Stress response and potential biomarkers in spinach (Spinacia oleracea L.) seedlings exposed to soil lead

Ecotoxicology and Environmental Safety 74 (2011) 41–47

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Stress response and potential biomarkers in spinach (Spinacia oleracea L.) seedlings exposed to soil lead$ Chengrun Wang a,b, Xueyuan Gu a, Xiaorong Wang a,, Hongyan Guo a, Jinju Geng a, Hongxia Yu a, Jian Sun a a b

State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Nanjing University, Nanjing 210093, China School of Life Science, Huainan Normal University, Huainan 232001, China

a r t i c l e in fo

abstract

Article history: Received 18 July 2008 Received in revised form 20 February 2009 Accepted 21 February 2009 Available online 8 October 2010

Oxidative stress and biochemical responses of spinach seedlings to soil Pb stress were investigated by pot experiments. The seedlings were exposed to 0–500 mg kg  1 extraneous Pb. After 30 days of germination, production of Od2  , HSP 70, HSP 60, superoxide dismutase (SOD) activities, carbonyl groups and lipid peroxidation was significantly induced by soil Pb. After 50 days, HSP 70 and HSP 60 decreased, and HSP 60 was significantly inhibited at 500 mg kg  1. The results indicated that Pb probably induced oxidative stress and proteotoxicity to the seedlings through Od2  accumulation, and that SOD, HSP 70 and HSP 60 were important defense mechanisms to alleviate the oxidative stress. It is found that Od2  , HSP 70 and HSP 60 were the most sensitive parameters and had the potential to act as biomarkers for early warning of soil Pb contamination. Concentrations of soil Pb, exposing time and combination of multiple parameters should be also taken into consideration when assessing soil Pb pollution by these biomarkers. & 2009 Elsevier Inc. All rights reserved.

Keywords: Spinach Superoxide radical (Od2  ) Oxidative stress Heat shock proteins (HSPs) Stress proteins Biomarker

1. Introduction Soil contamination by heavy metals is of widespread occurrence as a result of human, agricultural and industrial activities. Among them, lead (Pb) is a potential pollutant that readily accumulates in soils and sediments and its toxicity to plants has been widely investigated (Sharma and Dubey, 2005). Pb is a nonessential element for plants, but it can be easily taken up by roots of crop seedlings, transported to shoots and finally entered food chains. As a non-redox-active heavy metal, Pb can substitute essential metals or cofactors at enzyme-active sites, leading to an unbalance of essential trace metals and cellular redox status. The related phytotoxicity of Pb2 + is involved in the induction of reactive oxygen species (ROS) production, resulting in oxidative stress in growing plant parts (Verma and Dubey, 2003; Pallavi and Rama, 2005). Pb toxicity was also expressed as oxidized components in cells such as lipid peroxides, inactivated ¨ ¨ enzymes, damaged DNA and even dead cells (Schutzend ubel and Polle, 2002). Therefore, it is important to explore the physiological and biochemical mechanisms of oxidative stress and detoxification in plants, and to further develop early-warning methods for monitoring soil Pb contamination.

$

Assurance: Spinach seedlings were used as materials in this experiment.

 Corresponding author. Fax: + 86 25 83595222.

E-mail address: [email protected] (X. Wang). 0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2009.02.009

Biological monitoring is a direct test of biological responses to environmental contaminants, and has been proposed to complement the information given by chemical analyses (Flammarion et al., 2002). Thus, the use of biochemical or physiological parameters as biomarkers of ecotoxicity is under constant development and has the advantage of delineating effects before observed symptom (Che vre et al., 2003). Besides the antioxidative system, stress proteins (also called heat shock proteins, HSPs) are also activated in plant species under adverse conditions. HSPs consist of a series of proteins with various molecular weights, involving in homeostatic maintenance and accelerating repair or degradation of denatured proteins (Wang et al., 2004). Remarkably, HSP 70 and HSP 60 homologues work in concert and form an essential multistep pathway to correct conformation of protein structure (Hartl, 1996). Thus, induction of these two chaperones may indicate denaturing conditions of the ‘‘housekeeping’’ proteins in organisms (Downs et al., 2001) and a rescue of cells from oxidative stress due to damaged proteins. The conserved nature of HSPs from bacteria to plants and man renders them suitable as a biomarker of ecotoxicological research. In fact, HSP 70 can be produced in a dose-dependent way in response to most chemicals and at concentrations below the range of classical cytotoxicity testing (Bierkens et al., 1998). HSP 60 was found to respond to copper stress prior to reduction in growth scope measurements of Mytilus edulis (Sanders et al., 1991). HSP 70 gene induction is regarded to

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C. Wang et al. / Ecotoxicology and Environmental Safety 74 (2011) 41–47

be a criterion for toxicity of metallic pollution (Aı¨t-Aı¨ssa et al., 2003; Bhargav et al., 2008). Therefore, HSPs were conducted as biomarkers in terrestrial animals (Pyza et al., 1997; Nadeau et al., 2001; Bhargav et al., 2008) and aquatic animals (Hallare et al., 2006). Electron paramagnetic resonance (EPR) coupled with spintrapping technique overcomes limit of short half-lives of free radicals in vivo and has been used in biological research for direct detection of oxygen radicals in plant species (Lynch and Thompson, 1984; Selote et al., 2004). The amplitude of EPR spectrum for 1, 2-dihydroxybenzene-3, 5-disulfonic acid (Tiron) semiquinone is a specific and quantitative indicator of Od2  production (McRae and Thompson, 1983). Therefore, this method is capable of characterization and quantification of intracellular Od2  in plant species subjected to heavy metal stress. Our previous study showed that Pb-contaminated soil led to intracellular induction of HSP 70 and ROS as well as to activation of antioxidative enzymes, including their isoenzymes in tomato (Wang et al., 2008a) and Vicia faba seedlings (Wang et al., 2008b) subjected to soil Pb stress. Spinach seedlings are staple vegetable and widely cultivated in the world, which have many features to act as tested material in many experiments (Pavlı´kova´ et al., 2008; Xiao et al., 2008; Hada et al., 2001). The objectives of this study are (1) to investigate the possible changes of Od2  , HSP 70, HSP 60 and other related parameters, (2) to understand the toxicological mechanisms, and (3) to screen potential biomarkers and give some advice about their application in spinach seedlings subjected to Pb-contaminated soil.

leaves, immediately frozen in liquid nitrogen, were quickly ground into fine powder and homogenized in 50 mM CHES buffer (pH 8.6) containing 10 mM Tiron and 0.5% (v/v) Tween-20 under nitrogen in a hermetic box. Homogenates were centrifuged at 10,000g for 10 min at 4 1C and stored immediately in ice for EPR investigation. EPR spectra were recorded with a Bruker EMX 10/12 X-band spectrometer at room temperature. EPR instrument settings for all samples were as follows: center field, 3476 G; sweep width, 50 G; modulation frequency, 100 kHz; modulation amplitude, 1.0 G; receiver gain, 2  104; scans, 1 times; microwave power, 20 mW. The EPR signals were used to calculate the height of the first peak and interpreted as Od2  intensity.

2.4. Determination of SOD activity in leaves Enzyme extraction was prepared according to the method of Romero-Puertas et al. (2004). Soluble protein content was assayed according to the method of Bradford (1976) with BSA as standard. SOD (EC 1.15.1.1) activities were determined according to the method described by Garcı´a-Limones et al. (2002) and expressed as unit min  1 mg  1 protein. One unit of enzyme activity is defined as the amount of enzyme required to inhibit NBT reduction by 50%.

2.5. Determination of lipid peroxidation and carbonyl groups in leaves Lipid peroxidation was determined by measuring the concentration of 2thiobarbituric acid reacting substances (TBARS) according to the method of Verma and Dubey (2003). Lipid peroxidation was quantified and expressed as total TBARS in terms of mmol g  1 FW using an extinction coefficient of 155 mM  1 cm  1. Carbonyl groups were assayed using dinitrophenyl hydrazine according to the method of Levine et al (1994). Carbonyl content was calculated by the extinction coefficient of 22,000 M  1 cm  1. Soluble protein content was quantified by measuring absorbance at 280 nm according to a bovine serum albumin (BSA) standard curve (0.25–2.0 mg ml  1) dissolved in 6 M guanidine hydrochloride.

2. Materials and methods 2.1. Soil treatment and plant materials 2.6. Determination of HSP 70 and HSP 60 The soil was sampled from the suburb of Huainan city, Anhui Province, China. The background Pb content in the soil was 20.13 mg kg  1. The original organic matter in the soil was 1.027%, which was determined according to the method described by Walkley and Black (1934). Each pot was filled with 1.5 kg of dry soil. The soil was spiked with Pb (NO3)2 solution (dissolved in deionized water) and the extraneous Pb2 + in the soil were 12.5, 25, 125, 250 and 500 mg kg  1, respectively. The controls were spiked with deionized water. All soil samples were allowed to equilibrate naturally for one month. The final soil pH values ranged from 7.3 to 7.5. Spinach seeds were sown in each pot after sterilization and incubation. After germination, 50 uniform seedlings were reserved and cultivated under controlled conditions (18–20 1C, 70% relative air humidity, 13-h light/11-h dark cycles, photosynthetic active radiation of 200 mmol m  2 s). The seedlings were watered with deionized water daily and supplemented with Hoagland nutrient solutions (Lucretti et al., 1999) once a week. After 30 days, 40 seedlings in each pot were harvested and after 50 days, the rest of the seedlings were harvested. Biological parameters such as HSP 70, HSP 60, SOD activities, lipid peroxidation (MDA) and carbonyl groups in the leaves were investigated. All the experiments were performed in triplicate. 2.2. Determination of available Pb in soil Since the biomass of leaves was not sufficient to determine Pb content, available Pb in soil was determined, instead. Acetic acid (0.11 M) extraction, the first step of Community Bureau of Reference Programme three steps sequential extraction, was used to determine available Pb. A total of 0.5 g of dry soil was extracted with 20 ml acetic acid (0.11 M) by shaking at 20 1C for 16 h. Then, the mixture was centrifuged at 5000g for 20 min, followed by filtering through 0.45 mm membranes. The concentration of metal in filtrate was measured by an atomic absorption spectrophotomer (Solaar M series equipped with a GF95 graphite furnace and an FS95 autosampler, Thermo Elemental, Cambridge, UK). The detection limits for Pb were 0.07 mg l  1 and all the samples were higher than the detection limit. Certified standard samples and triplicates were performed to guarantee the precision of the methods and analyses. 2.3. Determination of Od2  in vivo in leaves Od2  levels were determined by EPR according to the method described by Lynch and Thompson (1984) with minor modifications. A total of 0.3 g of fresh

Total protein was extracted in 0.1 M Tris–HCl, pH 8.0, 10% (v/v) glycerol, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2% (v/v) Triton X-100, 2 mM DTT, 1 mM phenylmethylsulphonyl fluoride (PMSF) at 4 1C according to the method of Romero-Puertas et al (2004). The extraction was centrifuged at 12,000g for 25 min to remove precipitation. Soluble protein content was determined by Bradford (1976) with BSA as standard. Aliquots of total protein extract was mixed with lysis buffer, heated up to 95 1C for 5 min, cooled on ice and used for analysis of HSP 70 and HSP 60. Constant weight of total protein (21.9 mg per lane) with prestained molecular weight markers was analyzed by SDS-PAGE (Bio Rad MiniPROTEAN 3 mini gel system) (10% separating gel and 5% stacking gel). After electrophoresis, gels were either stained with Coomassie brilliant blue R250 or transferred onto PVDF membrane (Amersham Pharmacia) for immunoblot analysis (BioRad semi-dry transfer apparatus). The membrane was blocked using 8% (w/v) non-fat milk/TBS-T buffer (50 mM Tris, 150 mM NaCl, pH 7.5, containing 0.05% Tween-20) for 2 h. After washing in TBS-T buffer, mouse anti-HSP 70/HSC 70 monoclonal antibody (SPA-820, Stressgen, Victoria, BC, Canada) (dilution 1:5000 in 1% BSA/TBS-T), mouse anti-HSP 60 (SPA-807, Stressgen, Victoria, BC, Canada) (dilution 1:1000 in 1% BSA/TBS-T) and mouse anti-b-actin monoclonal antibody (MS-1295, Labvison, CA, USA) (dilution 1:100 in 1% BSA/TBS-T) were added, respectively, and incubated overnight at 4 1C. After washing, the PVDF membranes were incubated in secondary antibody (anti-mouse IgG conjugated with horseradish peroxidase, Stabizyme, USA) (dilution 1:5000 in 1% BSA/TBS-T) at room temperature for 2 h. Bands were visualized by ECL reagent (Amersham) and blots were exposed to X-ray film. Quantification of integrated density in bands was performed by Image J software (National Institutes of Health, USA) and related to respective internal standard (b-actin) with background subtraction. Three replicates in each treatment were performed in this experiment.

2.7. Statistical analysis All the statistical analyses were performed using SPSSs version 13.0 for Windowss (SPSS, Chicago, IL, USA). The data were all presented as mean 7 standard deviations of triplets. Differences were considered to be significant at po 0.05(*) and highly significant at po 0.01(**) by Dunnett’s t-test to compare treated groups with controls. Representative photographs from each treatment were presented.

C. Wang et al. / Ecotoxicology and Environmental Safety 74 (2011) 41–47

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3. Results

3.5. Effect of Pb on induction of HSP 70 and HSP 60 in spinach leaves

3.1. Available Pb in soil samples

HSP 70 and HSP 60 productions were investigated by SDSPAGE and western blotting (Fig. 4a). Both of HSP 70 and HSP 60 increased with the increase of the added Pb from 0 to 125 mg kg  1, and declined thereafter. Significant increases in both of HSP 70 and HSP 60 were found in all treatments compared to controls after 30 days of germination (Fig. 4b, po0.05 and po0.01). After 50 days of germination, HSP 70 enhanced slightly along with the increase of added Pb from 12.5 to 125 mg kg  1, and then decreased below the controls (Fig. 5). HSP 60 increased slightly with the increase of extraneous Pb from 0 to 25 mg kg  1, and then decreased hereafter and was significantly inhibited at 500 mg kg  1 (Fig. 5b, p o0.05).

The available Pb in soil increase with the increase of extraneous Pb (r ¼0.986, po0.01). Significant increases (po0.01) compared to the controls were shown when the added Pb increased up to 125 mg kg  1 in soil (Table 1). 3.2. Effects of Pb on Od2  production in spinach leaves The Od2  -EPR spectrum in the spinach seedlings exposed to 0– 500 mg kg  1 extraneous Pb after 30 days of germination is shown in Fig. 1a. The Od2  intensities increased with the increase of added Pb in soil and significant increase was shown at concentrations ranging from 25 to 500 mg kg  1 (po0.05, p o0.01) (Fig. 1b). A positive correlation was found between the Od2  production and available Pb (r ¼0.878, p o0.05). After 50 days of germination, the Od2  was still enhanced along with the added Pb, but declined slightly when the extraneous Pb increased up to 500 mg kg  1. Significant accumulations occurred at concentrations ranging from 125 to 500 mg kg  1 (Fig. 1b). Additionally, the trapping agent itself was examined simultaneously and showed slight Tiron-spin-adduct signals (Fig. 1a), suggesting that Tiron itself trapped a few residual Od2  in the air inside of the box or trapping solution. 3.3. Effects of Pb on SOD activities in spinach leaves Total activities of SOD enzymes after 30 and 50 days of germination in spinach seedlings were assayed spectrophotometrically. After 30 days, SOD activities were significantly enhanced when Pb concentration was up to 125 mg kg  1 (po0.05, p o0.01) (Fig. 2), indicating that more severe stress occurred. A significant correlation was found between the Od2  production and SOD activities (r¼ 0.992, po0.01). After 50 days, the SOD activities also increased with the added Pb, but tended to decline when the added Pb increased up to 500 mg kg  1. Significant enhancement was found only at concentrations ranging from 250 to 500 mg kg  1 (Fig. 2). A positive correlation was also found between the Od2  and SOD activities (r ¼0.854, po0.05). 3.4. Effects of Pb on protein carbonylation and lipid peroxidation in spinach leaves After 30 and 50 days of germination, production of lipid peroxidation increased generally along with the increase of added Pb (Fig. 3a). The contents of available Pb were positively correlated with levels of TBARS after 30 days (r¼ 0.800) and 50 days (r ¼0.841, po0.05). Meanwhile, the protein carbonylation increased with the increase of added Pb in soil (Fig. 3b). The available Pb was positively correlated with the production of carbonyl groups after 30 days (r ¼0.698) and 50 days (r ¼0.908, p o0.05), respectively. The levels of carbonyl groups were also highly correlated with the production of lipid peroxidation after 30 days (r ¼0.973, p o0.01) and 50 days (r¼0.935, po0.01).

4. Discussion Although Pb’s toxicology to plants has been widely studied ¨ ¨ (Saint-Denis et al., 2001; Schutzend ubel and Polle, 2002; Verma and Dubey, 2003), direct determination of Od2  in plant species subjected to soil heavy metal contamination by EPR technique was less reported. In the present study, the direct quantification of Od2  by EPR coupled with trapping agent Tiron under nitrogen atmosphere in a hermetic box can be interpreted as Od2  production produced endogenously in spinach seedlings subjected to Pb stress, since the amplitude of EPR-Tiron-Od2  semiquinone spectrum is a specific and quantitative indicator of Od2  production (McRae and Thompson, 1983). The results showed that the Od2  production increased with the increase of extraneous Pb or available Pb after two exposures (Fig. 1, Table 1). A significant correlation was found between the Od2  and available Pb after 30 days of germination (r ¼0.878, p o0.05). Pb caused Od2  accumulation indirectly (Qian et al., 2005), suggesting that the Od2  production was possibly mediated by Pb in the spinach seedlings absorbed from the soil. Since the biomass of leaves was scant to determine Pb content in the leaves, the available Pb in the soil was determined, instead. The results showed that the available Pb contents in the soil were significantly correlated with lipid peroxidation (r ¼0.841, po0.05) and protein carbonylation (r ¼0.908, p o0.05) after 50 days of germination (Fig. 3), suggesting that Pb was possibly involved in production of lipid peroxidation and protein oxidation in the seedlings. ROS can catalyze oxidation of proteins and accumulation of carbonyl groups, and mark them for degradation by proteases (Stadtman, 1992). Thus, it is proposed that Pb probably mediated lipid peroxidation and protein carbonylation via Od2  accumulation and/or its derivatives in the seedlings. SOD enzymes were all activated in the two exposing periods and SOD activities were found well correlated with Od2  production in both periods, reconfirming that SOD was responsible for dismutation of Od2  to H2O2 and O2. SOD was an important defense mechanism in spinach seedlings subjected to Pb stress. SOD was reported as a sensitive biomarker of oxidative stress of Cd and petroleum hydrocarbons (Sun and Zhou, 2008). In this study, SOD enhanced significantly in response to concentrations

Table 1 Available Pb corresponding to Pb-treated soil. Added Pb (mg kg  1 DW) Available Pb (mg kg  1 DW)

0 1.9 7 0.1

12.5 4.5 7 0.3

25 7.57 0.4

125 44 73**

Data are expressed as the mean 7standard deviation (n ¼3). Significant differences versus control are indicated as **p o0.01.

250 817 1**

500 172 7 9**

44

C. Wang et al. / Ecotoxicology and Environmental Safety 74 (2011) 41–47

18

mg/kg

30 days

16 SOD activity (U mg-1 Protein)

0

12.5

25

125

**

50 days

14

*

* *

12

*

10 8 6 4 2 0

0

12.5

25

125

250

500

Added lead in soil (mg kg-1)

250

Fig. 2. SOD activities in leaves of spinach seedlings cultivated in Pb-contaminated soil 30 and 50 days after germinations, respectively. Values are denoted as mean 7SD (n¼ 3). Significant differences versus control are indicated as following: *p o 0.05, **p o 0.01.

500

trapping agent 1000 30 days

**

**

EPR signal intensity (Arbitrary units)

50 days **

800 **

*

*

600 * 400

200

0 0

12.5

25

125

250

500

Added lead in soil (mg kg-1) Fig. 1. (a) Od2  -Tiron-spin-adduct EPR spectra 30 days after germination and (b) Od2  intensities in leaves of spinach seedlings cultivated in Pb-contaminated soil 30 and 50 days after germinations, respectively. Values are denoted as mean 7SD (n¼ 3). Significant differences versus control are indicated as following: *p o0.05, **p o 0.01.

of soil Pb more than 125 mg kg  1 after 30 days of germination, suggesting that SOD in spinach seedlings can be used as an indicator of soil Pb stress. The results also showed that, after 30 days of germination, both of HSP 70 and HSP 60 increased significantly in response to all treatments, and that the production of carbonyl groups was highly correlated with HSP 70 (r ¼0.968, p o0.01) and HSP 60 (r ¼0.999, p o0.01) at concentrations of Pb2 + from 12.5 to 125 mg kg  1 (Fig. 4b). Accumulation of oxidized protein could elicit expression of HSP genes (Craig and Gross, 1991). Hence, Pb probably induced the production of HSP 70 and HSP 60 following accumulation of carbonylated proteins in the seedlings. The increased HSP 70 and HSP 60 might cooperate to renature conformation of denatured proteins or to facilitate their degradation. Therefore, the increased production of HSP 70 and HSP 60 is one of the primary defense mechanisms to rescue cells from proteotoxicity in spinach seedlings subjected to soil Pb. At high concentrations of Pb, HSP 70 and HSP 60 tended to decline (Figs. 4b and 5b), which could be explained by a general pharmacological kinetics of HSP’s response and concomitant quenching of the stress response subjected to severe stress (Eckwert et al., 1997). Inhibition of HSPs normally indicates a more toxic response than enhancement of these proteins (Arts et al., 2004). Under these circumstances, denatured or damaged proteins might be aggregated to such an extent that the biological mechanisms in cells were disturbed, which can be evidenced by the increase of lipid peroxidation (Fig. 3b). After 50 days, the leaves of spinach seedlings exposed to higher Pb concentrations were shorter and thinner than those at lower concentrations (data not shown), indicating more severe stress at higher Pb concentrations. Being sensitive to unspecific stressors, HSPs can be used as a potential biomarker of the integrity of the superstructure in the organisms. HSP 70 or HSP 60 has been proposed as indicators of environmental stress in terrestrial and aquatic animals (Downs et al., 2001; Arts et al., 2004), as well as in a few of plant species (Manitaˇsvic´ et al., 2007; Rau et al., 2007; Saidi et al., 2007; Wang et al., 2008a,b). In addition, ZnCl2 and SeO2 were found to be the strongest inducers of HSP 70 in R. subcupifutu. HSP 70 could be induced at concentrations below the range of classical cytotoxicity testing, such as growth inhibition and lethality, etc.

C. Wang et al. / Ecotoxicology and Environmental Safety 74 (2011) 41–47

TBARS content (μmol g-1 FW)

4000

0

30 days

*

50 days

3500

*

*

*

2

3

4

5

6

kDa

**

170 130 95

3000 2500

72

2000

55

1500

43

1000

34

500 0

1

45

26 0

12.5

25

125

250

500

Added lead in soil (mg kg-1)

HSP70

18 30 days

**

**

14 **

β-actin

** **

12

3.0 HSP 60

10 8 6 4 2 0

HSP60

50 days

0

12.5

25

125

250

500

Added lead in soil (mg kg-1) Fig. 3. (a) Production of lipid peroxidation and (b) carbonyl groups in leaves of spinach seedlings cultivated in Pb-contaminated soil 30 and 50 days after germinations, respectively. Values are denoted as mean 7SD (n ¼3). Significant differences versus control are indicated as following: *p o0.05, **p o0.01.

(Bierkens et al., 1998). The present results showed that Od2  (at concentrations not less than 12.5 mg kg  1), HSP 70 and HSP 60 (not less than 25 mg kg  1) were significantly induced by soil Pb after 30 days of germination (Figs. 1b and 4b). Meanwhile, the total SOD activities, production of lipid peroxidation (TBARS content) and carbonyl groups accumulated significantly only when the added Pb increased to 125 mg kg  1 after 30 days of germination (Figs. 2 and 3). Combined with the existing literature, the present results indicate that Od2  , HSP 70 and HSP 60 in spinach seedlings responded more sensitively than other parameters to soil Pb stress and they have the potential to act as biomarkers of Pb-contaminated soil. Nevertheless, the sensitivity of HSP 70 and HSP 60 declined along with the exposing time, implying that their induction may be related to dose and exposing time. Excessive exposure might contribute to repressed expression levels even at moderate concentrations of Pb (Fig. 5), which probably mask the actual toxicological effect of contaminants. It needs to be noted that a

Intensities of HSP60 and HSP70 (vs control)

Carbonyl groups (nmol C=O mg-1 Pr)

16

**

HSP 70

2.5

**

30days 2.0 *

**

**

** **

**

** *

1.5

1.0

0.5

0.0

0

12.5

25

125

Added lead in soil (mg

250

500

kg-1)

Fig. 4. (a) Western blotting and (b) statistical analysis of HSP 60 and HSP 70 production in leaves of spinach seedlings cultivated in Pb-contaminated soil 30 days after germinations. 1–6 represent control, 12.5, 25, 125, 250 and 500 mg added Pb kg  1 soil, respectively. Values are denoted as mean 7 SD (n¼ 3). Significant differences from control are indicated as following: *po 0.05, **p o 0.01.

single biomarker alone cannot provide a reasonable assessment of soil pollution. A suite of physiological and biochemical parameters may act together at different biological levels to reflect the early integrated effects of heavy metal stress.

5. Conclusion Pb induced oxidative stress and proteotoxicity in spinach seedlings via protein oxidation and modification, which was

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C. Wang et al. / Ecotoxicology and Environmental Safety 74 (2011) 41–47

HSP70 HSP60 β-actin

1

3

2

4

5

6

1.6 Intensities of HSP60 and HSP70 (vs control)

HSP60 1.4

HSP70 50days

1.2 1.0 0.8 **

0.6 0.4 0.2 0.0

0

12.5

25

125

Added lead in soil (mg

250

500

kg-1)

Fig. 5. Western blotting (a) and statistical analysis of HSP 60 and HSP 70 production (b) in leaves of spinach seedlings cultivated in Pb-contaminated soil 50 days after germinations. 1–6 represent control, 12.5, 25, 125, 250 and 500 mg added Pb kg  1 soil, respectively. Values are denoted as mean 7 SD (n¼ 3). Significant differences from control are indicated as following: *p o 0.05, **p o0.01.

possibly mediated by Od2  accumulation. SOD, HSP 70 and HSP 60 are important components of the primary defense mechanisms to alleviate the oxidative stress. HSP 70, HSP 60 and Od2  inductions are the most potential biomarkers for early-warning of Pbcontaminated soil compared with the other parameters. Meanwhile, soil Pb concentrations, exposing time and a suite of biomarkers at different biological levels should also be taken into consideration when these biomarkers are employed in bioassay of soil Pb contamination.

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