bioaccumulation and oxidative stress in submerged macrophyte ceratophyllum demersum l. upon exposure...

Bioaccumulation and Oxidative Stress inSubmerged Macrophyte Ceratophyllumdemersum L. Upon Exposure to Pyrene

Ying Yin,1 Xiaorong Wang,1 Yuanyuan Sun,2 Hongyan Guo,1 Daqiang Yin1

1State Key Laboratory of Pollution Control and Resources Reuse, School of Environment,Nanjing University, Nanjing 210093, China

2Department of Hydrosciences, Nanjing University, Nanjing 210093, China

Received 15 March 2007; revised 10 August 2007; accepted 1 September 2007

ABSTRACT: Laboratory experiments were carried out to investigate pyrene bioaccumulation and its con-sequent biological responses in submerged macrophyte Ceratophyllum demersum. Plants were exposedto different levels (0.01, 0.02, 0.05, 0.07, 0.1 mg/L) of pyrene for 10 days, and the pyrene content, and totalfree radicals in plant were analyzed. The pyrene concentration in plant was highly correlated to exposureconcentration (R2 5 0.990). Electron paramagnetic resonance (EPR) analysis revealed that pyrene expo-sure significantly increased total free radicals in the plants. A strong positive correlation (R2 5 0.956)between O2

�2 generation and pyrene contents implied that pyrene exposure induced reactive oxygen spe-cies (ROS) and led to oxidative stress in C. demersum. The activities of antioxidant enzymes and the con-tents of glutathione were determined. Change in the contents of malondialdehyde (MDA) was also stud-ied. Results indicated that the bioaccumulation of pyrene resulted in the changes of the antioxidantdefense system and the production of ROS with the oxidative stress, ultimately induced damnification inC. demersum. # 2008 Wiley Periodicals, Inc. Environ Toxicol 23: 328–336, 2008.

Keywords: pyrene; bioaccumulation; Ceratophyllum demersum; electron paramagnetic resonance; freeradical; oxidative stress

INTRODUCTION

Free radical mediated damage in plants is one of several

mechanisms involved in the cytotoxic effect of many xeno-

biotics. Xenobiotics sometimes termed ‘‘protertogens,’’

which are relatively nontoxic, can be enzymatically bioacti-

vated in the embryo to highly toxic, electrophilic, or free

radical reactive intermediates (Juchau et al., 1992). If not

detoxified, electrophilic reactive intermediates can bind co-

valently to embryonic cellular macromolecules (e.g., pro-

teins, DNA), while free radical reactive intermediates can

react directly or indirectly with molecular oxygen to form

reactive oxygen species (ROS), such as superoxide anion

(O2�2), hydrogen peroxide (H2O2), and hydroxyl radicals

(�OH) (Wells et al., 2005).

Free radicals and ROS are produced under normal physi-

ological condition and can be detected in all plant tissues.

Under environmental stress, changes in free radical proc-

esses are expected to occur and these are in turn to affect

the radical scavenging ability of a plant (Pirker et al.,

2002). Namely, when ROS generation exceeds the capacity

of the cellular antioxidants, it will cause oxidative stress

Correspondence to: X. Wang; e-mail: [email protected]

Contract grant sponsor: the National Fundamental Research Founda-

tion of China.

Contract grant number: 2003 CB415002.

Contract grant sponsor: Natural Science Foundation of China.

Contract grant number: 20237010.

Published online 23 January 2008 in Wiley InterScience (www.

interscience.wiley.com). DOI 10.1002/tox.20330

�C 2008 Wiley Periodicals, Inc.

328

and significant oxidative damage to a plant. These cytotoxic

ROS can strongly disrupt normal metabolism through oxi-

dative damage of chlorophyll, lipids, protein, and nucleic

acids (Wise and Naylor, 1987; Herbinger et al., 2002).

Since internal O2 concentrations are high during photo-

synthesis, chloroplasts are especially prone to generate

ROS (Asada and Takahashi, 1987). Electrons leaked from

electron transport chains can react with O2 during normal

aerobic metabolism to produce ROS. Once produced, O2�2

will rapidly dismutate, either enzymatically or nonenzy-

matically, to yield H2O2 and O2. In addition, H2O2 and O2�2

may interact in the presence of Fe21 to yield the highly re-

active �OH (Imlay and Linn, 1988). ROS production and

subsequent oxidative damage may be an important mecha-

nism of toxicity in organisms exposed to xenobiotics (Liv-

ingstone, 2001). To protect them against oxidative stress,

plant cells produce both antioxidant enzymes, such as

superoxide dismutase (SOD), catalase (CAT), peroxidase

(POD), glutathione-S-transferase (GST), and nonenzymatic

antioxidants such as glutathione and ascorbate. Some of

these constitute good molecular bioindicators for contami-

nant-mediated oxidative stress (Shao et al., 2002). Also,

in vivo free radical formation has been cited as a contribut-

ing factor to the deleterious effects of many chemical pollu-

tants (Pedrajas et al., 1995).

Polycyclic aromatic hydrocarbons (PAHs) are ubiqui-

tous contaminants in aquatic and terrestrial environments.

The fate of these compounds is of concern due to their

toxic, mutagenic, and carcinogenic properties. There is an

increasing concern about the deleterious effects of PAHs on

estuarine and coastal ecosystems (Aas et al., 2000). Low-

molecular weight PAHs (�three benzene rings) degrade

easily in the environment, whereas high-molecular weight

(HMW) PAHs (�four benzene rings) are less bioavailable

and appear more resistant to biological degradation

(Giessing and Johnsen, 2005). Pyrene, a tetracyclic PAH,

has been studied extensively in the last two decades.

Because of its wide distribution in aquatic environments

and as a major component in PAH mixtures, pyrene is cho-

sen as a model HMW PAH to serve as an indicator of PAH

pollution (Zhang et al., 2004).

Submerged macrophytes (Ceratophyllum demersum)can cover large areas and dominate primary productivity in

aquatic environments. Their ability to accumulate toxic

metals from water has been well documented (Mishra et al.,

2006), but in general less is known about the effects of or-

ganic contaminants. The plants growing in polluted water

bodies can absorb toxic xenobiotics which thus enter into

the food chain, posing a serious threat to human health

(Gupta and Chandra, 1998). Few studies was undertaken on

organic xenobiotics (such as PAH) accumulation capacity

of C. demersum and its physiological responses to stress

particularly concerning oxidative stress protection.

The objectives of this study were to (1) determine the

bioaccumulation of pyrene by C. demersum, (2) investigate

the pyrene-induced oxidative stress in the plants, and (3)

elucidate the possible mechanisms of the oxidative damage

of pyrene. This was accomplished by determining pyrene

concentrations in C. demersum, assessed the levels of free

radical, superoxide anion content, and glutathione, and

activities of antioxidative enzymes. We also investigated

pyrene-induced damage in C. demersum by determining

malondialdehyde (MDA) content representing as lipid per-

oxidation in plant.

MATERIALS AND METHODS

Chemicals

Pyrene (98% purity) and bovine serum albumin (BSA)

were purchased from Aldrich Sigma Co., Florisil, metha-

nol, acetone, hexane, and dichloromethane (chromatogram

grade) were purchased from Sigma Chemical (St. Louis,

MO, USA). Other reagents (analytical grade) were pur-

chased domestically.

Plant Material and Growth Conditions

Plants of C. demersum were collected from unpolluted

water bodies and acclimated in 18 glass jars (4 plants per

jar) containing 4 L 10%-strength Hoaglands nutrient solu-

tion for 1 month under natural day and night cycle in shade

conditions. During the experiment, airflow was kept con-

stant, the pH value of water was 7.3 6 0.3, the temperature

was (206 2)8C, and the hardness of water was about 100 mg/

L as CaCO3. After acclimatization, plants were subjected to

different concentrations (0.01, 0.02, 0.05, 0.07, 0.1 mg/L) of

pyrene (3 jars per treatment), while three jars without addition

of pyrene were designed as a control group. Fifty percentage

of water was replaced daily by fresh pyrene solution to mini-

mize contamination from metabolic waste. After 10 days

treatment duration, all plants were collected and washed thor-

oughly with tap water and then distilled water. Plant tissues

were divided into several parts and used for the determination

of various parameters.

Determination of Pyrene Concentration

One portion of the plant samples were weighted, cut into

pieces, and homogenized with pure anhydrous sodium sul-

fate in a mortar at room temperature. Pyrene was extracted

from the homogenized mixture with acetone/hexane (3/2;

v/v) under sonication for 20 min. Supernatants were col-

lected after centrifugation. The samples were sonicated and

centrifuged three times, and supernatants were combined.

The supernatant was concentrated to about 1 mL by a rotary

evaporator, cleaned up using an anhydrous Na2SO4/Florisil

column, and eluted with dichloromethane. The elution was

evaporated gently to dryness and the residue was dissolved

329BIOACCUMULATION AND OXIDATIVE STRESS IN CERATOPHYLLUM DEMERSUM

Environmental Toxicology DOI 10.1002/tox

in methanol for high performance liquid chromatography

(HPLC) analysis.

Pyrene contents in the samples were analyzed by a Hew-

lett Packard (HP) 1100 HPLC equipped with a column Zor-

bax Eclipse XDB-C8 (4.6 mm i.d 3 150 mm length) and a

HP photodiode array detection (DAD), which operating

wavelength was at 238 nm (Gao et al., 2006). The samples

were eluted by methanol/water (85/15; v/v). The recovery

of analysis in the plant were 95.0% 6 3.63% (n 5 3). Py-

rene was not detected in the control samples.

Electron Paramagnetic Resonance Spectra

The leaf frozen in liquid nitrogen were lyophilized in a

Labconco freeze dryer and introduced into EPR-type quartz

tubes (3 mm diameter). Concentration of free radicals was

measured by an Electron paramagnetic resonance (EPR)

spectrometer recording signals with Bruker EMX 10/12 X-

band at room temperature. The measurement settings were

as follows: center field, 3470 G; scan range, 200 G; modu-

lation frequency, 100 kHz; modulation amplitude, 1.0 G;

receiver gain, 5 3 104; scans, two times; microwave power,

20 mW. The EPR signal obtained with these parameters

was used to calculate the height of the central peak, indica-

tive of intensity of free radicals (Garnczarska and Bednar-

ski, 2004).

Superoxide Anion Assay

The measurement of superoxide anion was performed by

determining the oxidation of hydroxylammonium chloride

(Wang and Luo, 1990), Superoxide anion reacts with

hydroxylammonium to form NO22, which further reacts

with p-aminobenzene sulponic acid anhydrous and a-naph-thylamine, producing a azo dyestuff with absorption at

530 nm.

Determination of Glutathione

Fresh plant material was homogenized in 0.1 M sodium

phosphate-5 mM EDTA buffer (pH 8.0) and 25% m-phos-phoric acid. The homogenate was centrifuged at 12 000 3 gat 48C for 15 min to obtain supernatant. Reduced glutathi-

one (GSH) and oxidized glutathione (GSSG) levels were

determined fluorometrically according to the method of

Hissin and Hilf (1976), and fluorescence intensity was

recorded at 420 nm after excitation at 350 nm on a Hitachi

fluorescence spectrophotometer (Model 850).

Activities of Antioxidant Enzymes

Fresh plant material was homogenized in 50 mM phosphate

buffer (pH 7.8) containing 1% polyvinyl pyrrolidine and 0.1

mM EDTA. The homogenate was centrifuged (10 000 3 g

for 20 min) at 48C and the supernatant was used for antioxi-

dant enzyme assays.

SOD activity was measured according to Beauchamp

and Fridovich (1971). One unit of enzyme activity was

defined as the quantity of SOD required to produce a 50%

inhibition of reduction of nitroblue tetrazolium (NBT).

CAT and POD activities were measured according to

Maehly and Chance (1954). One unit of enzyme activity

was defined as the change of absorbance in 60 s at 258C.GST activity was measured according to Habig et al.

(1974). Enzyme activity was determined by monitoring

changes in absorbance at 340 nm (e340 1-chloro-2,4-dinitro-benzene (CDNB) conjugation 5 9.6 mM21 cm21) for

2 min at constant temperature.

Protein was estimated by the method of Bradford

(1976), using BSA as a standard. All antioxidant enzyme

activities were expressed as units (U) mg21 protein. The

absorbance was measured with a Shimadzu UV-220

spectrophotometer.

Determination of MDA

MDA content was determined according to Peever and Hig-

gins (1989). Plant material (0.5 g) was homogenized in

5 mL of 10% (w/v) trichloric acid (TCA). The homogenate

was centrifuged at 4000 3 g for 15 min and 4 mL of 20%

TCA containing 0.6% (w/v) 2-thiobarbituric (TBA) was

added to 1 mL of supernatant. The mixture was heated at

958C for 15 min and then quickly cooled on ice. The con-

tents were centrifuged at 10 0003 g for 15 min and absorb-

ance of the supernatant at 532 and 600 nm was read. After

subtracting the nonspecific absorbance at 600 nm, the

MDA concentration was determined using its extinction

coefficient of 155 mM21 cm21.

Statistical Analysis

Data were expressed as means6 standard deviation and an-

alyzed using one-way ANOVA. Significant differences

were compared between groups at P � 0.05. Graphs were

prepared using Origin 7.0.

RESULTS

Bioaccumulation of Pyrene in Plants

Submerged macrophyte C. demersum plants were exposed

to 0.01–0.1 mg/L pyrene for 10 days. The concentrations of

pyrene in C. demersum significantly increased with increas-

ing exposure concentrations, showing a dose-dependent

effect (Fig. 1). Plant pyrene content (y) was linearly corre-

lated with the exposure concentration (x). The regress equa-tion is y 5 143.14x 1 1.976, R2 5 0.990.

330 YIN ET AL.


Electron Paramagnetic Resonance Spectra

Typical EPR spectra of the control and pyrene-treated sam-

ples are shown in Figure 2(A). The spectra consisted of a

single peak free radical signal with a g-value of 2.0036 60.0004 and linewidth of 0.8897 6 0.0456 mT. The signal

intensity in the treatment was significantly higher than the

control (P\ 0.05) [Fig. 2(B)]. It means that pyrene expo-

sure induced generation of free radicals with free radicals

increased by about 99.8% (0.1 mg/L) compared with the

control. The regression equation for the signal intensity (y)of the free radical and exposure concentration (x) is y 53607.7 Ln(x)1 6790.6, R2 5 0.981.

Superoxide Anion Production

Compared with the control group, O2�2 was induced in all

pyrene exposure groups (Fig. 3). Similar to the bioaccumu-

lation of pyrene, the concentrations of O2�2 in plant materi-

als increased significantly with higher exposure concentra-

tions, and showed a dose-dependent increase with the

exposed doses of pyrene. Regressive analysis demonstrates

a good dose-effect relationship between the O2�2 contents

(y) and the exposure concentration (x). The regress equationis y 5 1520.2x1 63.29, R2 5 0.956.

Activities of Antioxidant Enzymesand Glutathione Content

The antioxidant responses of plant to pyrene exposure are

shown in Figure 4. SOD activity was induced in three treat-

ments with pyrene concentrations �0.05 mg/L. When the

exposure concentration of pyrene was 0.02 mg/L, SOD ac-

tivity reached a maximum with an activation rate at 36.9%.

From 0.02 mg/L group on, SOD activity began to decrease

and reached the minimum in 0.1 mg/L group, with an inhi-

bition rate at 23.9%. POD activity exhibited a pattern simi-

lar to that of SOD, when the exposure concentration of py-

rene was 0.02 mg/L, POD activity reached the maximum.

From 0.02 mg/L group on, POD activity began to decrease

and reached the least value in 0.1 mg/L treatment, with an

inhibition rate at 56.6% (P\0.05).

Fig. 2. A EPR spectrum of free radicals in C. demersum af-ter pyrene exposure. B Free radical signal intensity inC. demersum during 10 days pyrene exposure. *Significantlydifferent from control, P\0.05.

Fig. 1. Pyrene accumulation in C. demersum exposed todifferent concentrations of pyrene for 10-day.



All CAT activities were induced significantly (P\ 0.05)

by pyrene. At 0.05 mg/L, CAT activity was at the maximum,

with an activation rate at 93.3%, and then CAT activity

slightly decreased at concentrations �0.07. GST activities

increased significantly following exposure of 0.01 mg/L of

pyrene, and reached a plateau thereafter (P\ 0.05), and then

rose sharply in 0.1 mg/L group (195% of the control).

Pyrene exposure significantly reduced GSH contents

(P \ 0.05) (Table I). Regressive analysis demonstrates a

good dose-effect relationship between the GSH contents (y)and the exposure concentration (x). The regress equation is

y 5 2237.6x 1 33.14, R2 5 0.951. Compared with control

group, GSH content reached the minimum in 0.1 mg/L

group, with an inhibition rate at 70%. GSSG content was

elevated significantly (P \ 0.05) in the treatment groups

and showed little change after pyrene concentration reached

0.02 mg/L. Total glutathione content in the exposure groups

was induced compared with the control, reaching the maxi-

mum in 0.02 mg/L group, and then began to decrease. The

GSH/GSSG ratio decreased continuously, and the ratios in

five exposure groups correlated well with the corresponding

exposure concentration, R2 5 0.928.

Lipid Peroxidation

Changes of MDA content are shown in Figure 5. The MDA

content in 0.01mg/L group was not significantly different

Fig. 4. Antioxidant defense enzyme activity in C. demersum after exposed to pyrene for10-day. *Significantly different from control, P\0.05.

Fig. 3. O2�2 production rate in C. demersum after exposed

to pyrene for 10-day.

332 YIN ET AL.


from that in the control group. From 0.02 mg/L group on,

MDA contents were significantly induced in a dose-depend-

ent manner by treatment with pyrene (P \ 0.05). Regres-

sive analysis demonstrates a good dose-effect relation

between the MDA contents (y) and the exposure concentra-

tion (x). The regress equation is y 5 8.8067x 1 0.4027,

R2 5 0.978.

DISCUSSION

Bioaccumulation factors of a contaminant are closely

related to its water solubility and octanol:water partition

coefficient (Kow). The inertness of PAHs, coupled with their

low water solubility and strong lipophilic character, lead to

their high accumulation levels in the environment. It is

known that PAHs are bioaccumulated in aquatic organisms,

such as many invertebrate species, phytoplankton, and fish

species (Baussant et al., 2001). The bioaccumulation of or-

ganic contaminants by organisms is a balance between pas-

sive uptake and depuration, and elimination of xenobiotic

via biotransformation. The rates of metabolism of PAHs

are generally significantly less than rates of uptake, result-

ing in marked PAH’s bioaccumulation (Livingstone, 1998).

In this study, pyrene was accumulated in submerged plant

tissue because of its higher lipophilicity (logKow 5 4.88)

and lower metabolic ability of submerged plant.

PAHs entered into organism, interacted with aromatic

hydrocarbon receptor (AhR) and induced cytochrome

P4501A (CYP1A), leading to enhanced free radicals pro-

duction (Livingstone, 2001). The EPR method can be used

for estimating the type and content of more stable free radi-

cals, which accumulate in tissue during oxidative stress.

The direct detection of total stable free radicals in C.demersum by EPR spectroscopy revealed that 10-day py-

rene exposure of C. demersum promoted the synthesis of

free radicals. The g-value of 2.0036 is similar to the signal

from abiotically stressed leaves of A. thaliana (Muckensch-

nabel et al., 2002). In contrast, the g-value of other plant tis-sues exposed to abiotic stresses e.g., roots of lupin exposed

to lead, or freeze-dried wheat leaves exposed to elevated

ozone concentrations (at 2.0053) were considerably higher

than those in the submerged plants exposed to pyrene in the

present work (Reichenauer and Goodman, 2001). The EPR

signal implied that the paramagnetic radicals were derived

from a quinone although the exact origin of these radicals

was not determined (Garnczarska and Bednarski, 2004).

Atherton et al. (1993) suggested that the precursors of radi-

cals might be either quinones involved in electron transport

pathways, simple phenolic secondary metabolites, or more

complex polyphenols. To resolve this problem, further

studies were needed using the spin-trapping or other

method to identify other radical species.

Free radical reactive intermediates react directly or indi-

rectly with molecular oxygen to form ROS. Superoxide

anion (O2�2), produced by xanthine oxidase, tryptophan

dioxygenase, diamine oxidase and activated neutrophils,

can be a source of additional harmful ROS (Di Giulio et al.,

1989). The one electron reduction of molecular oxygen

results in the form of superoxide radical anion as an inter-

mediate. O2�2 radical is toxic by products of oxidative me-

tabolism and can interact with H2O2 to form highly reactive

hydroxyl radicals (�OH), which is thought to be primarily

responsible for oxygen toxicity in the cell. The pre-

sent study showed that O2�2 radical accumulation in

TABLE I. The changes in GSH and GSSG of C. demersum after exposure to pyrene (mg/g leaf wet weight)

Exposure concentrations of pyrene (mg/L)

Group 0 1 2 3 4 5

GSH 35.96 5.54 27.86 7.08a 28.76 2.08a 21.6 6 0.52a 14.76 0.80a 10.76 4.72a

GSSG 5.496 2.09 23.26 4.14a 31.76 3.11a 32.5 6 7.78a 29.66 5.52a 33.36 6.85a

GSH 1 GSSG 41.46 9.09 51.06 4.35 60.46 0.49a 54.1 6 0.68a 48.26 11.13 44.06 11.59

GSH/GSSG 6.536 0.91 1.206 0.06a 0.916 0.24a 0.66 6 0.16a 0.636 0.07a 0.326 0.08a

Data are expressed as means6 sd, n 5 4.aSignificantly different from control, P\ 0.05.

Fig. 5. MDA concentration in C. demersum after exposedto pyrene for 10-day. *Significantly different from control,P\0.05.



C. demersum was significantly enhanced under exposure of

pyrene, as indicated by its high correlation coefficient

(R2 5 0.956).

Previous experiments suggested that PAH could induce

ROS such as superoxide anion radical and hydroxyl radical

in the liver of mice and fish (Sun et al., 2006). It has not

been reported whether PAH can induce ROS and oxidative

stress in aquatic plants. Our results clearly demonstrated

the action of pyrene in stimulating the production of ROS

in C. demersum (Figures 2 and 3). The control group

showed some free radicals from EPR spectra signal and

superoxide anion assay. This could be explained as the nor-

mal production of ROS during cellular function, while the

environmental pollutants may accelerate this process,

resulting in excessive production of ROS. To protect them

against oxidative stress, plant cells produce both antioxi-

dant enzymes and nonenzymatic antioxidants such as gluta-

thione and a-tocopherol. An important feature of these

enzymes and nonenzymatic antioxidants is their inducibil-

ity under oxidative stress. When plants live in a polluted

environment, a large number of free radicals will be pro-

duced, causing damage to the plant cells. Activities of anti-

oxidant enzymes in plants will change accordingly to

remove these free radicals. However, activities of antioxi-

dant enzymes generally increase at the early stage of plants

suffering pollution. Once the pollution becomes more

severe and injury symptoms of plants become more appa-

rent, activities of antioxidant enzymes in these plants will

decrease (Liao et al., 2005). Therefore, activities of antioxi-

dant enzymes in plant cells are often used to show the

injury degree of plants due to pollution.

SOD, the first enzyme to deal with oxyradicals, cata-

lyzes the dismutation of O2�2 to H2O2 and O2. Oruc and

Uner (2000) indicated that induction of SOD can occur dur-

ing high production of O2�2, therefore, an increase in SOD

activity indicates an increase of O2�2 production. It has

been shown that increased SOD activity may be considered

as circumstantial evidence for enhanced ROS production

(Mittler, 2002). In contrast, Dimitrova and Tsinova (1994)

reported that superoxide radicals by themselves or after

their transformation to H2O2 induce an oxidation of cystein

in enzymes and decrease SOD activity. Inhibition by SOD

alone does not constitute rigorous proof for involvement of

O2�2. In this study, SOD activity was induced in C. demer-

sum exposed to\0.05 mg/L pyrene. When pyrene concen-

trations were increased to[0.02 mg/L, SOD activity began

to decrease. This suggests when O2�2 generation exceeds

the elimination ability of SOD, O2�2 as well as other oxy-

radicals can inactivate enzymes.

In plants, CAT is located predominantly in peroxisomes

where it functions mainly to remove H2O2 formed during

photorespiration or during b-oxidation of fatty acids in

glyoxysomes while POD reduces H2O2 to H2O using sev-

eral reductants available to the cells and protects cells

against harmful concentration of hydro peroxides (Mittler,

2002). In our study, the activity of POD showed a trend

similar to that of SOD, the highest value being observed in

C. demersum exposed to 0.02 mg/L pyrene. Activities of

SOD and POD in plant cells were often used to show injury

degrees of plants due to pollution, once the level of pyrene

exceeding 0.02 mg/L, injury symptoms of plants became

more apparent. CAT activity, unlike SOD and POD, exhib-

ited a significant increase in all groups, with the two highest

concentrations showing a decreasing trend. It can be

assumed that CAT was more tolerant than SOD and POD

in C. demersum under pyrene exposure. GST catalyzes the

conjugation of various electrophilic compounds with tri-

peptide glutathione, the resulting conjugates being water

soluble and thus more easily excretable. GST played an im-

portant role in biotransformation of pyrene by microalgal

species (Lei et al., 2003). GST responded differently to dif-

ferent compound exposures. In our study, GST activities

were clearly increased in treated groups compared with

control. It suggested that GST was sensitive to the pyrene

exposure in C. demersum.GSH acts as a cellular reducing and protective reagent

against numerous toxic substances through the -SH group.

When plant cells make contact with pollutants, such as py-

rene, they remove them by conjugation with GSH directly or

by means of GSTs, which decreases GSH levels. Total gluta-

thione (tGSH) serve as a prospective biological index to indi-

cate contaminants exposure (Stein et al., 1992), due to its

function in resisting reactive oxygen toxicity. The most

obvious direct effect of certain pollutants is a decrease in

thiol status, i.e., the ratio of reduced to oxidized glutathione

(GSH/GSSG), due to either direct radical scavenging or

increased POD activity. The GSH/GSSG ratio could be a

useful index of the precarious state of the cell. To evaluate

the effect of pyrene as a cause of oxidative stress, tGSH lev-

els, and GSH/GSSG ratios were examined in C. demersum.In the present study, pyrene exposure induced a significant

decrease in GSH contents. GSSG contents changed along

with GSH contents, which indicated there is a transformation

trend from GSH to GSSG under oxidative stress. The genera-

tion of GSSG was higher than the reduction back to GSH,

then GSSG accumulated and tGSH was increased in plants

compared with control. The GSH/GSSG ratios decreased

continuously, which correlated well with the corresponding

exposure concentration, partially due to the high GSSG con-

tents. It may arose from the oxidation of GSH into GSSG, a

sign of oxidative stress by ROS. On the basis of GSH and

GSSG levels measured in numerous studies, Oost et al.

(1996, 2003) demonstrated that GSH and GSSG parameters

could not yet considered as valid biomarkers but GSH/GSSG

ratios may be a potential biomarker for oxidative stress. Our

results indicated that both GSH and GSH/GSSG ratio were

all sensitive to pyrene and more suitable as an indicator of

pyrene exposure than tGSH or GSSG parameter.

As a major oxidation production of peroxidized polyun-

saturated fatty acids, MDA content is an important

334 YIN ET AL.


indicator of lipid peroxidation in plant cells (Shalata and

Tal, 1998). According to Liu (2001), MDA contents in the

aquatic plant tissues, positively related to surfactant con-

centrations in the solutions, reflecting environmental pollu-

tion. Liao et al. (2005) also indicated that MDA content in

plant tissues was a useful index to evaluate pollution levels

and judge toxic effects of pollutants such as heavy metals

and acid rain. In our study, increased MDA upon pyrene ex-

posure revealed that the organism was in oxidative stress

from pyrene exposure. MDA content indicated the preva-

lence of free radical reactions in tissues, suggesting that py-

rene-induced membrane lipid peroxidation, which could be

attributed to the decreases in SOD and POD activities.

These decreased activities favored accumulation of O2�2

and H2O2, which could result in lipid peroxidation. This

process can be a vicious spiral with higher pyrene exposure

concentration, which enhanced toxic effects on plants.

In the present study, we found that pyrene was able to be

accumulated by submerged plant C. demersum, and

induced production of a large number of free radicals, and

led to oxidative damage to tissue of plants. Additionally,

the changes in the activities of antioxidant defense and lipid

peroxidation were observed. Among these parameters,

GSH levels and GSH/GSSG ratio were more suitable as

indicators in C. demersum under pyrene pollution condi-

tion. The bioaccumulation content of pyrene, superoxide

anion, GSH contents, GSH/GSSG ratio, and MDA contents

all had strong positive correlation with pyrene concentra-

tion. When pyrene concentration exceed 0.02 mg/L, the

SOD, POD activities and tGSH contents began to decrease,

and the MDA contents appeared significantly increase.

According to our result, it is suggested that further studies

are needed to determine the mechanisms of accumulation,

toxicity and stress resistance in aquatic plants upon expo-

sure to pyrene.

We thank Yunxia Sui for analysis of EPR spectra.

REFERENCES

Aas E, Baussant T, Balk L, Liewenborg B, Andersen OK. 2000.

PAH metabolites in bile, cytochrome P4501A and DNA adducts

as environmental risk parameters for chronic oil exposure: A labo-

ratory experiment with Atlantic cod. Aquat Toxicol 51:241–258.

Asada K, Takahashi M. 1987. Production and scavenging of acti-

vated oxygen radicals in photosynthesis. In: Kyle DJ, Osmond

CJ, Amtzen CJ, editors. Photoinhibition. Amsterdam: Elsevier.

pp 227–297.

Atherton NM, Hendry GAF, Mobius K, Rohrer M, Torring JT. 1993.

A free radical ubiquitously associated with senescence in plants:

Evidence for a quinine. Free Radic Res Commun 19:297–301.

Baussant T, Sanni S, Skadsheim A, Jonsson G, Børseth JF, Gaude-

bert B. 2001. Bioaccumulation of polycyclic aromatic com-

pounds. II. Modeling bioaccumulation in marine organisms

chronically exposed to dispersed oil. Environ Toxicol Chem

20:1185–1195.

Beauchamp C, Fridovich I. 1971. Superoxide dismutase:

Improved assay and an assay applicable to acrylamide gels.

Anal Biochem 44:276–287.

Bradford MM. 1976. A rapid and sensitive method for the quanti-

tation of microgram quantities of protein utilizing the principle

of protein-dye binding. Anal Biochem 72:248–254.

Di Giulio RT, Washburn PC, Wenning RJ, Winston GW, Jewell

CS. 1989. Biochemical responses in aquatic animals: A review

of determinants of oxidative stress. Environ Toxicol Chem

8:1103–1123.

Dimitrova MST, Tsinova V. 1994. Combined effect of zinc and

lead on the hepatic superoxide dismutase-catalase system in

carp (Cyprinus carpio). Comp Biochem Physiol C 108:43–46.

Gao YZ, Ling WT, Wong MH. 2006. Plant-accelerated dissipation

of phenanthrene and pyrene from water in the presence of a

nonionic-surfactant. Chemosphere 63:1560–1567.

Garnczarska M, Bednarski W. 2004. Effect of a short-term

hypoxic treatment followed by re-aeration on free radicals level

and antioxidative enzymes in lupine roots. Plant Physiol Bio-

chem 42:233–240.

Giessing AMB, Johnsen AR. 2005. Limited microbial degradation

of pyrene metabolites from the estuarine polychaete Nereisdiversicolor. Chemosphere 61:1281–1287.

Gupta M, Chandra P. 1998. Bioaccumulation and toxicity of mer-

cury in rooted-submerged macrophyte Vallisneria spiralis. En-viron Pollut 103:327–332.

Habig WH, Pabst MJ, Jakoby WB. 1974. Glutathione-S-transfer-ases: The first enzymatic step in mercapturic acid formation.

J Biol Chem 249:7130–7139.

Herbinger K, Tausz M, Wonisch A, Soja G, Sorger A, Grill D.

2002. Complex interactive effects of drought and ozone stress

on the antioxidant defence systems of two wheat cultivars. Plant

Physiol Biochem 40:691–696.

Hissin PJ, Hilf R. 1976. A fluorometric method for the direct

determination of oxidized and reduced glutathione in tissues.

Anal Biochem 74:214–226.

Imlay JA, Linn S. 1988. DNA damage and oxygen radical toxic-

ity. Science 240:1302–1309.

Juchau MR, Lee QP, Fantel AG. 1992. Xenobiotic biotransforma-

tion bioactivation in organogenesis-stage conceptal tissues:

Implications for embryotoxicity and teratogenesis. Drug Metab

Rev 24:195–238.

Lei AP, Wong YS, Tam NFY. 2003. Pyrene-induced changes of

glutathione-S-transferase (GST) activities in different microal-

gal species. Chemosphere 50:293–301.

Liao BH, Liu HY, Zeng QR, Yu PZ, Probst A, Probst JL. 2005.

Complex toxic effects of Cd21. Zn21, and acid rain on growth of

kidney bean (Phaseolus vulgaris L.). Environ Intern 31:891–895.

Liu HY. 2001.Studies of the harmful effects on aquatic plants and

the biodegradation of surfactants. Doctorate Dissertation,

Hunan Agricultural University, Changsha, China.

Livingstone DR. 1998. The fate of organic xenobiotics in aquatic

ecosystems: Quantitative and qualitative differences in biotrans-

formation by invertebrates and fish. Comp Biochem Physiol A

120:43–49.



Livingstone DR. 2001. Contaminant-stimulated reactive oxygen

species production and oxidative damage in aquatic organisms.

Mar Pollut Bull 42:656–666.

Maehly PC, Chance M. 1954. The assay of catalase and peroxi-

dases. In: Gluck D, editor. Methods of Biochemical Analysis.

New York: Interscience Publishers. pp 357–424.

Mittler R. 2002. Oxidative stress, antioxidants and stress toler-

ance. Trends Plant Sci 7:405–410.

Mishra S, Srivastava S, Tripathi RD, Kumar R, Seth CS, Gupta

DK. 2006. Lead detoxification by coontail (Ceratophyllumdemersum L.) involves induction of phytochelatins and antioxi-

dant system in response to its accumulation. Chemosphere

65:1027–1039.

Muckenschnabel I, Goodman BA, Williamson B, Lyon GD, Dei-

ghton N. 2002. Infection of leaves of Arabidopsis thaliana by

Botrytis cinerea: Changes in ascorbic acid, free radicals and

lipid peroxidation products. J Exp Bot 53:1–8.

Oost RVD, Goksøyr A, Celander M, Heida H, Vermeulen NPE.

1996. Biomonitoring aquatic pollution with feral eel (Anguillaanguilla). II. Biomarkers: Pollution-induced biochemical

responses. Aquat Toxicol 36:189–222.

Oost RVD, Beyer J, Vermeulen NPE. 2003. Fish bioaccumulation

and biomarkers in environmental risk assessment: A review.

Environ Toxicol Pharmacol 13:57–149.

Oruc EO, Uner N. 2000. Combined effects of 2,4-D and azinphos-

methyl on antioxidant enzymes and lipid peroxidation in liver

of Oreochromis niloticus. Comp Biochem Phys C 127:291–296.

Pedrajas JR, Peinado J, Lopez-Barea J. 1995. Oxidative stress in

fish exposed to model xenobiotics: Oxidatively modified forms

of Cu. Zn-superoxide dismutase as potential biomarkers. Chem

Biol Interact 98:267–282.

Peever TL, Higgins VJ. 1989. Electrolyte leakage, lipoxigenase,

and lipid peroxidation induced in tomato leaf tissue by specific

and non specific elicitors from Cladosporium fluvum. Plant

Physiol 90:867–875.

Pirker KF, Goodman BA, Pascual EC, Kiefer S, Soja G, Reichena-

uer TG. 2002. Free radicals in the fruit of three strawberry culti-

vars exposed to drought stress in the field. Plant Physiol Bio-

chem 40:709–717.

Reichenauer TG, Goodman BA. 2001. Stable free radicals in ozo-

nedamaged wheat leaves. Free Radic Res 35:39–101.

Shalata A, Tal M. 1998. The effect of salt stress on lipid peroxida-

tion and antioxidants in the leaf of the cultivated tomato and its

wild salt-tolerant relative Lycopersicon pennellii. Physiol Plant104:169–174.

Shao Y, Rudolf SSW, Richard YCK. 2002. Biodegradation and

enzymatic responses in the marine diatom Skeletonema costa-tum upon exposure to 2,4-dichlorophenol. Aquat Toxicol

59:191–200.

Stein JE, Collier TK, Reichert WL, Casillas E, Hom T, Varanasi

U. 1992. Bioindicators of contaminant exposure and sublethal

effects: Studies with benthic goldfish in Puget Sound, Washing-

ton. Environ Toxicol Chem 11:701–714.

Sun YY, Yu HX, Zhang JF, Yin Y, Shi HH, Wang XR. 2006. Bioac-

cumulation, depuration and oxidative stress in fish Carassius aur-atus under phenanthrene exposure. Chemosphere 63:1319–3027.

Wang AG, Luo GH. 1990. Quantitative relation of hydroxylamine

and superoxide anion radicals in plants. Chin Plant Physiol

Commun 6:55–57.

Wells PG, Bhuller Y, Chen CS, Jeng W, Kasapinovic S, Kennedy

JC, Kim PM, Laposa RR, McCallum GP, Nicol CJ, Parman T,

Wiley MJ, Wong AW. 2005. Molecular and biochemical mech-

anisms in teratogenesis involving reactive oxygen species. Tox-

icol Appl Pharmacol 207:S354–S366.

Wise RR, Naylor AW. 1987. Chilling-enhanced photooxidation

evidence for the role of singlet oxygen and endogenous antioxi-

dants. Plant Physiol 83:278–282.

Zhang Y, Zhu YX, Kwon KK, Park JH, Kim SJ. 2004. Novel

method for determining pyrene biodegradation using synchro-

nous fluorimetry. Chemosphere 55:389–394.

336 YIN ET AL.


bioaccumulation and oxidative stress in submerged macrophyte ceratophyllum demersum l. upon exposure...

Documents