influence of heavy metal stress on antioxidant status and dna

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 276417 6 pageshttpdxdoiorg1011552013276417

Research ArticleInfluence of Heavy Metal Stress on Antioxidant Status andDNA Damage in Urtica dioica

Darinka Gjorgieva1 Tatjana Kadifkova Panovska2 Tatjana Ruskovska1

Katerina BaIeva3 and TrajIe Stafilov3

1 Faculty of Medical Sciences Goce Delcev University Krste Misirkov Street bb PO Box 201 2000 Stip Macedonia2 Faculty of Pharmacy Ss Cyril and Methodius University 1000 Skopje Macedonia3 Institute of Chemistry Faculty of Natural Sciences and Mathematics Ss Cyril and Methodius University 1000 Skopje Macedonia

Correspondence should be addressed to Darinka Gjorgieva darinkagorgievaugdedumk

Received 2 April 2013 Accepted 20 May 2013

Academic Editor Brad Upham

Copyright copy 2013 Darinka Gjorgieva 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

Heavy metals have the potential to interact and induce several stress responses in the plants thus effects of heavy metal stresson DNA damages and total antioxidants level in Urtica dioica leaves and stems were investigated The samples are sampled fromareas with different metal exposition Metal content was analyzed by Inductively Coupled Plasma-Atomic Emission Spectrometer(ICP-AES) for total antioxidants level assessment the Ferric-Reducing Antioxidant Power (FRAP) assay was used and genomicDNA isolation from frozen plant samples was performed to obtain DNA fingerprints of investigated plant It was found that heavymetal contents in stems generally changed synchronously with those in leaves of the plant and extraneous metals led to imbalanceof mineral nutrient elements DNA damages were investigated by Random Amplified Polymorphic DNA (RAPD) technique andthe results demonstrated that the samples exposed to metals yielded a large number of new fragments (total 12) in comparisonwith the control sample This study showed that DNA stability is highly affected by metal pollution which was identified by RAPDmarkers Results suggested that heavy metal stress influences antioxidant status and also induces DNA damages in U dioica whichmay help to understand the mechanisms of metals genotoxicity

1 Introduction

Metals constitute one of the major groups of genotoxicenvironmental pollutants possessing serious threat to humanas well as environmental well-being Heavy metal stress in allliving organisms often results in the production of reactiveoxygen species (ROS) which are relatively reactive comparedto molecular oxygen and thus potentially toxic [1 2]

Tolerance to heavy metal stress has been correlated withefficient antioxidative defense system as shown by manyauthors [2ndash4] Among different present methods used toassess the total antioxidant capacity of plants one of themis the Ferric-Reducing Antioxidant Power (FRAP) assay ofBenzie and Strain [5]

Heavy metals also induce several cellular stress responsesand damage to different cellular components such as mem-branes proteins and DNA Recently advances in molecularbiology have led to the development of a number of selective

and sensitive assays for DNA analysis in ecogenotoxicologyDNA-based techniques like Random Amplified Polymor-phic DNA (RAPD) is used to evaluate the variation at theDNA level and differences can clearly be shown when com-paring DNA fingerprints from individuals exposed andornonexposed to genotoxic agents [6ndash10]

Monitoring the pollution status of the environment usingplants is one of the main topics of environmental biogeo-chemistry [11] Although heavy metals are naturally presentin soils contamination comes from different sources mostlyindustry (mainly nonferrous iron and steel and chemicalindustries) waste incineration agriculture (use of pollutedwaters for irrigation fertilizers and phosphates especiallypesticides containing heavy metals) combustion of fossilfuels and traffic [12]

Nettle (Urtica dioica Urticaceae) was chosen as theobject of this study because it is a widespread plant in

2 BioMed Research International

R Macedonia edible used in medicinal purposes and alsofrequently used as a model plant in different studies [13ndash15]The objective of the present study was to investigate thusexposure to different metals can induce direct DNA damageand significant changes in metal content in the plant and alsoendogenous total antioxidants level

2 Materials and Methods

21 Sampling Area Plant samples from the industrializedarea were taken from 10ndash100m around the lead and zincsmelting plant ldquoMHKZletovordquo inVeles area while for uncon-taminated controls samples were taken from PlackovicaMountain about 60 km from the city of Veles (Figure 1)Leaves and stems from plants were analyzedThe plants wereidentified and specimens are deposited at the Departmentof Pharmacognosy Faculty of Pharmacy Skopje Republicof Macedonia Element analysis FRAP analysis and DNAextraction were performed

22 Sample Preparation for Element Analysis All plant sam-ples not rinsed were air dried milled in a nonmetal micro-hammer and stored in clean paper bags 05 g was weighedand placed into PTFE vessels with 5mL HNO

3(69 Merck

Tracepur) and 2mL H2O2(30 mV Merck) mixture was

digested by microwave (MARS CEM XP 1500) with twosteps procedure at 180∘C Digests were filtered on filter paper(Munktell) quantitatively transferred in 25mL calibratedflasks diluted with demineralized water and analyzed byinductively coupled plasma-atomic emission spectrometer(ICP-AES) Varian 715-ES for selected metals Standards ofselected metals were set by dilution of stock standards whichwere prepared using analytical grade salts of metals (MerckMultielement standard 1000mgL) Samples were analyzed intriplicate All results were calculated on a dry weight basis(mg kgminus1 dw)

23 FRAP Assay The total antioxidant power of a freshlyprepared cooled and filtered infusion (5 g of dry leaves orstems100mL of boiling distilled water) of each sample wasmeasured using the FRAP assay In the FRAP assay reduct-ants (antioxidants) in the sample reduce Fe3+tripyridyltri-azine complex present in stoichiometric excess to the bluecolored ferrous form with an increase in absorbance at595 nm Samples were analyzed using microplate reader(ChemWell) at 600 nmThe antioxidant status is expressed as120583mol FeSO

4Lminus1 All values are means of triplicate analyses plusmn

SD

24 Genomic DNA Isolation Frozen plant samples were usedforDNA isolation 05 to 07 cmdisks of leaf tissuewere cattedwith standard one-hole paper punch Samples were kept onice while the procedure was done DNA extractions wereperformed using REDExtract-N-Amp Plant PCRKit (Sigma-Aldrich) following the instructions of themanufacturer Plantdisk was placed into a 15mL microcentrifuge tube with100 120583L extraction solution and incubated for 10 minutes at95∘C 100 120583L of dilution solution is added and vortexesExtract is stored at 2ndash8∘C until use

Figure 1 City of Veles and Plackovica Mountain as sampling areas

25 RAPD Amplification Methods PCR reactions were per-formed using REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich) PCR reactions were performed in reaction mix-tures of 20120583L containing 10 ng of genomic DNA 04120583Mprimer (Sigma-Aldrich) and 10120583L REDExtract-N-Amp PCRreaction mix The REDExtract-N-Amp PCR reaction mix isa ready mix containing buffer salts dNTPs and REDTaqDNA polymerase Sequences (51015840 rarr 31015840) from primer 1 to 7(with 60ndash70 GC content) used are GGTGCGGGAA (P1)GTTTCGCTCC (P2) GTAGACCCGT (P3) AAGAGCC-CGT (P4) AACGCGCAAC (P5) CCCGTCAGCA (P6)GGCACTGAGG (P7) respectively Amplifications were per-formed in a DNA thermocycler (Mastercycler personalEppendorf) programmed for 5min at 95∘C (initial dena-turing step) 45 consecutive cycles each consisting of 1minat 95∘C (denaturing) 1min at 36∘C (annealing) 2min at72∘C (extension) and followed by the last cycle for 5minat 72∘C (final extension step) Negative controls with waterwithout any template DNA were always included to monitorfor contamination After amplification electrophoresis ofRAPD reaction products was performed in 2 (wv) agarose(Agarose 1000 Invitrogen) using a TBE (TrisborateEDTA)buffer system (1 times TBE = 90mM tris base 90mM boric acidand 2mM EDTA) DNA bands were stained with ethidiumbromide for 10 minutes visualized and photographed underUV light (Biometra) All amplifications were repeated twicein order to confirm the reproducible amplification of scoredfragments Only reproducible and clear amplification bandswere scored for the construction of the data matrix

3 Results and Discussion

Veles area (around lead and smelting plant) was chosen asan investigated area because it is an important source oflead and zinc pollution in R Macedonia with estimatedlead emission of 83 tons per year according to the NationalEnvironmental Action Plan (NEAP) [16] and there wereseveral investigations in the region of Veles for heavy metalscontents [17ndash20] As shown in Table 1 varying amounts ofmetal contents were noted not only for the heavy metalsbut also for essential metals Levels of metals uptake and

BioMed Research International 3

Table 1 Elemental analysis of U dioica sampled from two different areas (in mg kgminus1 dry mass)

Plant organ investigated MetalsCa Cd Cu Mg Mn Na Ni Pb Zn

Location (unpolluted area) Plackovica MountainU dioica leaves Plackovica 23281 plusmn 4 ltLDlowast 673 plusmn 006 4295 plusmn 13 2966 plusmn 008 5233 plusmn 03 ltLD 373 plusmn 01 1424 plusmn 01U dioica leaves Veles 37725 plusmn 5 734 plusmn 004 113 plusmn 003 64124 plusmn 25 7471 plusmn 014 1381 plusmn 27 289 plusmn 004 1022 plusmn 04 4653 plusmn 06

Location (polluted area) VelesU dioica stems Plackovica 9245 plusmn 5 ltLDlowast 716 plusmn 006 3366 plusmn 14 1516 plusmn 008 4643 plusmn 05 ltLD ltLD 2227 plusmn 03U dioica stems Veles 17816 plusmn 16 667 plusmn 003 825 plusmn 003 5036 plusmn 12 2463 plusmn 005 8695 plusmn 08 ltLD 2479 plusmn 02 2295 plusmn 13lowastLD is limit of detection (001mg kgminus1)

Table 2 Total antioxidants level in U dioica sampled from twodifferent areas obtained with FRAP assay (in 120583mol FeSO4 L

minus1)

Plant organ investigated FRAP valuesLocation (unpolluted area) Plackovica

MountainU dioica leaves Plackovica 48455 plusmn 73U dioica leaves Veles 18496 plusmn 25

Location (polluted area) VelesU dioica stems Plackovica 961 plusmn 19U dioica stems Veles 640 plusmn 11

accumulation by plants increased with increasing metalconcentration in environment

Results for total antioxidants level in samples sampledfrom two different areas obtain with FRAP assay are pre-sented in Table 2

RAPD profile generated by samples exposed to heavymetals was different from those obtained using control DNAThe RAPD profiles are presented in Figures 2 and 3 A sum-mary of results obtainedwith the primer set usedwith controland toxic metals exposed DNA samples is shown in Table 3Polymorphism (119875 in ) was calculated as the following

119875 = [119886 + 119887

119888] sdot 100 (1)

where 119886 is the number of new bands detected in samples (dif-ferent from the control) 119887 is the number of disappeared bandsand 119888 is the total number of scored bands Polymorphism inRAPD profiles included disappearance of a normal band andappearance of a new band in comparison to the control

Content of 1022mg kgminus1 of Pb was observed in U dioicaleaves in samples taken from the area around the lead andzinc smelting plant These results are corresponding to thefact that most uptake of Pb has been demonstrated to bethrough the leaves and fact that various authors [21 22]refer to U dioica as a plant possess a high natural potentialfor hyperaccumulation and hypertolerance of lead U dioicastems from the same location showed also high Pb content(2479mg kgminus1) In contrast in control plants sampled fromPlackovica Mountain a content of 373mg kgminus1 was mea-sured in plant leaves which is in the normal range for Pb inplants 01ndash10mg kgminus1 dw according to Kabata-Pendias and

Pendias [23] while generally toxic concentrations of Pb aredefined as 30ndash300mg kgminus1 [24]

Also extremely high values are determined for Zn contentin U dioica leaves from the Veles region Zinc is an essentialelement in all organisms and is not considered to be highlyphytotoxic where toxicity limit for Zn (300ndash400mg kgminus1)depends on the plant species as well as on the growth stage[23] According to the above mentioned criteria investigatedplants in this area are exposed to highly phytotoxic doses ofPb and Zn

This is valid also for Cd which is very toxic metal and asfar as is known Cd is not a constituent of any metabolicallyimportant compound Obtained values for Cd content (734and 667mg kgminus1) in U dioica leaves and stems respectivelyare also in the phytotoxic rangeThe normal limits of Cd con-tent in plants are between 02ndash08mg kgminus1 and toxic concen-trations of Cd are in the range of 5ndash30mg kgminus1 [23 24]

Although Cu is an essential micronutrient for plantgrowth it can be more toxic than nonessential Pb to biotawhen extraneous Cu is present in soil environments Plantcontents for Cu above 25mg kgminus1 are considered toxic toplants [25] According to this criterion investigated regionsare not highly polluted by copper since its content in all plantspecies did not exceed the upper limit

Nickel as a heavy metal belongs to a group of essentialmicroelements to plants animals and humans and itsamounts exceeding optimum values show a toxic effect Nicontents in plants range from 05 to 5mg kgminus1 dry weight andthe values exceeding these limits are reported as toxic [25]so in respect of the fact that Ni-uptake relies upon plant spe-cies and that some of plants show hyperaccumulation effectsinvestigated plant do not belongs in this group

The results above indicated that the investigated plantcontains large amounts of essential metals and again plantssampled fromVeles area are rich in this metal compared withcontrol samples The abundance of Mg Ca and Na in theresult of this analysis was in agreement with previous find-ings that these three elements represent the most abundantmetal constituents in plants [26 27]

The ability of plants to increase antioxidative protection tocombat negative consequences of heavy metal stress appearsto be limited since many studies showed that exposure toelevated concentrations of redox reactive metals resulted indecreased and not in increased activities of antioxidativeenzymes This fact is also valid for U dioica as shown in


Table 3 Changes in the RAPD profiles (the number of bands and molecular sizesmdashbp) related to metals exposition compared to controlldquo+rdquo appearance of DNA bands andor ldquominusrdquo disappearance of DNA bands for all primers in the U dioica plants

Plant PrimersPrimer 1 Primer 2 Primer 3 Primer 4 Primer 5 Primer 6 Primer 7

U dioica Plackovica 0 2500 0 0 0 0 0

U dioica Veles (+) 3000 (+) 0 (+) 2500 2000 1250 (+) 5000 2500 (+) 5000 4000 3000 (+) 2000 (+) 3000 2500(minus) 0 (minus) 2500 (minus) 0 (minus) 0 (minus) 0 (minus) 0 (minus) 0

1 2 M 1 2 M 1 2 M

(1) (2) (3)

Figure 2 RAPD profiles of U dioica sampled from two different areas obtained with different primers ((1) primer 1 (2) primer 2 and (3)primer 3) (1) U dioica Plackovica and (2) U dioica Veles M is DNA marker

1 2 M 1 2 M 1 2 M 1 2 M

(1) (2) (3) (4)

Figure 3 RAPD profiles of U dioica sampled from two different areas obtained with different primers ((1) primer 4 (2) primer 5 (3) primer6 and (4) primer 7) (1) U dioica Plackovica and (2) U dioica Veles M is DNA marker


studies of many authors [13 28 29] As FRAP assay mea-sures only nonenzymatic (reductants) antioxidants in thesample there is an interesting relationship amongmetal con-tent and obtained FRAP value valid for all investigated met-als which are redoxmetals Results obtained fromFRAP assayin this study show that heavy metals induces oxidative stressin experimental model system which is evident from anti-oxidant levels (in 120583mol FeSO

4Lminus1) as in all cases levels for

total antioxidant activity in samples exposed to metals arelower than total antioxidant level in control sample Anti-oxidant systems and their significance for the acclimationof plants to air pollution and climatic stresses have beenreviewed frequently with emphasis on the responses of leaves[30ndash32] Exposure to heavy metals also provoked responsesof antioxidative systems but the direction of response isdependent on the plant species tissue analyzed the metalused for treatment and also intensity of the metal stress [233] However some common reaction patterns can be foundfor example decreasing activities of antioxidative enzymesafter metal exposition In most cases exposure to heavymetals Cd and some others as Cu Ni and Zn initiallyresulted in a severe depletion of glutathione (GSH) which isonly one exampleThis is a common response toCd caused byan increased consumption of GSH for phytochelatin produc-tion and their role in sequestering heavy metals which isa mechanism that contributes to the protection from metaltoxicity in different plants and in some fungi as well [34]In average the samples exposed to metals in our study showfor 619 lower antioxidant activity from the control sample(leaves) and 335 lower antioxidants level for the stems(Table 2) Evident are higher values for total antioxidants inplant leaves which is in accordance with previously pub-lished results [35] where leaves from the plants are noted asplant organs richest with the antioxidants that prevent DNAdamages induced by heavy metal stress

RAPD technique as PCR-based technique has been suc-cessfully used to detect DNA damage andmutations in plantsinduced by various types of toxic chemicals [8 9 36] Eachfragment in RAPD is derived from a region of the genomethat contains two short segments in inverted orientationon opposite strands that are complementary to the primerand sufficiently close together for the amplification process[37] Polymorphism observed in RAPD profiles includeddisappearance andor appearance of bands in comparisonwith control samples that were evaluated (Figures 2 and 3)

The RAPD profiles obtained exhibited bands between1250 and 5000 bp in length In a total of 14 bands scored13 were polymorphic (9286) we scored total 1 band withprimer 1 1 with primer 2 3 with primer 3 2 with primer 4 3with primer 5 1 with primer 6 2 with primer 7 by means ofstatistics there is an average of 186 bands per primer (Table3) Amplificationwith primer 1 andprimer 6 yielded only onefragment with each of the primers and we find this primersequence not suitable for fingerprinting Urticarsquos genome

The samples exposed to metals yielded a large numberof new fragments (total 12) compared with only one disap-peared fragment in the obtained RAPD profile New RAPDamplification products may be related to mutations (newannealing events) large deletions (bringing to pre-existing

annealing site closer) andor homologous recombination(two sequences that match the sequences of primer) [7 38]The high number of new appeared bands that was observedin samples exposed to metals suggests that long-term expo-sition to metals in high doses probably cause mutationson genomic level in U dioica plants These unique bandsclearly differentiated the samples exposed to heavy metalstress and would act as a marker for assessment of environ-mental exposition on metals

Accordingly to the results it may be noted that the nutri-ent imbalance leads to DNA damages mutations on genomiclevel in case of U dioica and also effects plant antioxidativedefense systemwhich contributed to the toxic effects in plantsexposed to the long-term high metal concentrations

4 Conclusions

Heavy metal stress can decrease total antioxidants level andinduce DNA damage in U dioica The changes occurring inplants RAPD profiles following exposition to heavy metalscan be successfully used as a sensitive tool for detectingmetal-induced DNA damage and showed potential as a reliableassay for genotoxicity The obtained results in this study sug-gested that the mineral nutrient imbalance DNA damagesand decreased antioxidants levels were involved in the metaltoxicity inU dioicawhich may help to understand the mech-anisms of metals genotoxicity in plants

Conflict of Interests

The authors declare that they do not have any kind of conflictof interests andor any financial benefit from any trademarkmentioned in the paper

References

[1] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seedl-ingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006

[2] F van Assche and H Clijsters ldquoEffects of metals on enzymeactivity in plantsrdquo Plant Cell amp Environment vol 13 no 3 pp195ndash206 1990

[3] V Dixit V Pandey and R Shyam ldquoDifferential antioxidativeresponses to cadmium in roots and leaves of pea (Pisum sativumL cvAzad)rdquo Journal of Experimental Botany vol 52 no 358 pp1101ndash1109 2001

[4] S Verma andR S Dubey ldquoLead toxicity induces lipid peroxida-tion and alters the activities of antioxidant enzymes in growingrice plantsrdquo Plant Science vol 164 no 4 pp 645ndash655 2003

[5] I F F Benzie and J J Strain ldquoThe ferric reducing ability ofplasma (FRAP) as a measure of ldquoantioxidant powerrdquo the FRAPassayrdquo Analytical Biochemistry vol 239 no 1 pp 70ndash76 1996

[6] D Savva ldquoUse of DNA fingerprinting to detect genotoxiceffectsrdquo Ecotoxicology and Environmental Safety vol 41 no 1pp 103ndash106 1998

[7] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques appliedto genotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006


[8] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquoBiotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006

[9] G KekecM S Sakcali and I Uzonur ldquoAssessment of genotoxiceffects of boron on wheat (Triticum aestivum L) and Bean(Phaseolus vulgaris L) by Using RAPD analysisrdquo Bulletin ofEnvironmental Contamination and Toxicology vol 84 no 6 pp759ndash764 2010

[10] D Gjorgieva T Kadifkova-Panovska S Mitrev et al ldquoAssess-ment of the genotoxicity of heavy metals in Phaseolus vulgarisL as a model plant system by Random Amplified PolymorphicDNA (RAPD) analysisrdquo Journal of Environmental Science andHealth A vol 47 no 3 pp 366ndash373 2012

[11] J Diatta W Grzebisz and K Apolinarska ldquoA study of soilpollution by heavy metals in the city of Poznan (Poland)using dandelion (Taraxacum officinale Web) as a bioindicatorrdquoElectronic Journal of Polish Agricultural Universities vol 6 no2 01 2003

[12] European Environmental Agency Soil Pollution by Heavy Met-als Europersquos Environment The Dobris Assessment Office despublications Luxembourg 1995

[13] I Gulcin O I Kufrevioglu M Oktay and M EBuyukokuroglu ldquoAntioxidant antimicrobial antiulcer and ana-lgesic activities of nettle (Urtica dioica L)rdquo Journal of Ethno-pharmacology vol 90 no 2-3 pp 205ndash215 2004

[14] A Tahri S Yamani A Legssyer et al ldquoAcute diuretic natri-uretic and hypotensive effects of a continuous perfusion ofaqueous extract ofUrtica dioica in the ratrdquo Journal of Ethnophar-macology vol 73 no 1-2 pp 95ndash100 2000

[15] J J Lichius and C Muth ldquoThe inhibiting effects ofUrtica dioicaroot extracts on experimentally induced prostatic hyperplasiain the mouserdquo Planta Medica vol 63 no 4 pp 307ndash310 1997

[16] National Environmental Action Plan (NEAP) Ministry ofUrban planning construction and environment Skopje Mace-donia 1996

[17] L Barandovski M Cekova M V Frontasyeva et al ldquoAtmo-spheric deposition of trace element pollutants in Macedoniastudied by the moss biomonitoring techniquerdquo EnvironmentalMonitoring and Assessment vol 138 no 1ndash3 pp 107ndash118 2008

[18] T Stafilov R Sajn Z Pancevski B Boev M V Frontasyevaand L P Strelkova Geochemical Atlas of Veles and the EnvironsFaculty of natural Sciences and mathematics-Skopje SkopjeMacedonia 2008

[19] T Stafilov R Sajn Z Pancevski B BoevM V Frontasyeva andL P Strelkova ldquoHeavy metal contamination of topsoils arounda lead and zinc smelter in the Republic of Macedoniardquo Journalof Hazardous Materials vol 175 no 1ndash3 pp 896ndash914 2010

[20] D Gjorgieva T Kadifkova-Panovska K Baceva and T StafilovldquoAssessment of heavymetal pollution in Republic ofMacedoniausing a plant assayrdquo Archives of Environmental Contaminationand Toxicology vol 60 no 2 pp 233ndash240 2011

[21] A P Murphy M Coudert and J Barker ldquoPlants as biomarkersfor monitoring heavymetal contaminants on landfill sites usingsequential extraction and inductively coupled plasma atomicemission spectrophotometry (ICP-AES)rdquo Journal of Environ-mental Monitoring vol 2 no 6 pp 621ndash627 2000

[22] M Grubor ldquoLead uptake tolerance and accumulation exhib-ited by the plants Urtica dioica and Sedum spectabile in con-taminated soil without additivesrdquoArchives of Biological Sciencesvol 60 no 2 pp 239ndash244 2008

[23] A Kabata-Pendias and H Pendias Elements in Soils and PlantsCRC Press Boca Raton Fla USA 2nd edition 1992

[24] A Kloke D C Sauerbeck andH Vetter ldquoThe contamination ofplants and soils with heavy metals and the transport of metalsin terrestrial food chainsrdquo inChangingMetal Cycles andHumanHealth J O Nriagu Ed pp 113ndash141 Springer Berlin Germany1984

[25] S E Allen Chemical Analyses of Ecological Material BlackwellScientific Publications Oxford UK 2nd edition 1989

[26] A Maiga D Diallo R Bye and B S Paulsen ldquoDeterminationof some toxic and essential metal ions in medicinal and edibleplants from Malirdquo Journal of Agricultural and Food Chemistryvol 53 no 6 pp 2316ndash2321 2005

[27] M M Ozcan and M Akbulut ldquoEstimation of minerals nitrateand nitrite contents of medicinal and aromatic plants used asspices condiments and herbal teardquo Food Chemistry vol 106 no2 pp 852ndash858 2008

[28] S Meir J Kanner B Akiri and S Philosoph-Hadas ldquoDeter-mination and involvement of aqueous reducing compounds inoxidative defense systems of various senescing leavesrdquo Journalof Agricultural and Food Chemistry vol 43 no 7 pp 1813ndash18191995

[29] Y S Velioglu GMazza L Gao and B D Oomah ldquoAntioxidantactivity and total phenolics in selected fruits vegetables andgrain productsrdquo Journal of Agricultural and Food Chemistry vol46 no 10 pp 4113ndash4117 1998

[30] G Noctor and C H Foyer ldquoAscorbate and glutathione keepingactive oxygen under controlrdquo Annual Review of Plant Biologyvol 49 pp 249ndash279 1998

[31] K Asada ldquoThe water-water cycle in chloroplasts scavengingof active oxygens and dissipation of excess photonsrdquo AnnualReview of Plant Biology vol 50 pp 601ndash639 1999

[32] A Schutzendubel and A Polle ldquoPlant responses to abioticstresses heavy metal-induced oxidative stress and protectionby mycorrhizationrdquo Journal of Experimental Botany vol 53 no372 pp 1351ndash1365 2002

[33] O Shainberg B Rubin H D Rabinowitch Y Libal and E Tel-Or ldquoAcclimation of beans to oxidative stress by treatment withsublethal iron levelsrdquo Journal of Plant Physiology vol 157 no 1pp 93ndash99 2000

[34] T Ishikawa Z-S Li Y-P Lu and P A Rea ldquoThe GS-X pumpin plant yeast and animal cells structure function and geneexpressionrdquo Bioscience Reports vol 17 no 2 pp 189ndash207 1997

[35] T Gichner ldquoDNA damage induced by indirect and direct actingmutagens in catalase-deficient transgenic tobacco cellular andacellular comet assaysrdquo Mutation Research vol 535 no 2 pp187ndash193 2003

[36] S Cenkci M Yildiz I H Cigerci M Konuk and A BozdagldquoToxic chemicals-induced genotoxicity detected by randomamplified polymorphic DNA (RAPD) in bean (Phaseolus vul-garis L) seedlingsrdquo Chemosphere vol 76 no 7 pp 900ndash9062009

[37] C C Hon Y C Chow F Y Zeng and F C C Leung ldquoGene-tic authentication of ginseng and other traditional Chinesemedicinerdquo Acta Pharmacologica Sinica vol 24 no 9 pp 841ndash846 2003

[38] H de Wolf R Blust and T Backeljau ldquoThe use of RAPD inecotoxicologyrdquoMutation Research vol 566 no 3 pp 249ndash2622004

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MEDIATORSINFLAMMATION

of


R Macedonia edible used in medicinal purposes and alsofrequently used as a model plant in different studies [13ndash15]The objective of the present study was to investigate thusexposure to different metals can induce direct DNA damageand significant changes in metal content in the plant and alsoendogenous total antioxidants level

2 Materials and Methods

21 Sampling Area Plant samples from the industrializedarea were taken from 10ndash100m around the lead and zincsmelting plant ldquoMHKZletovordquo inVeles area while for uncon-taminated controls samples were taken from PlackovicaMountain about 60 km from the city of Veles (Figure 1)Leaves and stems from plants were analyzedThe plants wereidentified and specimens are deposited at the Departmentof Pharmacognosy Faculty of Pharmacy Skopje Republicof Macedonia Element analysis FRAP analysis and DNAextraction were performed

22 Sample Preparation for Element Analysis All plant sam-ples not rinsed were air dried milled in a nonmetal micro-hammer and stored in clean paper bags 05 g was weighedand placed into PTFE vessels with 5mL HNO

3(69 Merck

Tracepur) and 2mL H2O2(30 mV Merck) mixture was

digested by microwave (MARS CEM XP 1500) with twosteps procedure at 180∘C Digests were filtered on filter paper(Munktell) quantitatively transferred in 25mL calibratedflasks diluted with demineralized water and analyzed byinductively coupled plasma-atomic emission spectrometer(ICP-AES) Varian 715-ES for selected metals Standards ofselected metals were set by dilution of stock standards whichwere prepared using analytical grade salts of metals (MerckMultielement standard 1000mgL) Samples were analyzed intriplicate All results were calculated on a dry weight basis(mg kgminus1 dw)

23 FRAP Assay The total antioxidant power of a freshlyprepared cooled and filtered infusion (5 g of dry leaves orstems100mL of boiling distilled water) of each sample wasmeasured using the FRAP assay In the FRAP assay reduct-ants (antioxidants) in the sample reduce Fe3+tripyridyltri-azine complex present in stoichiometric excess to the bluecolored ferrous form with an increase in absorbance at595 nm Samples were analyzed using microplate reader(ChemWell) at 600 nmThe antioxidant status is expressed as120583mol FeSO

4Lminus1 All values are means of triplicate analyses plusmn

SD

24 Genomic DNA Isolation Frozen plant samples were usedforDNA isolation 05 to 07 cmdisks of leaf tissuewere cattedwith standard one-hole paper punch Samples were kept onice while the procedure was done DNA extractions wereperformed using REDExtract-N-Amp Plant PCRKit (Sigma-Aldrich) following the instructions of themanufacturer Plantdisk was placed into a 15mL microcentrifuge tube with100 120583L extraction solution and incubated for 10 minutes at95∘C 100 120583L of dilution solution is added and vortexesExtract is stored at 2ndash8∘C until use

Figure 1 City of Veles and Plackovica Mountain as sampling areas

25 RAPD Amplification Methods PCR reactions were per-formed using REDExtract-N-Amp Plant PCR Kit (Sigma-Aldrich) PCR reactions were performed in reaction mix-tures of 20120583L containing 10 ng of genomic DNA 04120583Mprimer (Sigma-Aldrich) and 10120583L REDExtract-N-Amp PCRreaction mix The REDExtract-N-Amp PCR reaction mix isa ready mix containing buffer salts dNTPs and REDTaqDNA polymerase Sequences (51015840 rarr 31015840) from primer 1 to 7(with 60ndash70 GC content) used are GGTGCGGGAA (P1)GTTTCGCTCC (P2) GTAGACCCGT (P3) AAGAGCC-CGT (P4) AACGCGCAAC (P5) CCCGTCAGCA (P6)GGCACTGAGG (P7) respectively Amplifications were per-formed in a DNA thermocycler (Mastercycler personalEppendorf) programmed for 5min at 95∘C (initial dena-turing step) 45 consecutive cycles each consisting of 1minat 95∘C (denaturing) 1min at 36∘C (annealing) 2min at72∘C (extension) and followed by the last cycle for 5minat 72∘C (final extension step) Negative controls with waterwithout any template DNA were always included to monitorfor contamination After amplification electrophoresis ofRAPD reaction products was performed in 2 (wv) agarose(Agarose 1000 Invitrogen) using a TBE (TrisborateEDTA)buffer system (1 times TBE = 90mM tris base 90mM boric acidand 2mM EDTA) DNA bands were stained with ethidiumbromide for 10 minutes visualized and photographed underUV light (Biometra) All amplifications were repeated twicein order to confirm the reproducible amplification of scoredfragments Only reproducible and clear amplification bandswere scored for the construction of the data matrix

3 Results and Discussion

Veles area (around lead and smelting plant) was chosen asan investigated area because it is an important source oflead and zinc pollution in R Macedonia with estimatedlead emission of 83 tons per year according to the NationalEnvironmental Action Plan (NEAP) [16] and there wereseveral investigations in the region of Veles for heavy metalscontents [17ndash20] As shown in Table 1 varying amounts ofmetal contents were noted not only for the heavy metalsbut also for essential metals Levels of metals uptake and







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ToxinsJournal of

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AddictionJournal of






Autoimmune Diseases




Journal of


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4 Conclusions




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Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






1 2 M 1 2 M 1 2 M

(1) (2) (3)


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4 Conclusions




References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










4 Conclusions




References












































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

influence of heavy metal stress on antioxidant status and dna

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