 3.00 ± ± 2.44 SOD (U/ml) 0.37 ± ± 0.16 G-Px (U/L) 2.22 ± ± 0.42 GST (mu/l) 90 ± ± 56 ents had dis tant me tas ta ses, me a su red pa ra me ters we re not analyzed with res pect to me tas ta sis. The re la ti ons of me a su red pa ra me ters (8-OHdG, SOD, GPx, and GST) and tu mor gra de and tu mor si ze we - re exa mi ned. Tu mor si ze fo und to be ne ga ti vely cor re la ted with GST ac ti vity (r: -0.43, P< 0.01). DIS CUS SI ON In ag re e ment with the hypot he sis that oxi da ti ve stress is lin ked to can cer, 15 it was previously shown that 8-OHdG le vel in uri nary blad der tu mors was shown to be sig ni fi cantly hig her when compared to ne igh bo ring non-can ce ro us tis su es OHdG le v- els we re al so fo und to be hig her in le u kocy tes 17 of pa ti ents with uri nary blad der can cer. In the se previ o us stu di es, se rum le vel of 8-OHdG was not me - a su red and the re la ti ons bet we en 8-OHdG le vel and tu mor gra de, sta ge, me tas ta sis and tu mor si ze we re not exa mi ned. Ex cep ti o nally, Ak cay et al. 17 re por ted that le u kocy te 8-OHdG le vel was not cor re la ted with the cli ni cal gra ding and his to logy in the pa ti - ents with blad der can cer. This is the first study inves ti ga ting 8-OHdG le vel in se rum. The aim of the pre sent study was to in ves ti ga te whet her se rum le - vel of 8-OHdG had a pre dic ti ve va lu e in pa ti ents who un der went re sec ti on of for uri nary blad der Turkiye Klinikleri J Med Sci 2011;31(3) 555

4 Dinçer ve ark. car ci no ma. In the light of pre vi o us da ta, 16,17 we had ex pec ted higher 8-OHdG le vel in the se rum. We sup po sed that if 8-OHdG le vel was high in tu mor tis su e it might al so in cre a se in se rum, and se rum le - vel of 8-OHdG might ref lect the oxi da ti ve stress on DNA, and it might be related to the tu mor cha rac - te ris tics. Ho we ver, alt ho ugh se rum 8-OHdG le vel was hig her in the pa ti ent gro up, it did not re ach a sig ni fi cant le vel. Con si de ring that the only mec ha - nism for the ap pe a ran ce of 8-OHdG in se rum is the re pa ir of 8-OHdG re si du es by DNA re pa ir systems, unc han ged se rum 8-OHdG le vel makes one to suppose that DNA re pa ir may be im pa i red du ring the car ci no ge ne sis. Va ri o us de fects in DNA re pa ir result in dif fe rent forms of can cer. In de ed, mis match re pa ir de fects that ha ve be en de ter mi ned in so me kinds of gas tric and co lo rec tal can cer, 18,19 and uro e - pit he li al can cers of the ure ter (and blad der to les ser ex tent) has be en sug ges ted to sha re a number of cha rac te ris tics of mis match re pa ir de fi ci ency-dri - ven tu mo ri ge ne sis OHdG le vel did not ex hi bit sig ni fi cant chan ges with res pect to tu mor gra de and pre sen ce of lo cal in va si on. Se rum le vel of 8-OHdG was not fo und to be cor re la ted with tu mor gra de and si ze. Ta ken to get her, all tho se da ta show that se rum le vel of 8-OHdG is not a prog nos tic mar ker for uri nary blad der can cer. Ho we ver, as li mi ta ti on of the pre sent study, tu mor vo lu me was he te ro ge - no us in the pa ti ent gro up and the study population was small. In or der to cla rify prog nos tic po ten ti al of se rum le vel of 8-OHdG, furt her stu di es in lar ger gro ups are ne e ded. An ti o xi dant system is highly comp lex and mul ti fac to ri al. It inc lu des va ri o us enz ymes and small mo le cu les and they may not ex hi bit har mo - ni o us chan ge. Sci en ti fic da ta for SOD ac ti vity in uri nary blad der can cer pa ti ents is extremely li mi - ted and con tra dic tory. As com pa red to tho se of can cer-fre e ad ja cent tis su e, SOD ac ti vity in can ce - ro us blad der tis su e was fo und to be dec re a sed by Du rak et al. 21 and de ter mi ned to be in cre a sed by Sa vic- Ra do je vic et al. 22 As far as we know, SOD acti vity in pe rip he ral cir cu la ti on has not be en exa - mi ned in uri nary blad der can cer pa ti ents so far. Dec re a sed se rum SOD ac ti vity de ter mi ned in the pre sent study may be a ref lec ti on of po or an ti o xi - Tıbbi Biyokimya dant de fen ce in uri nary blad der can cer pa ti ents. In fact, ex pres si on of so me ge nes can be al te red du ring the ma lig nant trans for ma ti on. In uri nary blad der can cer, the ex pres si ons of a number of ge nes may be down-re gu la ted, and SOD may be one of them. In cre a sed GPx ac ti vi tiy was de ter mi ned in can ce ro us blad der tis su es when com pa red to cancer-fre e ad ja cent tis su es. 22,23 GPx ac ti vity was al so me a su red in eryt hrocy tes. Arı kan et al. 24 re por ted dec re a sed GPx ac ti vity, Yal cın et al. 25 re por ted incre a sed GPx ac ti vity in eryt hrocy tes of pa ti ents with uri nary blad der can cer as com pa red to controls. Se rum GPx ac ti vity has not be en exa mi ned in uri nary blad der can cer pa ti ents pre vi o usly. In the pre sent study, se rum GPx ac ti vity did not pre s- ent a sig ni fi cant dif fe ren ce bet we en uri nary bladder pa ti ents and con trols. The most in te res ting fin ding of this study was the in cre a sed GST ac ti - vity in the pa ti ent gro up, and its ne ga ti ve as so ci a - ti on with tu mor si ze. En han ced GST ac ti vity in uri nary blad der tu mors as com pa red to can cer-fre - e ad ja cent tis su e has be en sug ges ted by early stu di - es. 26,27 Fi ve clas ses of so lub le GSTs are known in hu mans, inc lu ding alp ha, mu, pi, the ta and ze ta. 28 GST mu1 (GSTM1) is in vol ved in the de to xi fi ca ti - on of polyc yclic aro ma tic hydro car bons fo und in to bac co smo ke and in so me in dus tri al che mi cals which are known as car ci no ge nic agents. Ma jo rity of the stu di es investigating the re la ti on bet we en blad der can cer and GSTM1 ha ve re por ted an in cre - a sed risk when there is a lack of GSTM1 ac ti - vity. 29,30 In cre a sed GST ac ti vity in uri nary blad der can cer pa ti ents may be a re sult of up- re gu la ti on of GST ge ne du ring the car ci no ge ne sis. Al ter na ti vely, this in cre a se may be a cel lu lar adap ti ve res pon se or de fen se mec ha nism aga inst ma lig nant trans for ma - ti on. Alt ho ugh GST ac ti vity was hig her in the pati ents with low gra de tu mor, it did not re ach a sig ni fi cant le vel. Ho we ver, GST ac ti vity was sig ni - fi cantly hig her in the pa ti ents wit ho ut lo cal in va - si on as com pa red to pa ti ents with lo cal in va si on. Furt her mo re, GST ac ti vity was ne ga ti vely cor re la - ted with tu mor si ze. Ta ken al to get her, the se fin d- ings sup port our sug ges ti on that in cre a sed GST ac ti vity is a de fen si ve res pon se to ma lig nant transfor ma ti on in early steps. 556 Turkiye Klinikleri J Med Sci 2011;31(3)

5 Medical Biochemistry In conc lu si on, in the con trary to pre vi o us da - ta ob ta i ned from can ce ro us uri nary blad der tis su - es, se rum 8-OHdG le vel is not sig ni fi cantly dif fe rent from con trols in uri nary blad der can cer pa ti ents. Alt ho ugh an ti o xi dant ba lan ce is dis tur - bed, chan ges in an ti o xi dant enz yme does not ha - ve a prog nos tic va lu e. The most pro mi sing an tio xi dant enz yme as a prog nos tic mar ker is GST beca u se of its in cre a sed ac ti vity and its ne ga ti ve asso ci a ti on with tu mor si ze in the uri nary blad der can cer pa ti ents. Is in cre a sed GST ac ti vity spe si fic for uri nary blad der can cer or a com mon con di ti - on in all can cers? This is of in te rest. Our syudy was a pre li mi nary study with so me li mi ta ti ons such as he te ro ge nity of tu mor sta ge and a small pa ti ent population. Mo re de ta i led stu di es in lar - ger and ho mo ge no us gro ups sho uld be held to cla - rify the prog nos tic po ten ti al of se rum le vel of 8-OHdG and SOD, GPx, GST ac ti vi ti es in uri nary blad der can cer pa ti ents. Ack now led ge ments Dinçer et al This work was sup por ted by The Re se arch Fund of Istan bul Uni ver sity (Pro ject num ber: BYP-1757). 1. Zhong W, Ober ley LW, Ober ley TD, Yan T, Do mann FE, St Cla ir DK. In hi bi ti on of cell growth and sen si ti za ti on to oxi da ti ve da ma ge by ove rex pres si on of man ga ne se su pe ro xi de dis mu ta se in rat gli o ma cells. Cell Growth Differ 1996;7(9): Ames BN. Mu ta ge ne sis and car ci no ge ne sis: en do ge no us and exo ge no us fac tors. En vi ron Mol Mu ta gen 1989;14(Suppl 16): Troll W, Wi es ner R. The ro le of oxy gen ra di - cals as a pos sib le mec ha nism of tu mor promo ti on. An nu Rev Phar ma col To xi col 1985; 25: Hus sa in SP, Har ris CC. 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