Induction of Oxidative Stress and Anatomical Changes by Polycyclic Aromatic Hydrocarbons in Medicago sativa L.

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1 JCHR (218) 8(1), M. Mirzaie et al / Journal of Health Risks 7() (217) Journal of Chemial Health Risks ORIGINAL ARTICLE Indution of Oxidative Stress and Anatomial Changes y Polyyli Aromati Hydroarons in Mediago sativa L. Leyla Jafari 1, Maryam Khoshsokhan-Mozaffar *1, Elaheh Vatankhah 2 1 Department of Biology, Basi siene faulty, Qom Branh, Islami Azad University, Qom, Iran 2 Department of Biology, Faulty of Sienes, University of Zanjan, Zanjan, Iran (Reeived: 23 Novemer 217 Aepted: 29 Feruary 218) KEYWORDS Anthraene; Environmental pollution; Mediago sativa; Oxidative stress; PAHs ABSTRACT: In this study effet of anthraene on germination, anatomy and oxidative stress in Mediagosativa was evaluated. Seed germination, length and weight of seedlings were measured after seven days of treatment (2 and 4 mmol L -1 ). After twelve days, anatomial hanges and ativity of Superoxide Dismutase, Polyphenol Oxidase, Asorate Peroxidase, Glutathione Transferase, Solule Peroxidase, Malondialdehyde in shoots and roots, as well as hlorophyll ontent of aerial parts, were determined. Also, morphologial hanges during the growth in omplete plants were studied. The results showed that, anthraene had no signifiant effet on seed germination, ut redued the length of seedlings and the weight of them. The ativity of mentioned enzymes in the shoot and often in the roots, in 4 mmol L -1 anthraene signifiantly was inreased ompared to the ontrol plants. Anthraene treatment dereased signifiantly Malondialdehyde levels in shoot, while it inreased signifiantly in roots and this treatment had no signifiant effet on hlorophyll a and ontents. Periderm diameter inreased in treated roots and xylem extent redued in treated shoots. It seems that the low water soluility of anthraene, also, the low sensitivity of alfalfa to PAHs, partially stailize the plant to low onentration of Anthraene. INTRODUCTION By inreasing environmental pollution, the study of aioti stress responses in plants has eome ever more important in agriulture, forest management, and eosystem restoration strategies [1-3]. Organi pollutants aumulate in vegetation [4, 5] and an ause health prolems [6, 7]. Some of the studied stresses fators are inlude: heavy metals, ozone, UV (Ultraviolet) light, salinity, and drought. The polyyli aromati hydroarons (PAHs) pollutants are poorly studied as stress induers. PAHs are organi ompounds omposed of multiple aromati rings, whih make these moleules highly persistent in the environment. Many PAHs in the environment are typially found as omplex mixtures. Lower-temperature omustion, suh as toao smoking or wood-urning, tends to generate low moleular weight PAHs, whereas high-temperature *Corresponding Author: m.khoshm@gmail.om, m-khoshsokhan@qom-iau.a.ir (M. Khoshsokhan-Mozaffar) 19

2 L. Jafari et al / Journal of Chemial Health Risks 8(1) (218) industrial proesses typially generate PAHs with higher moleular weights [8]. Hydroaron aumulation in the rhizosphere an hallenge vegetation eause growth parameters suh as protein, arohydrate, and lipid omposition are related to photosyntheti ativity of plants. PAH metaolism generates reative eletrophili metaolites, whih are the atual arinogeni ompounds that ause DNA (Deoxy nuleotide adenosine) damage [9]. Furthermore, PAHs trigger the prodution of reative oxygen speies and ell death in animal [1-12] and plant [13-15] ells. Alkio et al. (25) and Liu et al. (29) in separate works treated Araidopsis speies with phenanthrene. They indiated that Morphologial hanges and oxidative stress symptoms appeared due to phenanthrene treatment. In another study, inreasing of anthraene remediation y transgeni toao plants was shown. Furthermore, high expression of fungal GST (Glutathione Transferase) was indiated as an index of anthraene tolerane [14]. Also, various plant speies suh as Carexgrailis [16], Popolusspp. [17-19], Spirodella puntate [2], have een shown to tolerate and phytodegrade xenoioti pollutants [21]. Generally, xenoioti detoxifiation in plants involves transformation, onjugation (for example with gluose or glutathione) and sequestration in the vauole or ell wall [22]. However, the plant genes responsile for PAH uptake, degradation, and onjugation are largely unknown. Mehanisms of the PAH toxiity to plants are poorly understood, and the phytotoxiity appears to vary depending on the partiular PAH and plant speies [23, 24]. The ojetive of this study was to investigate the anthraene (a persistent 3-ring polyaromati hydroaron present in dyes, wood preservatives) effet on physiologial, morphologial and anatomial hanges of Mediago sativa (as a forage and eonomi plant) in hydroponi system. MATERIALS AND METHODS Seed germination, length and weight of seedlings Seeds of Mediago sativa L. with germination potential were surfae sterilized with.5% sodium hypohlorite for 2 min, thoroughly rinsed with deionized water and then germinated in rolls of neutral PH germtest paper under laoratory ondition in Islami Azad University of Qom. Anthraene was dissolved in aetone to prepare 1 mol L -1 anthraene stok. This stok was used for treatments. The seeds divided to four groups with four repliates: ontrol, solvent (aetone), 2 mmol L -1 anthraene and 4 mmol L -1 anthraene [14]. After seven days germination perent, length, dry and fresh weight of seedlings was measured. Plant materials, growth and treatment onditions After seven days, the seedlings that normally germinated, were seleted and transferred to pots ontaining hydroponi ulture and kept there for 24 days, in order to adapt to hydroponi onditions. Growth environment temperature was set at aout 27±3. In order to prevent the penetration of light, the pots were overed with aluminum foil. It should e noted, plants do not ome into ontat with aluminum. Medium pots were replaed every five days and every day the amount onsumed from the pots was added. The ulture was aerated with an air pump. Hoagland modified nutrient solution (1/2) ontains maro and miro elements are as follows (ph =6):(in mmol L -1 ):Ca(NO 3 ) 2.4H 2 O:2.5; KNO 3 :2.5; MgSO 4.7H 2 O:1; KH 2 PO 4 :.5, (Values in ppm): Fe-EDTA:3; H 3 BO 3 :.5; Mn (MnCl 2.):.5; Zn(ZnCl 2 ):.5 Cu(CuCl 2.2H 2 O):.2; Mo(Na 2 MoO 4 ):.2 After 24 days of adaptation, the plants were treated with anthraene at onentrations of 2 mmol L -1 and 4 mmol L -1 for 12 days. Eah of the four groups had five repetitions (pots) and eah pot ontained ten plants. The appearane of plants and roots were oserved during the 52

3 L. Jafari et al / Journal of Chemial Health Risks 8(1) (218) growth stages. After the twelfth day, treatment and ontrol samples (5 repetitions eah) were harvested. A ath of them were washed thoroughly with deionized water, frozen in liquid N 2 and kept at 8 C for further analytial experiments. The seond ath were fixated in FAA solution for anatomial studies. Anatomial Studies Cross setions of root and shoot in four groups were made y hand using ommerial lades. The setions were stained with douled armine- methylene lue. Cortial thikness, the numer of vasular undles, et. were oserved using Nikon optial mirosope. Biohemial Analysis All iohemial analyzes were arried out on the shoot and root separately (exept hlorophyll on aerial parts). 2 mg of fresh tissues were homogenized in 3 ml of.1 mol L -1 potassium phosphate uffers, ph 6.8. Then, the homogenate was entrifuged at 12, g for 2 min and the supernatant was used as soure of rude enzyme. All steps to otain enzyme preparation were arried out at 4ºC. The ativity of SOD (superoxide dismutase) was determined y adding 5 μl the rude enzyme preparation to a solution ontaining 13 mmol L -1 metionine, 75 μmol L - 1 p-nitro lue tetrazolium hloride (NBT), 1 nmol L -1 EDTA and 2 μmol L -1 rioflavin in a 5 mmol L -1 sodium phosphate uffer, ph 7.8 [25]. The reation took plae in a hamer under illumination the lue formazane produed y NBT photoredution was measured y the inrease in asorane at 56 nm. The lank mixture had the same omposition ut it was kept in the dark. One SOD unit was defined as the amount of enzyme required to inhiit 5% of the NBT photoredution. Ativity of APX (Asorate peroxidase) was measured aording to the method of Nakano and Asada [26]. In rief, samples were homogenized in 1 ml of 5 mmol L -1 Na-phosphate uffer (ph = 7.8) ontaining 5 mmol L -1 asorate, 5 mmol L -1 DTT (Dithiothreitol), 5 mmol L -1 EDTA (Ethylene diamide tetra aeti aid), 1 mmol L -1 NaCl and 2% (w/v) PVP. The homogenate was entrifuged as 15g for 15 min at 4 C. The reation was initiated y adding H 2 O 2. The reation rate was monitored y the derease in asorane at 29 nm. Protein ontents were determined y the method of Bradford [27] using BSA (Bovine serum alumin) as standard. PO (Peroxidase) ativity was determined y adding 1 μl of the rude enzyme preparation, diluted in the proportion of 1:25 (v/v), to 4.9 ml of a solution ontaining 25 mmol L -1 potassium phosphate uffer, ph 6.8, 2 mmol L -1 pyrogallol and 2 mmol L -1 H 2 O 2. After inuation of the solution at 25C for 1 min, the reation was stopped y adding.5 ml of 5% (v/v) H2SO4 and the asorane was read at 42 nm [28]. The enzyme ativity was alulated using the molar extintion oeffiient of 2.47 mmol L -1 m -1. The ativity of PPO (Polyphenol oxidase) was determined using the same methodology desried efore for PO ut omitting H 2 O 2 from the reation mixture. The enzyme ativity was alulated as desried efore for PO. GSTs ativity was assayed spetrophotometrially at 25 C with redued glutathione (GSH) and 1- hloro-2, 4-dinitroenzene (CDNB) as sustrates. This was done y wathing an inrease in asorane at 34nm [29]. Malondialdehyde onentration was measured y the method of De Vos et al. (1991). MDA forms olor omplexes y omining with thioarituri aid (TBA) its onentration eing measured y a spetrophotometer [3]. The ontent of Chlorophylls a and was estimated y the method desried y Horwitz et.al (199). Chlorophyll extration from leaf material was arried out with 8% (v/v) aetone. The asorane of resulting supernatant was reorded at 664 and 647 nm. The onentra- 53

4 Weight (g) Germination perent Seedling length (mm) L. Jafari et al / Journal of Chemial Health Risks 8(1) (218) tions of Chls a and were alulated, using the following formulae [31]: (SPSS ver. 16) and differenes among treatments were evaluated y Dunan Test (p.5). Chl a = [12.7 (D 663) 2.69 (D 645)] V/1 W RESULTS Chl = [22.9 (D 645) 4.28 (D 663)] V/1 W STATISTICAL ANALYSIS Experiments followed a randomized omplete lok design. Three explants per pot and three repliations per treatment were tested. Analysis of variane was performed y the General Linear Model proedure Effet of anthraene on Seeds The results showed that anthraene treatment didn t hange germination perent, ut seedling length and iomass (in 4 mmol L -1 ) dereased ut dry and fresh weight inreased in 2 mmol L -1 after seven days of treatment (Figures 1 & 2). Figure 1. The effet of Anthraene treatment on germination and seedling length in (a) ontrol () aetone () 2 mmol L -1 anthraene (d) 4 mmol L -1 anthraene after 7 days. (n=4) Germination Seedling length a 2 solvent solvent Weight of seedlings Fresh weight mean Dry weight mean.2.1 solvent.2.4 Figure 2. (a) Germination perent, () Seedling length and () Fresh and dry seedling weights of Mediago sativa under anthraene treatment. (n=4). error ars represent ± SD. Symols * and represent signifiant differenes at p.5 ompared to the ontrol and solvent (=aetone) groups, respetively. 54

5 L. Jafari et al / Journal of Chemial Health Risks 8(1) (218) Plant growth responses against anthraene Plants whih were treated with anthraene showed great stress features, e.g. the vitality and growth of the aerial shoots fell due to anthraene stress (Figure 3). Anthraene engulfed the roots so that they lost their steady state (Figure 4). The symptoms of stress inreased, oviously, in 4 mmol L -1 anthraene. Also, the roots treated with anthraene inreased numer of hairs (Figure 5). Plants grown at a onentration of 2 mmol L -1 anthraene, were ale to ope with stress. At 4 mmol L -1 anthraene, alfalfa seemed to have survived for more than two weeks. Figure 3. Aerial parts of plants in the 8th day of treatment. (a) ontrol () Aetone () 2 mmol L -1 anthraene (d) 4 mmol L -1 anthraene. Figure 4. Roots of plants on the 8th day of treatment: (left) ontrol (right) treated with 4 mmol L -1 anthraene. The arrows show the overage of anthraene around the roots. Figure 5. The root on the 8th day: (left) ontrol and (right) treated with anthraene with 2 and 4 mmol L

6 Rooting and ullusing perent L. Jafari et al / Journal of Chemial Health Risks 8(1) (218) Effet of Anthraene on Anatomial hanges Anthraene inreased the thikness of periderm around the roots, reating to stress with a ork-like overing, while the ross setions of the ontrol group did not display the suerin layer around the root epidermis (Figure 6). The length of undles in stem started to derease after anthraene treatment (Figure 7) a a d d f d e f late June late August Rooting Cullusing Interation effets time and IBA hormone (mg l -1 ) ( Figure 6. A ross-setion of Mediago sativa roots (1X). a: ontrol : solvent (=aetone) : 2 mmol L -1 anthraene treatment d: 4 mmol L -1 anthraene treatment. ep: the epidermis, pa: parenhyma, en: endoderm, p: periyle, ph: phloem, x: xylem, suer: suerin. Figure 7. A view of the largest undle in Mediago sativa leaf (4X) after 12 days. a: ontrol : solvent (=aetone) : 2 mmol L -1 anthraene treatment d: 4 mmol L -1 anthraene treatment. ph: phloem, x: xylem, s: slerenhyma. The lines show the extent of xylem ells. Effet of Anthraene on antioxidant responses SOD: The results of measuring the ativity of the superoxide dismutase in aerial shoots of Mediago sativa revealed that anthraene treatment led to an inrease in the regular ativity of this enzyme, signifiantly. This inreasing at 4 mmol L -1 of anthraene was more ovious in shoot. Also, SOD ativity elevation was oserved in the roots of the treatment groups (Figure 8a). SPO: anthraene treatment led to an inrease in the regular ativity of this enzyme in aerial organs. This elevation in the stems at 4 mmol L -1 was ovious. Aording to the results, ativity hanging of solule peroxidase in roots in the treated groups was not signifiant at the.5 p. (Figure 8). PPO: The results of the enzyme in the stem indiated 56

7 L. Jafari et al / Journal of Chemial Health Risks 8(1) (218) that ompared to ontrol plants, the enzyme ativity at 2 and 4 mmol L -1 anthraene is signifiantly inreased (Figure 8). APX: Aording to the results of ativity of the enzyme APX in shoots of alfalfa in 4 mmol L -1 anthraene ompared to the ontrol treatment, has inreased as well. in roots treated in oth 2 and 4 mmol L -1 anthraene the ativity of this enzyme inreased signifiantly (Figure 8d). GST: The results of measuring of GST in shoots of alfalfa showed that treatment of anthraene on the glutathione transferase had no effet, signifiantly, treatment inreased the enzyme ativity in the roots (Figure 8e). MDA: In shoots of alfalfa, treated with 2 and 4 mmol L - 1 anthraene dereased MDA signifiantly and in ontrast, the results of the measurements MDA in roots showed that treatment of anthraene inreases MDA, ompared to ontrol plants (Figure 8f). Chlorophyll ontent Changes of hlorophyll a and ontent in treated plants were not signifiant ompared with ontrol groups (Figure 8g). 57

8 The Content of Chl a, (mg/g FW) Lipid peroxidation rate M Mda /g FW APX ativity ΔA29/mg protein Root numer SOD ativity ΔA56/mg protein PPO ativity ΔA47/mg protein M. Khoshsokhan-Mozaffar et al / Journal of Chemial Health Risks (218)8(1) SOD ativity Shoot Root SPO ativity a 1.5 a a 8 7 solvent.2.4 solvent.2.4 GST ativity ΔA34/mg protein PPO ativity ΔA41/mg protein d PPO ativity solvent.2.4 d d APX ativity solvent.2.4 e GST ativity.5 solvent.2.4 ppm 1ppm 2ppm 4ppm 6ppm ppm 1ppm 2ppm 4ppm 6ppm late June late August solvent.2.4 f MD g Chl a, ontent Chl a Chl Intration effets time and hormone solvent.2.4 Figure 8. The effets of two onentrations of Anthraene on (a) SOD () SPO () PPO (d) APX (e) GST (f) MDA (g) Chl a,. in shoot and root system after 12 days. Error ars represent ± SD. (n=5). Symols * and represent signifiant differenes at p.5 ompared to the ontrol and solvent (=aetone) groups, respetively. 58

9 L. Jafari et al / Journal of Chemial Health Risks 8(1) (218) DISCUSSION Past studies reported phytotoxiity of PAHs to various plant speies. Alkio et al. [13] doumented several effets of phenanthrene to Araidopsis thaliana inluding inhiition of growth and root development and indution of leaf lesions. In another study, Zhung et al. [32] showed Polynulear aromati hydroarons differentially influene growth of various on emergent wetland speies. In other words, the different result is eause of the different sensitivity of the plants speies. In most studies, PAHs have no signifiant effet on germination [33, 34]. Paskova et al. [33], For instane, with the effet of PAHs and N-heteroyli derivatives (NPAHs) on plants, showed only NPAHs have a negative effet on germination, ut PAHs do not affet seed germination. In onordane with those, present investigation indiated anthraene treatment did not effet on seed germination (Figure 1) ut susequent growth of seedlings suh as length, fresh and dry weight signifiantly dereased, espeially in 4 mmol L -1 anthraene (Figure 2). Aording to this researh, Smith et al. (26) reported that germination of seven plant speies from grasses and legumes in soil polluted with PHAs was unaffeted ut susequent growth was signifiantly diminished[34]. Therefore, germination studies alone would not predit the suess of susequent growth of the speies tested in the ranges of PHAs levels. Also, the iomass of seedlings at 2 mmol L -1 anthraene had signifiant inreasing in ompared with ontrol plants (Figure 2). It would appear that low onentration of anthraene simulates the growth of seedlings ut moreonentration of anthraene inhiitsthe growth. Fan et al. (28) reported that alfalfa root and shoot growth and dry weight were redued in soil polluted with pyrene [35]. Also, Ahmadi et al. (213) proposed that the iomass of three plant speies was dereased with inreasing the pollution level [36]. Alkio et al. (25) reported that the transript levels of expansin protein in phenanthrene treated Araidopsis plants were redued [13]. Expansins have known in ell wall loosening and ell enlargement. The redution in expansin suggests thagrowth redution may largely e due to inhiition of ellenlargement. Despite the importane of PAHs stresses in Plants, limited information is availale aout the effet of PAHs stress on anatomial responses. In this study, anthraene was affeted root and stem morphology and anatomy (Figures. 3-7). On the hydroponi ultivation of alfalfa with phenanthrene treatment, Flooet al. (22) reported that morphologial symptoms suh as leaf hlorosis and nerosis, lak of living plant ells and lak roots with exessive ranhing an e oserved [37]. It seems that plants try to inrease their ompatiility against stress, through developing lateral roots and root hairs to asor water and minerals, so that their resistane is enhaned [38]. Furthermore, ondensed anthraene around root hairs (espeially in 4 mmol L -1 ) ats as a arrier of water and nutrients. The severity of stress aused damage to the shoot at 4 mmol L -1 anthraene. Anatomial analysis indiated that anthraene treatment inreased the thikness of periderm (a suerin layer) around root epiderm layer while suh suerin layer was not oserved in roots of ontrol plants (Figure 6). It appears that presene of the periderm around the root was resulted more resistane against stress. Also, the diameter of xylem vessel undles in stem of anthraene-exposed plants was dereased and this redution in 4 mmol L -1 anthraene was higher (Figure 7). Zhan et al. (215) suggested that higher onentration of phenanthrene in roots resulted the larger driving fore for its transport from root to shoot [39], it appears that redution of xylem vessel undles diameter with inreasing of anthraene onentration an e a defensive mehanism against the massive transfer of anthraene from root to shoot. The effets of PAH Toxiants on oxidative stress in plants, are less doumented. Our data, together with 59

10 L. Jafari et al / Journal of Chemial Health Risks 8(1) (218) some reports, indiated that PAHs exposure auses an oxidative stress response in plants [5, 15]. In this regards, plant and animal ells respond similarly to PHAs exposure, as shown y the prodution of H 2 O 2 and loalized ell death in Araidopsis[13]. To prevent damage to ellular omponents y ROS, plant have developed a omplex antioxidant system suh as SOD, CAT (atalase), PO, APX and glutathione related enzymes [15]. Our results showed that anthraene treatment inreased signifiantly the SOD, APX and PPO ativities in root and shoot of M. sativa ut just, inreasing of PO in shoot system and glutathione transferase (GST) ativities in root was signifiant (Figure 8). GST, a family of enzymes responsile for detoxifiation of a road range of xenoiotis inluding heriides y onjugating them with glutathione, may e a useful andidate for detoxifiation of PHAs[14]. It has een proposed that oth PPO and PO play important roles in the metaolism of aromati ompounds in soil and water [37, 4]. Also, ativity of antioxidant enzymes suh as SOD, PO, CAT and APX inreased with phenanthrene in Araidopsis [15]. Aording to our results, it appears upregulation of antioxidant enzymes in shoot and root system of M. sativa an limit hemial damage due to high levels of ROS. Also, our data indiated that ativities of root enzymes often inreased signifiantly in oth anthraene onentrations (2 and 4 mmol L -1 ). Baldyga et al. (25) reported that anthraene was present in all pea plant organs with its greatest amount in the roots. Therefore, high onentration of anthraene in root auses the eginning of an earlier oxidative stress response [41]. Anthraene treatment dereased signifiantly MDA level in shoot of M. sativa in oth onentrations ompared to the ontrol plants while it inreased signifiantly in root. High aumulation of anthraene and overoming antioxidant system in roots auses inreasing of lipid peroxidation and MDA level. Also, low MDA level in shoot an e ahieved y anthraene aumulation in the alfalfa root and effiieny of photosyntheti system, as well as, modulated onentrations of antioxidant enzymes. Despite that Liu et al. (29) reported that MDA level in leaf tissue of Araidopsis after phenanthrene treatment inreased signifiantly. In the present study, no signifiant differenes in total hlorophylls, hl a and hl ontents of alfalfa leaves of plant exposed to anthraene in ompare to nonexposed ones oserved. Our results agree with data reported y Floo et al. (22). He expressed that total hlorophyll ontents of alfalfa leaves of plants exposed to 5 mg L - 1 phenanthrene didn t hange signifiantly at the end of experiment. Inreased inhiition of photosynthesis during photomodifiation of anthraene was reported in the aquati higher plant Lemnagia [42]. Aording to otain results, it appears that the photosyntheti apparatus of alfalfa an tolerate 2 mmol L -1 anthraene. However, in oth onentrations (2 & 4 mmol L -1 ), oxidative stress enzymes inreased. Anthraene did not have a signifiant effet on germination, hlorophyll ontent, photosyntheti effiieny and plant vigor in aerial part at the 2mM anthraene. CONCLUSIONS The findings of this study support the some hypotheses. First, the sensiility of alfalfa, like some other memers of Faaeae family is not very high. Seond, low water soluility of anthraene makes it less aessile to the plant roots and onsequently, third, alfalfa an tolerate oxidative stress of 2 mmol L -1 anthraene and it an e as a monitoring plant for this PAH. This result ould e applied for developing tolerane to realitrant environmental pollutants suh as anthraene using transgeni approah and phytoremedation of PAHs. ACKNOWLEDGEMENTS This work done as a M.S. thesis and was supported y Islami Azad University, Qom ranh. 6

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