Chromium induced lipid peroxidation in the plants of Pistia stratiotes L.: role of antioxidants and antioxidant enzymes

Chemosphere 58 (2005) 595–604 www.elsevier.com/locate/chemosphere

Chromium induced lipid peroxidation in the plants of Pistia stratiotes L.: role of antioxidants and antioxidant enzymes Sarita Sinha *, Rohit Saxena, Shraddha Singh Ecotoxicology and Bioremediation, Environmental Sciences Division, National Botanical Research Institute, Rana Pratap Marg, Lucknow, UP 226 001, India Received 16 February 2004; received in revised form 29 July 2004; accepted 30 August 2004

Abstract In the plant, Pistia stratiotes L., the effect of different concentrations of chromium (0, 10, 40, 80 and 160 lM) applied for 48, 96 and 144 h was assessed by measuring changes in the chlorophyll, protein, malondialdehyde (MDA), cysteine, non-protein thiol, ascorbic acid contents and superoxide dismutase (SOD), ascorbate peroxidase (APX) and guiacol peroxidase (GPX) activity of the plants. Both in roots and leaves, an increase in MDA content was observed with increase in metal concentration and exposure periods. In roots, the activity of antioxidant enzymes viz. SOD and APX increased at all the concentrations of Cr at 144 h than their controls. The GPX activity of the treated roots increased with increase in Cr concentration at 48 and 96 h of exposures, however, at 144 h its activity was found declined beyond 10 lM Cr. The level of antioxidants in the roots of the treated plant viz. cysteine and ascorbic acid was also found increased at all the concentrations of Cr at 144 h than their respective controls, however, an increase in the non-protein thiol content was recorded up to 40 lM Cr followed by decrease. The chlorophyll content decreased with increase in Cr concentrations and exposure periods. However, the protein content of both roots and leaves were found decreased with increase in Cr concentrations at all the exposure periods except an increase was recorded at 10 lM Cr at 48 h. In Cr treated plants, the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) for leaves chlorophyll and protein contents were 40 and 80 lM Cr, respectively after 48 h exposure while NOEC and LOEC for root protein content were 10 and 40 lM, respectively after 48 h. The analysis of correlation coefficient data revealed that the metal accumulation in the roots of the plant was found positively correlated with antioxidant parameters except SOD after 48 h of exposure, however, negatively correlated with most of all the parameters studied at 144 h in both part of the plant. It may be suggested from the present study that toxic concentrations of Cr cause oxidative damage as evidenced by increased lipid peroxidation and decreased chlorophyll and protein contents. However, the higher levels of enzymatic and non-enzymatic antioxidants suggest the reason for tolerating higher levels of metals. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Chromium; Pistia stratiotes; Antioxidants; Antioxidant enzymes; Lipid peroxidation

*

Corresponding author. Tel.: +91 522 205831 35x221; fax: +91 522 205839/5836. E-mail address: sinha_sarita@rediffmail.com (S. Sinha).

0045-6535/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.08.071

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S. Sinha et al. / Chemosphere 58 (2005) 595–604

1. Introduction Chromium is a transition metal located in group VI B of the periodic table. Although, it is able to exist in several oxidation states, the most stable and common forms of Cr are the trivalent and hexavalent species, which display quite different chemical properties. Chromium is the 7th most abundant element on earth and 21st in the crustal rock. Its widespread use in leather tanning, pigments, electroplating and alloys has converted Cr in a serious pollutant of air, soil and water. It is a hazardous heavy metal, causing membrane damage, ultra structural changes in the organelles, impaired metabolic activities and growth retardations (Vazquez et al., 1987; Kimbrough et al., 1999). Chromium has been demonstrated to stimulate formation of free radicals (FR) and reactive  oxygen species (ROS) such as superoxide radicals ðO
2 Þ,  hydrogen peroxide (H2O2) and hydroxyl radicals ( OH) either by direct electron transfer involving metal cations or as a consequence of metal mediated inhibition of metabolic reactions (Stohs and Bagchi, 1995). Their presence cause oxidative damage to the biomolecules such as lipids, proteins and nucleic acids (Kanazawa et al., 2000). In the plants, metal induced lipid peroxidation has been reported (De Vos et al., 1991), which profoundly alters the structure of membranes and consequently modifies their enzymatic and transport activities. To scavenge ROS and to avoid oxidative damage, plants posses a complex system of enzymatic and non-enzymatic antioxidants. Antioxidants such as ascorbate, cysteine, and thiols act as free radicals scavengers in aerobic cells. Furthermore, carotenoids also have important antioxidant effects in photosynthetic systems (Halliwell, 1987). Antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX) and guiacol peroxidase (GPX) play an important role in scavenging reactive oxygen species produced under oxidative stress (Harris, 1992), thereby protects potential cell injury against tissue dysfunction (Miquel, 1989). The enzyme  SOD dismutates superoxide radical (O
2 ) to H2O2 and oxygen. Peroxidases, which are located in cytosol, vacuole, cell wall as well as in extracellular space and use various substrates as electron donor, utilize H2O2 in the oxidation of various inorganic and organic substrates. Aquatic flora occupies an important position in food chain as primary producer and regulator of oxygen level and plays a significant role in the biogeo-chemical cycling of the elements. Most of the water bodies are contaminated with heavy metals and aquatic plants are reported to accumulate trace metals (thousand to several thousand folds) which other wise are toxic to biota (StCyr et al., 1994). Pistia stratiotes L., (Common name: water lettuce, Family Araceae), a free floating aquatic macrophyte has been selected for the present study due to its medicinal use and also as a food source. In view of the importance and luxuriant growth of the plant in

metal contaminated water bodies, it is worthwhile to study metal induced oxidative stress and likely alteration in the behaviour of non-enzymatic and enzymatic antioxidants under repeated metal exposure.

2. Materials and methods 2.1. Plant material and experimental setup The plants of P. stratiotes L. were collected and maintained in large hydroponic tubs in the field Laboratory. For experimental studies, healthy plants of P. stratiotes were further acclimatized in 10% Hoagland solution for 6 weeks under laboratory conditions. Care was taken to select the plants of uniform size and wt (8–10 g). The plants were treated with four different concentrations of Cr (10, 40, 80, 160 lM) under standard physiological conditions providing 16 h light (115 lmol M
2 s
1) 8-h dark photoperiod at 25 ± 2 °C for 48, 96 and 144 h. The plants were refeeded with initial metal concentration at every 48 h. The different concentrations of Cr were prepared using K2Cr2O7 (Cr VI – Merck). The plants of P. stratiotes (one plant per beaker, 8–10 g fw) were kept in 250 ml beakers (200 ml solution) containing four concentrations of Cr in 10% Hoagland solution along with one set of control (10% Hoagland solution) both in triplicate. Plants were harvested after 48, 96 and 144 h and the blotted roots and leaves were used for biochemical studies. 2.2. Metal uptake Harvested plants were washed thoroughly with distilled water for total metal accumulation. The roots and leaves of the plants were separated manually, dried in an oven at 80 °C for one week and kept for metal analysis. Dried plant tissues were digested in HNO3 (70%) using Microwave Digestion System MDS P 2000 and Cr content was estimated by GBC Avanta Atomic Absorption Spectrophotometer using air–acetylene gases at 357.9 nm wavelength. 2.3. MDA content The level of lipid peroxidation was measured in terms of malondialdehyde (MDA) content by thiobarbituric acid (TBA) reaction. The plant samples (roots and leaves both, 500 mg each) were homogenized in 3 ml of 0.2% trichloroacetic acid (Heath and Packer, 1968). 2.4. Estimation of antioxidants 2.4.1. Extraction of enzymes Plant tissues, both leaves and roots (200 mg each) was homogenized in 2 ml of 100 mM potassium phosphate

S. Sinha et al. / Chemosphere 58 (2005) 595–604

buffer, pH 7.5 containing 1 mM of EDTA in presence of pinch of polyvinyl polypyrrolidone (PVP). The homogenate was centrifuged at 12,000g for 15 min at 4 °C. All steps in the preparation of enzyme extract were carried out at 0–4 °C. This supernatant was used to measure the activities of superoxide dismutase, ascorbate peroxidase and guiacol peroxidase. 2.4.2. Superoxide dismutase (EC 1.15.1.1) The activity of superoxide dismutase was measured in the roots and leaves of the plant by the method of Nishikimi and Rao (1972), using the enzyme extract. Inhibition of 50% shows the expression of one Unit (1 U) enzyme. A system devoid of enzymes served as control. 2.4.3. Ascorbate peroxidase (EC 1.11.1.11) The Ascorbate Peroxidase activity was measured in the root and leaves of the P. stratiotes by the method of Nakano and Asada (1981), estimating the rate of ascorbate oxidation at 290 nm. The activity was calculated using the extinction coefficient of 2.8 mM
1 cm
1. 2.4.4. Guiacol Peroxidase (EC 1.11.1.7) Guiacol peroxidase was measured in plant parts, following the method of Curtis, 1971, modified by Kato and Shimizu (1987). Activity was calculated using the extinction coefficient of 26.6 mM
1 cm
1 at 470 nm for oxidized tetraguiacol polymer. One unit of peroxidase activity was defined as the calculated consumption of 1 lmol of H2O2 min
1 g
1 fresh weight. 2.4.5. Cysteine content Free cysteine content in roots and leaves of the plants (250 mg each) was measured by the method of Gaitonde (1967). 2.4.6. Non-protein thiol content Non-protein thiol content in the roots and leaves of the plant (250 mg) was measured spectrophotometrically with EllmanÕs reagent (Ellman, 1959). 2.4.7. Ascorbic acid content Ascorbic acid content in the plant samples was estimated by the method of Keller and Schwager (1977). 2.5. Estimation of chlorophyll and protein contents The blotted fresh parts of the plants were used for the estimation of chlorophyll (leaves) and protein (roots and leaves) contents. Chlorophyll content in the leaves (100 mg) of treated and control plants were extracted in 80% chilled acetone and estimated by the method of Arnon (1949) using GBC Cintra 10e Spectrophotometer. Protein content in the roots and leaves was measured by

597

the method of Lowry et al. (1951) using bovine serum albumin as the standard protein. 2.6. Statistical analysis The whole experiment was set up in the randomized block design. All the data were subjected to an analysis of variance (ANOVA) using Microsoft Excel 2000 followed by least significant difference (LSD) to test for significance compared to control (p < 0.1) or between various treatments/exposures. In tables and figures, the values are marked for the significance level as compared to control (Gomez and Gomez, 1984). 2.7. Quality control and quality assurance Analytical data quality of metals was ensured through repeated analysis (n = 6) of EPA quality control samples (Lot TMA 989) for metals (Cd, Cr, Cu, Pb) in water and the results were found to be within ±2.89% of certified values. For plants, recoveries of metals from the plant tissues were found to be more than 98.8% as determined by digesting three samples each from an untreated plant with known amount of metals. The blanks were run in triplicate to check the precision of the method with each set of samples.

3. Results The accumulation of Cr in the roots and leaves of P. stratiotes at various concentrations and exposure periods was presented in Table 1, which increased in concentration (p < 0.01) and exposure (p < 0.01) dependent manner. The analysis of the results revealed that the primary site of Cr accumulation was the root and the amount of Cr translocated into the leaves was found less by the order of magnitude. The formation of malondialdehyde (MDA) content was considered as a general indicator of lipid peroxidation, which raised markedly in the Cr treated plants of P. stratiotes over control values (Table 2). Compared to control, concentration of MDA was found increased in all the Cr concentrations and exposure periods in the roots and leaves of the metal treated plants, indicating enhanced lipid peroxidation. Maximum increase of 133.41% at 160 lM after 48 h in the roots and 106% at 40 lM after 96 h in the leaves was observed. In both parts of the plant, the MDA content were found positively correlated with metal accumulation after 48 h and negatively correlated after 144 h (Table 5). Amongst various enzymes involved in the abolishment of ROS, superoxide dismutase (SOD) can be considered as a key enzyme. SOD activity of the treated Pistia roots (Fig. 1A) was found to decrease with

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S. Sinha et al. / Chemosphere 58 (2005) 595–604

Table 1 Accumulation of Cr (lg g
1 dw) in the roots and leaves of P. stratiotes at different concentrations and exposure periods Conc. (lM)

Plant parts

Exposure periods (h) 48

96

144

10

Roots Leaves Roots Leaves Roots Leaves Roots Leaves

93.23 ± 4.31 24.32 ± 2.74 134.14 ± 10.94* 42.23 ± 3.64* 230.61 ± 10.77* 63.45 ± 3.29* 256.72 ± 15.40* 72.48 ± 4.6*

157.29 ± 16.30 25.90 ± 1.05 181.93 ± 9.39* 62.33 ± 3.19* 243.48 ± 21.37* 81.79 ± 8.22* 665.54 ± 47.39* 211.63 ± 11.47*

297.64 ± 24.75 42.93 ± 5.73 375.53 ± 22.84* 86.59 ± 4.85* 383.54 ± 26.11* 106.16 ± 5.59* 846.77 ± 27.94* 296.88 ± 18.60*

40 80 160

All values are means of triplicates ±SD. The plants were refeeded with initial metal concentration at every 48 h. LSD (p < 0.01): Metal accumulation conc; roots = 51.20, leaves = 10.3, expo.; roots = 44.34, leaves = 8.93. *Significant (p < 0.01) compared to 10 lM Cr.

Table 2 Effect of Cr on MDA content (mmol g
1 fw) of roots of P. stratiotes at different concentrations and exposure periods Conc. (lM)

Exposure periods (h) 48

96

144

Control

4.10 ± 0.38 (4.34 ± 0.67) 6.73 ± 0.08* (5.12 ± 0.29) 7.77 ± 0.18* (7.68 ± 0.72)* 7.89 ± 0.76* (7.75 ± 0.65)* 9.57 ± 1.54* (8.63 ± 0.62)*

4.60 ± 0.60 (4.65 ± 0.82) 8.51 ± 0.85* (7.99 ± 0.27)* 10.07 ± 0.49* (9.55 ± 2.12)* 7.49 ± 0.41* (7.09 ± 0.61)* 6.64 ± 0.33* (4.71 ± 0.55)

5.20 ± 1.09 (4.21 ± 0.74) 9.66 ± 0.68* (8.12 ± 0.52)* 6.65 ± 0.91* (6.35 ± 0.58)* 5.79 ± 0.88 (5.14 ± 0.17) 5.48 ± 0.76 (4.94 ± 0.23)

10 40 80 160

Values in parenthesis are the MDA content in leaves. All the values are mean of three replicates ±SD. The plants were refeeded with initial metal concentration at every 48 h. LSD (p < 0.01): MDA content, conc.; roots = 0.87, leaves = 1.01, expo.; roots = 0.68, leaves = 0.79. *Significant (p < 0.01) compared to control.

increase in metal concentrations at 48 h, compared to control and negatively correlated with metal accumulation (Table 5). However, at 96 and 144 h, the SOD activity of the roots of P. stratiotes increased substantially at all the metal concentrations and registered higher values when compared to controls with maximum increase of 37.1% at 80 lM after 96 h. Further, significant increase in the SOD activity of the leaves (Fig. 1B) was recorded at lowest Cr concentration (10 lM) followed by significant decrease at all the metal concentrations and exposure periods, compared to their respective controls. SOD activity of the leaves were found negatively correlated with metal accumulation (Table 5). As a member of ascorbic acid-glutathione cycle, APX plays a crucial role in eliminating poisonous H2O2 from plant cells. In roots of P. stratiotes, the APX activity was found higher at all the Cr concentrations and exposure periods than their controls (Fig. 1C) with maximum increase of 85% at 160 lM after 48 h. It was positively correlated with

metal accumulation (Table 5). The APX activity in the leaves (Fig. 1D) of the plant increased with higher Cr concentrations at 48 h of exposure than its control. However, it increased up to 40 lM and 10 lM Cr at 96 and 144 h, respectively, followed by decrease as compared to their respective controls. The maximum decrease of 82% was found in APX activity in the leaves at 96 and 144 h as compared to their respective controls and negatively correlated with metal accumulation. The GPX activity in the roots (Fig. 1E) of P. stratiotes was found higher at all the concentrations of Cr at 48 and 96 h, compared to their respective controls and positively correlated with metal accumulation (Table 5). However, at 144 h, it increased non-significantly at lowest Cr concentration (10 lM), followed by decrease as compared to control and negatively correlated with metal accumulation of the leaves (Table 5). A decrease in the GPX activity of the leaves (Fig. 1F) was recorded at all the Cr concentrations at initial exposure period (48 h),

S. Sinha et al. / Chemosphere 58 (2005) 595–604 80

45

b

A

c

b

Roots

40

b

60 c

b

50 40

c

c a

a

a

a

30 48h 96h 144h

20 10

Cysteine content

Cysteine content

70

a

B

a

30

a

25 20

b

15

c 48h 96h 144h

10

10

40

80

160

0

c

c

a

b c

10

40

80

160

60

80

C c

b b

50

Roots b

50

40

a

30

c c

48h 96h 144h

20 10

b

D

Leaves b

a a

SH content

c

60

SH content

b

Cr concentrations (µM)

Cr concentrations (µM)

b

40 b

c

30

c

a

20

c c

a

48h 96h 144h

10

a

a

0

0 0

10

40

80

0

160

Cr concentrations (µM)

b

E

a

b a c

80 40

b

Roots

48h 96h 144h

0 0

10

40

80

40

80

160

200

a

b c

c

120

a

10

Cr concentrations (µM)

160

Cr concentrations (µM)

Ascorbic acid content

200

Ascorbic acid content

b

0 0

160

Leaves

35

5

0

70

599

F

b

c

160

c c

120 b

a

80

a b

b a

c

Leaves 48h 96h 144h

40 0 0

10

40

80

160

Cr concentrations (µM)

Fig. 1. The effect of Cr treatment on cysteine (nmol g
1 fw) (A, B), SH (lmol g
1 fw) (C, D) and ascorbic acid (lg g
1 fw) (E, F) contents in the roots and leaves of P. stratiotes. All the values are mean of three replicates ±SD. LSD (p < 0.01): cysteine, conc.; roots = 2.44, leaves = 0.63, expo.; roots = 1.89, leaves = 0.49. SH, conc.; roots = 2.41, leaves = 1.46, expo.; roots = 1.87, leaves = 1.30. Ascorbic acid, conc.; roots = 9.49, leaves = 2.47, expo.; roots = 7.35, leaves = 7.90. Significant (p < 0.01) compared to control, a = 48 h, b = 96 h, c = 144 h.

compared to control. However, it increased in a concentration and duration dependent manner at 96 and 144 h, compared to their respective controls and has shown positive correlation with metal accumulation (Table 5). Cysteine content of the roots (Fig. 2A) of P. stratiotes increased significantly with elevation in chromium levels and exposure periods. Correlation coefficient analysis showed positive correlation between metal accumu-

lation of the roots with cysteine content. The maximum increase of 76% was found at 40 lM after 96 h in root cysteine content. However, the cysteine content of the leaves (Fig. 2B) increased up to 40 lM Cr at 48 h as compared to control followed by decrease. A decrease in the cysteine content of the leaves was recorded with increase in the concentration of Cr at 96 h (39.2%) and 144 h (43%) of exposure periods, compared to their respective

600

S. Sinha et al. / Chemosphere 58 (2005) 595–604 60

45

SOD activity

b

40

b

Root b s

c

c

c

30

a

40

SOD activity

A

50

b

a

20 48h 96h 144h

10

35

c a

20

40

a

15 48h 96h 144h

80

0

160

Roots a

140 c b

b c

b a

150

APX activity

APX activity

c a

c b

250

48h 96h 144h

100 50

D

40

80

160

Leaves

b a

100

a c

80

b

a c b

60 48h 96h 144h

40 20 0

0

10

40

80

0

160

10

40

80

160

Cr concentrations (µM) 100

160 b

E

b

F

b Roots

80

120 b 100 80

a

60 40

0

10

c c

60

b a

40

Leaves

c

c b a

b a

b

a

48h 96h 144h

20

48h 96h 144h

20

GPX activity

GPX activity

c

120

Cr concentrations (µM)

0

10

Cr concentrations (µM) 160

C

200

b a c

0

10

300

140

c b

b

5

400

0

Leaves

a

25

Cr concentrations (µM)

350

c

30

10

0 0

B

c 0 40

80

160

Cr concentrations (µM)

0

10

40

80

160

Cr concentrations (µM)

Fig. 2. The effect of Cr treatment on SOD (U g
1 fw) (A, B), APX (lmol min
1 g
1 fw) (C, D) and GPX (lmol min
1 g
1 fw) (E, F) activity in the roots and leaves of P. stratiotes. All the values are mean of three replicates ±SD. LSD (p < 0.01): SOD, conc.; roots = 2.91, leaves = 1.81, expo.; roots = 2.02, leaves = 1.40. APX, conc.; roots = 10.18, leaves = 10.12, expo.; roots = 7.84, leaves = 7.84. GPX, conc.; roots = 6.60, leaves = 5.96, expo.; roots = 5.11, leaves = 4.61. Significant (p < 0.01) compared to control, a = 48 h, b = 96 h, c = 144 h.

controls. In contrast to roots, leaves cysteine content was found negatively correlated with metal accumulation at all the exposure periods (Table 5). The thiol content of the P. stratiotes roots (Fig. 2C) was found to increase at all the concentrations of Cr after 48 h, compared to control and positively correlated with metal accumulation (Table 5). However, it increased up to 80 lM (41.8%) at 96 h and 40 lM Cr concentrations (53.6%) at 144 h followed by decrease, compared to their respective controls. Compared to control, the SH con-

tent of the leaves (Fig. 2D) was found increased up to 10 and 40 lM Cr at 48 and 144 h respectively, followed by decrease. However, it increased significantly at all the concentrations of Cr at 96 h of exposure, compared to control with maximum increase of 122% at 40 lM. The ascorbic acid content of the roots and leaves (Fig. 2E and F) of Cr treated plants of P. stratiotes exhibited increase at all the Cr concentrations and exposure periods as compared to their respective controls except decrease (7.83%) in ascorbic acid content of the roots at

S. Sinha et al. / Chemosphere 58 (2005) 595–604 Table 3 Effect of Cr on total chlorophyll content (mg g
1 fw) of P. stratiotes at different concentrations and exposure periods Conc. (lM)

Control 10 40 80 160

Exposure periods (h) 48

96

144

0.896 ± 0.03 (0.285 ± 0.03) 1.060 ± 0.08* (0.345 ± 0.02)* 0.899 ± 0.01 (0.299 ± 0.03) 0.808 ± 0.01* (0.277 ± 0.01) 0.699 ± 0.03* (0.244 ± 0.03)

0.893 ± 0.02 (0.286 ± 0.01) 0.861 ± 0.02* (0.289 ± 0.01) 0.792 ± 0.03* (0.264 ± 0.02)* 0.754 ± 0.04* (0.256 ± 0.02)* 0.707 ± 0.02* (0.229 ± 0.02)*

0.895 ± 0.03 (0.275 ± 0.01) 0.799 ± 0.04* (0.267 ± 0.01) 0.690 ± 0.05* (0.252 ± 0.01)* 0.635 ± 0.02* (0.237 ± 0.03)* 0.511 ± 0.02* (0.216 ± 0.04)*

Values in the parenthesis are the carotenoid content (mg g
1 fw) in the leaves. All values are means of triplicates ±SD. The plants were refeeded with initial metal concentration at every 48 h. LSD (p < 0.01): conc.; total chl = 0.04, carotenoid = 0.02, expo.; total chl = 0.03, carotenoid = 0.02. *Significant (p < 0.01) compared to control.

160 lM Cr at 96 h. The ascorbic acid content of the roots and leaves were found positively correlated with metal accumulation after exposure of 48 and 96 h, whereas, negatively correlated at 144 h (Table 5). Results (Table 3) indicated a decline in the total chlorophyll content of the treated leaves with increase in concentration of metal and exposure period, except at 10 and 40 lM Cr at 48 h of treatment, compared to control. It has been observed that the plants exposed for a period of 144 h caused 42.91% decrease in total chlorophyll content of the leaves at 160 lM Cr than the control. The chlorophyll content in the plants is considered as one of the sensitive parameter under stress condition particularly metal toxicity. Thus, the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC) values were calculated based on concentration effect relationship. The NOEC and LOEC for metal and leaves chlorophyll content were 40 and 80 lM, respectively at 48 h. The carotenoid content increased up to 40 lM Cr at 48 h and 10 lM at 96 h followed by decrease at higher metal concentrations as compared to their respective controls. At 144 h, the carotenoid content of the leaves declined sharply at all the Cr concentrations than control with maximum decrease of 78%. Both chlorophyll and carotenoid contents in the leaves were observed negative correlation with metal accumulation at all the exposure periods (Table 5). The exposure of the plants of P. stratiotes to all the concentrations of Cr resulted in decrease in the protein content of the roots and leaves (Table 4), except at lowest metal concentration (10 lM Cr) at 48 h, compared to their respective controls. The protein content was found negatively correlated with metal accumulation in both

601

parts of the plant at all the exposure periods (Table 5). The NOEC and LOEC for metal and roots protein content were 10 and 40 lM, respectively after 48 h while NOEC and LOEC for metal and leaves protein content were 40 and 80 lM, respectively at 48 h.

4. Discussion Results of the present study indicated that the plants of P. stratiotes accumulated significant quantities of Cr in its roots than leaves when exposed to various Cr concentrations. This is in agreement with the earlier reports on rooted aquatic plants, which tend to accumulate Cr from the externally supplied metal solution (Gupta et al., 1994; Sinha et al., 2002; Suseela et al., 2002) and registered significantly higher values in the roots than their upper parts. High accumulation of the metal in the fine roots of the plant agrees with the earlier reports (Sinicrope et al., 1992). Qian et al. (1999) also reported highest concentration of Cr in the plant roots and least level in shoots among ten elements studied in the twelve aquatic plants including P. stratiotes. This is probably due to binding of metal to the ligands and thus reducing its mobility from roots to aerial parts. In the present study, the influence of repeated metal exposures is quite marked showing an increase in metal accumulation, which has been reported earlier (Sinha et al., 2002, 2003). Heavy metal toxicity is considered to induce the production of reactive oxygen species and may result in significant damage to cellular constituents. Membrane lipids and proteins are especially prone to attack by free radicals and are considered reliable indicators of oxidative stress in plants (Halliwell and Gutteridge, 1993; Palma et al., 2002). Chromium induced oxidative stress in P. stratiotes was evident from the increased lipid peroxidation in its roots and leaves, which is in agreement with the other studies carried out in hydroponics (Gallego et al., 1996). Cells are normally protected against ROS by the operation of intricate antioxidant systems, comprising both enzymatic systems such as superoxide dismutase (SOD), ascorbate peroxidase (APX), guiacol peroxidase (GPX) and non-enzymic systems, acting as free radical scavengers such as ascorbic acid, thiols and cysteine (Halliwell and Gutteridge, 1993). SOD, the first enzyme in detoxifying process, con radicals to H O . Chromium mediated verts O
2 2 2 enhancement in activity of SOD found in this study may be due to either direct effect of this metal on the SOD gene or to an indirect effect mediated via an in radicals (Chongpraditnum crease in the level of O
2 et al., 1992). Peroxidases are known to play a significance role in oxidative stress conditions and it has been shown that peroxidase activity can be used as a potential biomarker for sublethal metal toxicity in examined plant

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Table 4 Effect of Cr on protein content (mg g
1 fw) of roots of P. stratiotes at different concentrations and exposure periods Conc. (lM)

Exposure periods (h) 48

96

144

Control

14.81 ± 0.26 (15.87 ± 0.85) 15.31 ± 0.43* (20.48 ± 0.26)* 11.53 ± 0.38* (16.92 ± 0.24)* 10.02 ± 0.49* (13.26 ± 0.22)* 9.73 ± 0.62* (9.85 ± 0.93)*

14.32 ± 0.35 (15.44 ± 0.49) 12.67 ± 0.26* (15.43 ± 0.26) 11.19 ± 0.16* (14.81 ± 0.52) 10.27 ± 0.17* (14.39 ± 0.44)* 8.05 ± 0.19* (10.98 ± 0.31)*

14.63 ± 0.31 (15.32 ± 0.31) 12.04 ± 0.42* (15.05 ± 0.42) 10.47 ± 0.43* (14.43 ± 0.43)* 9.20 ± 0.65* (12.30 ± 0.65)* 6.46 ± 0.30* (8.67 ± 1.37)*

10 40 80 160

Values in parenthesis are the protein content in leaves. All the values are mean of three replicates ±SD. The plants were refeeded with initial metal concentration at every 48 h. LSD (p < 0.01): protein content, conc.; roots = 0.46, leaves = 0.82; expo.; roots = 0.36 leaves = 0.035. *Significant (p < 0.01) compared to control.

Table 5 Correlation coefficient of various parameters studied in roots and leaves with metal accumulation in P. stratiotes at different exposure periods Parameters

Plant parts

MDA

Roots Leaves Roots Leaves Roots Leaves Roots Leaves Roots Leaves Roots Leaves Roots Leaves Roots Leaves Leaves Leaves

Protein Cysteine
SH Ascorbic SOD APX GPX Chlorophyll Carotenoid

Exposure periods (h) 48

96

144

0.94 0.95
0.90
0.69 0.95
0.39 0.83
0.78 0.99 0.95
0.69
0.84 0.98 0.96 0.71
0.59
0.69
0.55

0.73
0.30
0.95
0.31 0.87
0.93 0.86 0.22 0.78 0.39 0.96
0.77 0.97
0.55 0.96 0.81
0.92
0.97


0.11
0.22
0.96
0.97 0.54
0.89
0.32
0.73
0.03
0.11
0.12
0.86 0.74
0.78
0.87 0.84
0.93
0.93

species (Radotic et al., 2000). In plants, the detoxification of H2O2 has been known to be an important function of the peroxidases that use ascorbate as the hydrogen donor (Hegedus et al., 2001). The APX activity was found to increase in the plants of P. stratiotes with increasing concentrations of externally supplied metal (Cr). The induction of APX activity in plants is also reported in Ceretophyllum demersum under copper stress (Devi and Prasad, 1998), Cu treated Phaseolus vulgaris (Gupta et al., 1999) and Cd treated green or green-

ing plants of barley (Hegedus et al., 2001). Among H2O2 destroying enzymes, GPX activity was found to increase in roots and leaves of the Cr treated P. stratiotes plants. Shah et al. (2001) also reported similar findings showing increase in GPX activity in the metal treated plants. The increase in the activity of GPX levels could be the consequence of either the microenvironment or the tissue specific gene expression in the treated plants (Hegedus et al., 2001). Cysteine, a –SH containing amino acid is a key constituent of phytochelatins and plays an important role in metal detoxification. The results of the present study conform with the findings of Sinha et al. (1996, 1997) as they have reported an increase in cysteine content of the plants exposed to low level of metal followed by decrease with increase in its concentration. The decrease in cysteine content in the leaves of P. stratiotes at higher concentrations of Cr might be due to decreased activities of sulphate reduction enzymes, ATP sulphurylase and adenosine 5-phosphosulphate sulphotransferase under metal stress (Nussbaum et al., 1988). Varying responses to Cr induced oxidative stress might be related to the concentration of thiolic groups, since they are consequently able to counteract oxidative stress. Further, the antioxidant property of thiols depends on the oxidation of SH group of the tripeptide to disulphide form (Toppi and Gabbrielli, 1999). The increase in the thiol content in treated plant of P. stratiotes is in agreement with the earlier findings of Rai et al. (1995), which may be due to inactivation of reactive metal by a cytoplasmic detoxification mechanism. Ascorbic acid plays a prominent role in scavenging free oxy-radicals. Similar to the present study, the enhanced levels of ascorbic acid were also reported in zinc treated Brassica juncea (Asada and Takahashi, 1987) and Hg exposed Bacopa monnieri (Sinha et al., 1996).

S. Sinha et al. / Chemosphere 58 (2005) 595–604

Inhibition of chlorophyll biosynthesis is well reported in plants under metal stress (Sinha et al., 2002, 2003), which may be due to reduced d-aminolevulinic acid dehydratase (ALAD) activity (Padmaja et al., 1990). Carotenoid is a part of photosynthetic pigment, playing an important role in protection of chlorophyll pigment under stress conditions. Carotenoids are known to quench the photodynamics reactions leading to loss of chlorophylls, replace peroxidation and collapse of membrane in chloroplasts (Knox and Dodge, 1985). In the present study, an increase in the carotenoid content was recorded, which is also reported in Cr treated Vallisneria spiralis (Vajpayee et al., 2001). The present study revealed the stimulation of protein content in the roots and leaves of P. stratiotes up to certain concentrations of chromium, however, protein content decreased at higher metal concentrations. The decrease in protein contents in the treated plant at higher concentrations of Cr was probably due to adverse effects of reactive oxygen species, which may be due to degradation of a number of proteins (Davies, 1987). Palma et al. (2002) reported that proteolysis is also an important component together with protein oxidation in oxidative stress situation induced by senescence and heavy metals. Thus, the observed relation between Cr induced oxidative stress and antioxidant capacity in P. stratiotes suggest the tolerance capacity of the plants to the metal depends on the balance of the factors favoring oxidative stress and factors reducing oxidative stress. Based on the present work, it can be concluded that toxic concentrations of Cr cause oxidative damage as evidenced by increased lipid peroxidation and decreased chlorophyll and protein contents. In Cr treated plants, the NOEC and LOEC for leaves chlorophyll and protein contents were 40 and 80 lM Cr, respectively after 48 h of exposure, while the NOEC and LOEC for metal and roots protein content were 10 and 40 lM, respectively. The analysis of correlation coefficient data revealed that the metal accumulation in the roots of the plant was found positively correlated with all the antioxidant parameters except SOD after 48 h of exposure, however, negatively correlated with most of all the parameters studied except cysteine content and APX activity in the roots and GPX activity in the leaves after 144 h in both parts of the plant. However, the higher levels of enzymatic and non-enzymatic antioxidants suggest the reason for tolerating higher levels of metals. The level of metals in the plants may be analysed before its use in medicine.

Acknowledgement We thank the Director, P. Pushpangadan, National Botanical Research Institute, Lucknow (India) for providing required research facilities. S. Singh and R. Sax-

603

ena are grateful to CSIR, New Delhi for the award of Senior Research Fellowship.

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