BIOAKUMULACJA METALI CIÊ KICH I ODPOWIED FIZJOLOGICZNA ROŒLIN Silene vulgaris Moench (Garcke) Z TERENÓW ZANIECZYSZCZONYCH METALAMI CIÊ KIMI

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1 E C O L O G I C A L C H E M I S T R Y A N D E N G I N E E R I N G A Vol. 16, No Aleksndr NADGÓRSKA-SOCHA 1, Ryszrd CIEPA 1, Mrt KANDZIORA 1 nd Alin KAFEL 2 HEAVY METALS BIOACCUMULATION AND PHYSIOLOGICAL RESPONSES TO HEAVY METAL STRESS IN POPULATIONS OF Silene vulgris Moenh (Grke) FROM HEAVY METAL CONTAMINATED SITES BIOAKUMULACJA METALI CIÊ KICH I ODPOWIED FIZJOLOGICZNA ROŒLIN Silene vulgris Moenh (Grke) Z TERENÓW ZANIECZYSZCZONYCH METALAMI CIÊ KIMI Astrt: In this study we hve evluted the hevy metls umultion in the leves of Silene vulgris nd its influene on glutthione, free mino ids nd nthoynins onentrtions. Glutthione is tripeptide ( -Glu-Cys-Gly) whih is involved in mny metoli proesses of plnt ell. It is relted to the sequestrtion of xenoiotis nd hevy metls nd it is lso n essentil omponent of the ntioxidnt system, whih keeps retive oxygen speies under ontrol. The umultion of nthoynin pigments in the leves n e indued y environmentl nd nthropogeni stressors, suh s pollution, osmoti stress, nd nutrients defiieny. The positive role of nthoynins s well s mino ids nd orgni ids in metl sequestrtion is suggested. Smples of Silene vulgris leves were olleted in My nd July from the hevy metls ontminted sites from the nerest viinity of non-ferrous metl smelter Szopienie t the distne of 50, 250, 450 m, from zin wstes hep in Ktowie nd from former lmine site in D¹row Górniz (the South of Polnd). In the previous study we noted tht the nerest viinity of non-ferrous metl smelter Szopienie ws more polluted thn the other res. The highest Zn, Cd nd P umultion in the leves of Silene vulgris ws noted in the plnts olleted in the nerest viinity of the non-ferrous metl smelter Szopienie. The free mino ids onentrtions hve differed in the vegettive seson. It ws higher in the most polluted sites (50 nd 250 m) in July. Higher ontent of the redued form of glutthione ws estimted in the plnt leves olleted in the distne of 250 m from the emitter in July thn t the eginning of the study. Also the highest nthoynins umultion in Silene vulgris leves ws notied in July nd in plnts from the most polluted re (250 m). The estimtions, espeilly glutthione ontent, seem to e promising in eophysiologil reserh onneted with hevy metls stress in plnts growing in the postindustril res. Keywords: hevy metls, Silene vulgris, glutthione, mino ids, nthoynins 1 Deprtment of Eology, Fulty of Biology nd Environment Protetion, The University of Silesi, ul. Bnkow 9, Ktowie, Polnd, tel , emil: oln@hog.pl 2 Deprtment of Animl Physiology & Eotoxiology, Fulty of Biology nd Environment Protetion, The University of Silesi, ul. Bnkow 9, Ktowie, Polnd.

2 390 Aleksndr Ndgórsk-Soh et l The metllurgil proessing of zin, led, opper nd iron is responsile for onsiderle mounts of hevy metls introdued into environment (espeilly plnt emissions, postindustril dumps) [1 5]. Metllophyte Silene vulgris is speifi plnt to the lmine res nd it is pioneer plnt in the nerest viinity of non-ferrous metl smelter [6 11]. Suh plnts re tolernt of high hevy metls onentrtion nd hve high hevy metls umultion ility [7, 11 13]. Severl plnt speies suh s Silene vulgris, Crdminopsis renos, Plntgo leolt hve een shown to e good ioinditors of soil ontmintion with led nd zin [3, 6]. Mny investigtions were undertken to determine plnts defense ginst hevy metls. The investigtors looked for good stress nd defense inditors. Glutthione is involved in the ellulr defense ginst the toxi influene of xenoiotis s well s metl tions [14, 15] The positive role of nthoynins next to mino ids nd orgni ids in metl sequestrtion is suggested [16, 17]. Anthoynins re ssoited with the enhned tolerne to effets of hilling nd freezing, to hevy metl ontmintion, to dessition nd to wounding [18]. The im of this work ws to determine the hevy metl ioumultion in leves of Silene vulgris nd to define the responses (glutthione, free mino id nd nthoynin ontent) of Silene vulgris to environmentl metls exposure. Mteril nd methods The investigtion ws rried out in My nd July in The leves from Silene vulgris (Moenh) Grke plnts (from 20 individuls) were olleted in the nerest viinity of non-ferrous plnt Szopienie t the distnes of 50, 250, 450 m, on the zin smelter spoil hep in Ktowie We³nowie (H) nd from the former lmine site in D¹row Górniz (C). In order to determine the hevy metl onentrtion, the plnt mteril ws wshed in tp nd in distilled wter, dried t 105 o C to onstnt weight nd ground to fine powder, then minerlized nd dissolved in 10 % HNO 3. After filtrtion Zn, P nd Cd ontents were mesured using flme Atomi Asorption Spetrometry (AAS) [19]. The qulity of the nlytil proedure ws ontrolled y using the smples of the referene mteril in eh series of nlysis. (Certified Referene mteril CTA-OTL-1 Orientl Too Leves). Anthoynins ws estimted ording to method of Fuleki nd Frnis [20]; Giusti nd Wrolstd [21] y extrting the pigment in methnol/hcl (99/1, v/v). Glutthione (redued nd oxidtive forms) ws extrted from plnts y homogenizing plnt mteril (0.5 g) t 4 o C in ie old 5 % trihloroeti id with the ddition of N-phosphte uffer (ph 7.5) nd then entrifuged t g for 10 min nd determined ording to method of Anderson [22] nd Nishimoto et l [23]. The superntnt ws used for GSH nd GSSG dermintions y the DTNB-GSSG redutse reyling proedure. GSSG ws determined fter GSH hd een removed y 2-vinylpyridine derivtiztions. Chnges in the sorne of the retion mixtures were mesured t = 412 nm. Free mino ids were extrted from plnts y homogenizing plnt mteril (0.5 g) t 4 o C in 80 % etone nd determined with the usge of ninhydrin method of Keller [24]. All plnt smples were rried out in six replitions. The dt ws proessed using the softwre

3 Hevy Metls Bioumultion nd Physiologil Responses to Hevy Metl Stress Sttisti to ompute signifint sttistil differenes etween smples (p < 0.05) ording to Tukey s multiple rnge test nd to ompute Person s orreltion oeffiient. Results nd disussion The highest ioumultion of the investigted metls ws found in the nerest viinity of non-ferrous plnt Szopienie. Cd (50, 250 m) nd Zn (ll investigted res) were ove the toxi level, P mounts umulted in Silene vulgris leves were in the toxiity rnge [25] (Tle 1). In the erlier study in these stnds the delined plnt iomss ws notied. However, the highest metl fitoextrtion ws found for plnts in the distne of 450 m [13]. Anthoynins n e formed s retion to lots of dverse environmentl onditions. At nutrient shortge, suh s N- nd P-defiieny, surplus of rohydrtes n e stored s nthoynins [26]. The highest onentrtion of nthoynins ws found in the plnt leves olleted in the distne of 250 m from the emitter in July (Fig. 1). Anthoynins n e ssoited with enhned resistne to the hevy metls ontmintion. The hnges of plnt olours (due to inresed nthoynins onentrtion) my e the first indition of insuffiient detoxifition of metls resulting in deregultion of plnt physiology. However these symptoms my e not neessrily relted to surplus ut lso to the shortge of metls [27]. Under strong light (in environmentl ondition in July in our study), however, the nthoynins serve s useful optil filter diverting exessive high energy qunt wy from n lredy sturted photosyntheti eletron trnsport hin. Chloroplsts irrdited with light tht hs first pssed through red filter hve een shown to generte fewer superoxide rdils, therey reduing the propensity for struturl dmge to the photosystems. 5 4 V VII mg/g d.m. 3 2 d H C Investigted stnds Fig. 1. Men nthoynins onentrtion in leves of Silene vulgris. Vlues with the sme letter (for the month) re sttistilly the sme for p < 0.05

4 392 Aleksndr Ndgórsk-Soh et l Tle 1 Men hevy metls onentrtion in Silene vulgris leves. Vlues with the sme letter (in the olumns) re sttistilly the sme for p < 0.05 Metl Zn P Cd Site My SD July SD My SD July SD My SD July SD 50 m m m H d 0.8 C 846.1d d d d e 0.4

5 Hevy Metls Bioumultion nd Physiologil Responses to Hevy Metl Stress Most likely elevted onentrtion of nthoynins does not indite high surplus of hevy metls. Gould emphsized [28], tht nthoynins offer multifeted, verstile nd effetive protetion to plnt under stress. Higher onentrtions of free mino ids were found in My rther thn in July, in plnt leves, where smller metl ioumultion ws noted. However the free mino id ontent inresed in the plnt leves olleted in most polluted site in the distne of 50 m from the emitter during vegettive seson in July (Fig. 2). Amino ids nd prtiulrly phytoheltins nd glutthione ply n importnt role in metl inding [17]. Lesko nd Srdi showed tht dmium tretment t higher onentrtion used the highest umultion of totl mino id ontent in the shoots nd roots of whet seedlings [29]. Positive orreltion etween metl onentrtion nd free mino id ontent ws found in our study (Correltion oeffiient were 0.8 for Zn, 0.9 for Cd nd 0.7 for P). Hevy metls tretment modified free mino id omposition nd onentrtion. It my e suggested tht proline is involved in detoxifition of hevy metls [14, 17]. Metl tolernt popultions of Deshmpsi nd Silene hve een shown to hve higher onstitutive ontent of proline s ompred with nontolernt ounterprts. Allysum, when exposed to nikel produe histidine, proportionlly to n pplied Ni dose [32]. The further reserh is needed to find out tht free mino id ould e inditor of metl stress in lortory nd in the environmentl onditions. 7 6 V VII 5 d mg/g d.m d H C Investigted stnds Fig. 2. Men free mino ids ontent in Silene vulgris leves in My nd July. Vlues with the sme letter (for the month) re sttistilly the sme for p < 0.05 The higher onentrtions of the redued form of glutthione were oserved in the leves of Silene vulgris in July rther thn in My. The inresed onentrtion of glutthione in plnt tissues in the distne of 250 m suggested positive role of this tripeptide in plnt metl defense (Figs. 3, 4). However, strong positive orreltion ws noted only with Zn (orreltion oeffiient ws 0.84). The inresed ontent of GSSG forms ws oserved in My. The inresed GSSG level filittes formtion of mixed disulfides (protein gluthtiontion), whih indues hnges in redox sttus of thiols

6 394 Aleksndr Ndgórsk-Soh et l 200 GSH 150 GSSG M/g f.w d H C Investigted stnds Fig. 3. Men glutthione ontent in leves of Silene vulgris in My. Vlues with the sme letter (for the glutthione form) re sttistilly the sme for p < GSH GSSG 210 M/g f.w d H C Investigted stnds Fig. 4. Men glutthione ontent in leves of Silene vulgris in July. Vlues with the sme letter (for the glutthione form) re sttistilly the sme for p < 0.05 [30]. Bruns et l pointed tht the intrellulr sequestrtion of hevy metls y GSH is fvored in mosses treted with Cd [31]. Glutthione is relted to the sequestrtion of xenoiotis nd hevy metls nd is lso n essentil omponent of the ellulr ntioxidtive defense system, whih keeps retive oxygen speies (ROS) under ontrol [15]. Glutthione ould e suitle ndidte s stress mrker. Strong evidene hs indited tht the elevted GHS onentrtion is onneted with the plnt ility to metl indued oxidtive stress [32, 33].

7 Hevy Metls Bioumultion nd Physiologil Responses to Hevy Metl Stress Conlusions Zin ws ioumulted in the highest mount in Silene vulgris leves on ll investigted stnds. The highest ioumultion of ll investigted metls (Zn, Cd nd P) ws notied for Silene vulgris leves olleted in the distne of 50 nd 250 m from the smelter. Positive orreltion etween hevy metl onentrtions nd free mino id ontents in Silene vulgris leves ws found. It is very likely tht free mino ids were involved in plnt hevy metls defense. The prolem needs further investigtions. The nthoynins ontent inrese in July in the plnt leves from the most polluted re ould e n effetive protetion to the plnt under stress, however not only hevy metls. The glutthione ontent ws the highest in the plnt leves olleted from the distne of 250 m from the smelter, where high levels of hevy metl onentrtions in plnt leves ws noted. It seems to e promising in further investigtions. Referenes [1] Godzik B.: Hevy metls ontent in plnts from zin dumps nd referene res. Polish Bot. Stud., 1993, 5, [2] Grodziñsk K. nd Szrek- ukszewsk G.: H³dy ynkowo-o³owiowe w okolih Olkusz przesz³oœæ, terÿniejszoœæ i przysz³oœæ. KOSMOS. Prolemy nuk przyrodnizyh, 2002, 51(2), [3] Rostñski A.: Zwrtoœæ metli iê kih w gleie i roœlinh z otozeni niektóryh emitorów zniezyszzeñ n Górnym Œl¹sku. Arh. Environ. Protet., 1997, 23(3 4), [4] Guw-Przepiór E. nd Turnu K.: Arusulr Myorrhiz nd Plnt Suession on Zin Smelter Spoil Hep in Ktowie-We³nowie. At So. Bot. Polon., 2001, 70, [5] Tokrsk-Guzik B., Rostñski A. nd Klotz S.: Roœlinnoœæ h³dy poynkowej w Ktowih We³nowu. At Biol. Siles. Florystyk: geogrfi roœlin, 1991, 36(19), [6] Wierzik M. nd Rostñski A.: Miroevolutionry hnges in eotypes of lmine wste hep ner Olkusz, Polnd: A Review. At Biol. Crov. Ser. Bot., 2002, 44, [7] Heflik M., Ndgórsk-Soh A. nd Ciep³ R.: Hevy metls umultion nd its effets on Silene vulgris plnts grown in metl ontminted sites. Eol. Chem. Eng., 2006, 13(7), [8] Koszelnik-Leszek A.: Budow lszki liœiowej orz zwrtoœæ hromu, niklu i ynku w Silene vulgris (Moenh) Grke i w gleie n h³dzie odpdów serpentynitowyh w Wirkh (Dolny Œl¹sk). Zesz. Prol. Post. Nuk Roln., 2007, 520, [9] Heflik M., Kndzior M., Ndgórsk-Soh A. nd Ciep³ R.: Aktywnoœæ kwœnyh fosftz u roœlin wystêpuj¹yh n terenh o podwy szonej zwrtoœi metli iê kih. Ohr. Œrodow. Zso. Nturl., 2007, 32, [10] Kndzior M., Heflik M., Ndgórsk-Soh A. nd Ciep³ R.: Syntez zwi¹zków ogtyh w grupy -SH jko odpowiedÿ n podwy szone stê enie metli iê kih u roœlin Silene vulgris (Cryophyllee). Ohr. Œrodow. Zso. Nturl., 2007, 33, [11] Ernst W.: Biovilility of hevy metls nd deontmintion of soils y plnts. Appl. Geohem., 1996, 11, [12] Wierzik M. nd Pnufnik D.: The dpttion of Silene vulgris to growth on lmine wste hep (S. Polnd). Environ. Pollut., 1998, 101, [13] Ndgórsk-Soh A. nd Ciep³ R.: Phytoextrtion of Zn, P, Cd with Silene vulgris Moenh (Grke) growing in the postindustril re. Eol. Chem. Eng., in press. [14] Wu F., Chen F., Wei K. nd Zhng G.-P.: Effet of dmium on free minoid, glutthione nd sori id onentrtions in two rley genotypes (Hordeum vulgre L.) differing in dmium tolerne. Chemosphere, 2004, 57,

8 396 Aleksndr Ndgórsk-Soh et l [15] Tusz M., Sireij H. nd Grill D.: The glutthione system s stress mrker in plnt eophysiology: is stress response onept vlid? J. Exp. Bot., (404), [16] Clemens S.: Moleulr mehnisms of plnt metl tolerne nd homeostsis. Plnt, 2001, 212(4), [17] Shrm S. nd Dietz K.: The signifine of mino ids nd mino id-derived moleules in plnt responses nd dpttion to hevy metl stress. J. Exp. Bot., 2006, 57(4), [18] Gould K.: Nture s Swiss Army Knife: The Diverse Protetive roles of Anthoynins in Leves. J. Biomed. Biotehnol., 2004, 5, [19] Ostrowsk A., Gwliñski S. nd Szzui³k Z.: Metody nlizy i oeny w³œiwoœi gle i roœlin. Instytut Ohrony Œrodowisk, Wrszw 1991, [20] Fuleki T. nd Frnis F.: Quntittive Methods for Anthoynins. 1. Extrtion nd Determintion of Totl Antoynin in Crnerries. J. Food Si., 1968, 33, [21] Giusti M. nd Wrolstd R.: Chrteriztion nd Mesurements of Anthoynins y UV-Visile Spetrosopy. [In:] Current Prototools in Food Anlytil Chemistry. John Wiley & Sons, New York [22] Anderson M.E.: Determintion of glutthione nd gluthtione disulfide in iologil smples. Meth. Enzymol., 1985, 113, [23] Nishimoto M., Eerhrt B.T., Sorn H.R., Krone C., Vrnsi U. nd Stein J.E.: Effets omplex mixture of hemil ontminnts on hepti glutthione, L-ysteine nd gmm glutmyl ysteine in English sole (Pleuronetes vetulus). Environ. Toxol. Chem., 1995, 14(3), [24] Keller H.: Histologishe und physiologishe Untersuhungen n Forstpflnzen in einem Fluoroshdensgeiet. Ztg. Forst., 1976, 127(3), [25] Allowy B.J. nd Ayres D.: Chemizne podstwy zniezyszzeni œrodowisk. WN PWN, Wrszw 1999, 237. [26] Ernst W., Nelissen H. nd Ten Bokum W.: Comintion toxiology of metl-enrihed soils: physiologil responses of Zn- nd Cd-resistnt eotype of Silene vulgris on polymetlli soils. Environ. Exp. Bot., 2000, 43, [27] Khn H.R., Mdonld G.K. nd Rengel Z.: Chikpe genotypes differ in their sensitivity to Zn defiieny. Plnt Soil, 1998, 198, [28] Gould K.: Nture s Swiss Army Knife: The Diverse Protetive Roles of Anthoynins in Leves. J. Biomed. Biotehnol., 2004, 5, [29] Lesko K. nd Simon-Srkdi L.: Effet of dmium stress on mino id nd polymine ontent of whet seedlings. Periodi Polytehn., Ser. Chem. Eng., 2002, 46(1 2), [30] W³odek L.: Benefiil nd hrmful effets of thiols. Polish J. Phrmol., 2002, 54, [31] Bruns I., Sutter K., Neumnn D. nd Kruss G.-J.: Glutthione umultion speifi response of mosses to hevy metl stress. [In:] Sulfur Nutrition nd Sulfur Assimiltion in Higher Plnts. P. Hupt (Ed.), Bern, Switzerlnd 2000, [32] Kukkol E., Rutio P. nd Huttunen S.: Stress inditions in opper- nd niel-exposed Sots pine seedlings. Environ. Exp. Bot., 2000, 43, [33] Anderson M.E.: Glutthione n overview of iosynthesis nd modultion. Chemio-Biologil Intertion, 1998, , BIOAKUMULACJA METALI CIÊ KICH I ODPOWIED FIZJOLOGICZNA ROŒLIN Silene vulgris Moenh (Grke) Z TERENÓW ZANIECZYSZCZONYCH METALAMI CIÊ KIMI Ktedr Ekologii, Wydzi³ Biologii i Ohrony Œrodowisk Uniwersytet Œl¹ski Astrkt: Przeprowdzono dni nd kumulj¹ Zn, P i Cd w liœih Silene vulgris i wp³ywie tyh metli n zwrtoœæ gluttionu, wolnyh minokwsów i ntoyjnów. Gluttion jest tripeptydem ( -Glu-Cys-Gly), który jest zng owny w oronê przed ksenoiotykmi i metlmi iê kimi i jest podstwowym komponentem komórkowej ntyoksydyjnej orony, utrzymuj¹ym rektywne formy tlenu (ROS) pod kontrol¹. Akumulj rwników ntoyjnowyh mo e yæ powodown przez œrodowiskowe i ntropo-

9 Hevy Metls Bioumultion nd Physiologil Responses to Hevy Metl Stress genne zynniki stresowe, tkie jk zniezyszzenie, stres osmotyzny, niedoór sk³dników od ywzyh. Sugeruje siê pozytywn¹ rolê ntoyjn ook, wolnyh minokwsów i kwsów orgniznyh w oddzielniu metli iê kih. Liœie roœlin Silene vulgris zierno w mju i lipu z terenów zniezyszzonyh metlmi iê kimi (z njli szego s¹siedztw Huty Metli Nie elznyh Szopienie w odleg³oœi 50, 250, 450 m, z h³dy poynkowej w Ktowih We³nowu orz terenu po eksplotji glmnu w D¹rowie Górnizej). W poprzednih dnih wykzno njwiêksze zniezyszzenie metlmi iê kih n terenie njli ej po³o onym przy Huie Metli Nie elznyh Szopienie. Njwiêksz¹ kumuljê Zn, Cd, P stwierdzono w liœih roœlin Silene vulgris ziernyh w njli szym s¹siedztwie emitor (50, 250 m). Stê enie wolnyh minokwsów ró ni³o siê podzs sezonu wegetyjnego. Zwrtoœi y³y wiêksze n njrdziej zniezyszzonym terenie w lipu ni n poz¹tku dñ. Wiêksz¹ zwrtoœæ zredukownej formy gluttionu oznzono w liœih roœlin ziernyh w odleg³oœi 250 m od emitor w lipu ni n poz¹tku dñ. Znotowno tk e njwiêksz¹ kumuljê ntoyjnów w liœih roœlin w lipu i u roœlin z njrdziej zniezyszzonego terenu (250 m). Szzególnie oznznie zwrtoœi gluttionu wydje siê yæ oieuj¹e w ekofizjologiznyh dnih nd stresem wywo³ywnym przez metle iê kie u roœlin n terenh poprzemys³owyh S³ow kluzowe: metle iê kie, Silene vulgris, gluttion, wolne minokwsy, ntoyjny