differential response of antioxidative defense system of anabaena doliolum under arsenite and...

Journal of Basic Microbiology 2009, 49, S63–S72 S63

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Differential response of antioxidative defense system of Anabaena doliolum under arsenite and arsenate stress

Ashish Kumar Srivastava2, Poonam Bhargava1, Riti Thapar1 and Lal Chand Rai1

1 Molecular Biology Section, Laboratory of Algal Biology, Center of Advanced Study in Botany, Banaras Hindu University, Varanasi, India

2 Department of Botany, School of Life Sciences, Mizoram University, Tanhril Campus, Aizawl, India

This study offers first hand information on the arsenite (As(III)) and arsenate (As(V))-induced

oxidative stress and changes in antioxidative defense system of Anabaena doliolum. A re-

quirement of 58 mM As(V) as compared to only 11 mM As(III) to cause 50% reduction in

growth rate suggests that As(III) is more toxic than As(V) in the test cyanobacterium. In

contrast to above, oxidative damage measured in terms of lipid peroxidation, electrolyte

leakage and peroxide content were significantly higher after As(V) than As(III) treatment as

compared to control. Similarly all the studied enzymatic parameters of antioxidative defense

system except glutathione reductase (GR) and non-enzymatic parameters except glutathione

reduced (GSH) showed greater induction against As(V) than As(III). Interestingly, higher

increase in non-enzymatic counterpart than enzymatic in both the stresses suggests that

detoxification is mainly managed by former than the later. This confirms the belief of

pronounced stimulation of the antioxidative defense system by As(V) than As(III). In con-

clusion, the cyanobacterium may survive better in As(V) than As(III) contaminated fields

because of its low toxicity and pronounced induction of antioxidative defense system.

Keywords: Anabaena doliolum / Antioxidative defense system / Arsenate / Arsenite / Effective concentration / Reactive oxygen species

Received: September 19, 2008; accepted: February 04, 2009

DOI 10.1002/jobm.200800301

Introduction*

Arsenic contamination of groundwater is turning into a

gravest natural disaster encompassing different coun-

tries of South-Eastern Asia. Contrary to Bhopal and

Chernobyl tragedies, however, the arsenic crisis in the

Indian subcontinent is more of a natural catastrophe.

Arsenic enters into the biosphere from the geochemical

weathering of rocks, and volcanic and microbial ac-

tivities. Notwithstanding above mining, combustion,

smelting, use of fertilizers, pesticides (arsenicals) and

disposal of industrial wastes have increased the envi-

ronmental pervasiveness of arsenic. In India and Ban-

gladesh the natural concentration of arsenic in drink- Correspondence: Prof. L.C. Rai, Molecular Biology Section, Laboratory of Algal Biology, Center of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India E-mail: [email protected]; [email protected] Phone: +91-542-2307146 Fax: +91-542-2368174

ing water is >50 µg l–1 and may be as high as 400 µg l–1

[1] in arsenic contaminated paddy fields. Chaturvedi [2]

has reported that concentrations of arsenite and arse-

nate range between approximately 47.2–86.2% and

13.1–52.2%, respectively where the mean arsenic

concentration in the top soil layer (top 70 mm) was

0.69 mg kg–1, while that at the bottom layer was

0.37 mg kg−1. Further arsenite was the most predomi-

nant species, followed by arsenate [2].

Arsenate being a structural analogue of phosphate

uncouples oxidative phosphorylation, inhibits ATPase

in bacteria and other organisms and replaces phospho-

rus in DNA and inhibits the DNA repair mechanism [3],

while As(III) reacts with functional groups of cysteine

and imidazolium nitrogens of histidine residues [4]

inactivating a host of enzymes the most important

being pyruvate dehydrogenase [5], glutathione reduc-

tase, pyruvate oxidase, choline oxidase, transaminase

etc. [3]. Both As(V) as well as As(III) are also known to

S64 A. K. Srivastava et al. Journal of Basic Microbiology 2009, 49, S63–S72


induce oxidative stress in plants by generating reactive

oxygen species (ROS) which can seriously disrupt pig-

ments, proteins, lipids and nucleic acids thus affecting

the normal cellular metabolism [3]. The balance between

ROS production and their scavenging determines the

survival of the organism. Under stress condition, organ-

isms have to activate the antioxidative defense system to

cope with enhanced production of ROS. This system is

widely studied in cyanobacteria under salt, heavy

metal, temperature and UV-B stress [6]. Further, arsenic

toxicity has been variously studied in higher plants [7]

and algae [3]. Surprisingly cyanobacteria have remained

neglected in terms of arsenic effects. Further, no at-

tempt seems to have been made to compare the toxicity

of As(III) and As(V) on the photosynthetic pigments,

and antioxidative defence system of cyanobacteria.

While As(III) can freely enter the cell by passive dif-

fusion across the cell membrane [8], As(V) uses the

phosphate transporters and therefore encounters seri-

ous competition with phosphate to enter into the cell

[9]. Besides, the phosphate transporters have a much

lower affinity for As(V) than phosphate [10], and thus

As(V) can enter the cell only if present at high concen-

trations in the external medium. Once inside the cell

As(V) is known to induce oxidative stress not only via

uncoupling of oxidative phosphorylation and disrupt-

ing the pH gradient but also by generating ROS during

its conversion into As(III) [10]. It is pertinent to mention

that As(III) formed either from As(V) or those entering

directly into the cell have the same fate of either get-

ting effluxed through aquaglyceroporins or converted

to less toxic methyl and dimethyl arsenate. In view of

the transport across the plasma membrane and direct

effect of As(V) on the oxidative phosphorylation, which

exploits interaction with molecular oxygen, we hy-

pothesized that As(III) may be more toxic than As(V),

however, later may cause more oxidative damage than

former and therefore induction of antioxidative defense

system. Moreover, since cyanobacteria, the inhabitants

of rice fields, have tremendous ability to tolerate very

high level of arsenic (7.5 × 106 µg l–1) [11], effective con-

centrations (EC50, EC100) of As(III) and As(V) were deter-

mined and used to study the toxic impact of arsenic on

cyanobacteria.

In order to bridge the gap in literature on the arsenic

toxicity in cyanobacteria and to test the above hypothe-

sis, specific growth rate, chlorophyll and phycocyanin

contents, lipid peroxidation, electrolyte leakage, perox-

ide content and the enzymatic and non-enzymatic anti-

oxidants of A. doliolum, one of the most important in-

habitants of rice fields of Varanasi [6], were measured

under arsenate and arsenite stress separately.

This study is important in the sense that the results

generated would provide critical information regarding

the cyanobacteria’s ability to tolerate arsenic in soil.

This study will also shed light on the effect of arsenic

on nitrogen fixation (an important parameter for soil

fertility) in the soil.

Materials and methods

Test organism and culture conditions The diazotrophic cyanobacterium Anabaena doliolum

Bhardawaja was isolated from the rice field of Banaras

Hindu University, Varanasi and grown axenically in a

modified Allen and Arnon [12] medium containing

MgSO4 ⋅ 7 H2O (0.246 g l–1), NaCl (0.234 g l–1), CaCl2

(0.055 g l–1), K2HPO4 (0.346 g l–1), MnCl2 ⋅ 4 H2O (0.5 mg l–1),

Na2MoO4 ⋅ 2 H2O (0.01 mg l–1), H3BO3 (0.5 mg l–1), CuSO4

⋅ 5 H2O (0.02 mg l–1), CoCl2 and ZnSO4 ⋅ 7 H2O (0.05 mg l–1)

buffered with Tris/HCl at 24 ± 2 °C under 72 µmol pho-

ton m–2 s–1 PAR (photosynthetically active radiation)

with a photoperiod of 14:10 h (light: dark) at pH 7.5.

The cultures were shaken by hand two to three times

daily.

Experimental design, stress application and biochemicals The experiments were done in three sets. Cells of

A. doliolum were (i) untreated, (ii) treated with arsenite

(As(III)) and (ii) treated with arsenate (As(V)). Arsenite

and arsenate autoclaved separately were added directly

into the sterilized medium to achieve the desired con-

centration. All the selected parameters were evaluated

at regular interval of 24 h up to 72 h. All the experi-

ments were conducted in triplicate using exponential

phase cultures and repeated at least twice to confirm

the reproducibility of the results. All biochemicals were

procured from Sigma Chemical Co. USA.

Measurement of survival and growth To measure the survival cyanobacterial cells were

treated with different concentrations of arsenite (0.0–

15 mM) and arsenate (0.0–110 mM) separately. Growth

was estimated by measuring optical density of the

cyanobacterial cultures at 663 nm in a UV-Vis spectro-

photometer (Systronics, India) up to 72 h using refer-

ence blank of basal culture medium. Specific growth

rate was calculated using the equation: µ = [ln (n2/n1)]/

[t2 – t1] where µ stands for specific growth rate and n1

and n2 are absorbance of culture suspension at the be-

ginning (t1) and the end (t2) of selected time interval.

Effective concentrations (EC50, EC100) were determined

Journal of Basic Microbiology 2009, 49, S63–S72 Anabaena and arsenic stress S65


using data of the specific growth rate of the cyanobac-

terium under both the stresses as mentioned in Guil-

lard [13].

Protein estimation Total cell protein was estimated using the method of

Bradford [14]. 100 µl cell extract was mixed with 1 ml

of Bradford reagent, which was prepared by adding

2 ml of 0.2% Coomassie Brilliant blue G-250 and 4 ml of

orthophosphoric acid in 14 ml of Millipore water. Ab-

sorbance was recorded at 595 nm.

Photosynthetic pigments determination For extraction of photosynthetic pigments, a known

volume of cyanobacterial culture was centrifuged and

the pellet was suspended in a desired volume of acetone

(80%). After overnight incubation at 4 ºC the suspen-

sion was centrifuged and the supernatant was used for

measuring the chlorophyll a (Chl-a) and carotenoid

(CAR) contents. The residue so obtained was suspended

in 30 mM Tris-HCl buffer (pH 7.5) and sonicated. The

resulting suspension was recentrifuged and super-

natant was used for the measurement of phycocyanin

(PC) pigment. For estimation of pigments, absorbance

were recorded with the help of a UV-Vis spectropho-

tometer at 665, 480 and 610 nm for Chl-a, CAR and PC,

respectively. Quantitative estimation of photosynthetic

pigment in terms of g l–1 was done using formula:

C = D/dα where α is absorption coefficient, D is optical

density, d is inside path length of the spectrophotome-

ter in cm and C is concentration of pigment in g l–1. The

value of α for Chl-a is 82.04 [15], for CAR 200 [16], and

for PC 7.5 [17].

Lipid peroxidation Oxidative damage of lipid was measured in terms of

the total content of 2-thiobarbituric acid-reactive sub-

stances (TBA) and expressed as equivalent of MDA

(malondialdehyde) using method of Cakmak and Horst

[18] with minor modifications. These reactive sub-

stances were extracted in 3 ml of 0.1% (w/v) trichloro-

acetic acid (TCA) at 4 °C following centrifugation at

13,000 × g for 2 min. An aliquot of 0.5 ml from the

supernatant was added to 1.5 ml TBA (0.5% in 20%

TCA). Samples were incubated at 90 °C for 20 min and

the reaction was stopped under ice bath. Centrifugation

at 1000 × g for 5 min was performed and absorbance of

the supernatant was measured at 532 nm and corrected

for nonspecific turbidity by subtracting the absorbance

at 600 nm. The concentration of MDA was calculated

using its extinction coefficient (155 mM–1 cm–1).

Electrolyte leakage The relative intactness of the plasma membrane was

measured as percent leakage of electrolytes as de-

scribed by Gong et al. [19]. The pellets so collected were

washed three times with Milli ‘Q’ water to remove any

extracellularly adsorbed arsenite or arsenate. These

were then suspended in 25 ml of Milli ‘Q’ water and

incubated in a water bath at 30 °C for 2 h. The suspen-

sion medium was used for measuring the initial electri-

cal conductivity (EC1) by digital conductivity meter. The

samples were then boiled at 100 °C for 15 min to re-

lease all the electrolytes, cooled and the final electri-

cal conductivity (EC2) was measured. The percent leak-

age of electrolytes was calculated using the formula

(EC1/EC2) × 100.

Peroxide assay The total peroxide was measured according to Sagisaka

[20]. The cell pellets suspended in cell lysis buffer were

subjected to sonication. A volume of 5% TCA was added

and the resulting suspension was centrifuged. About

1.6 ml of the resulting supernatant was mixed with

0.4 ml 50% TCA, 0.4 ml 10 mM ferrous ammonium

sulphate and 0.2 ml 2.5 M potassium thiocyanate. This

was then centrifuged and the absorbance of the super-

natant was measured at 480 nm. A standard curve was

used for measuring the concentration of the peroxide.

Assay of enzymatic antioxidants Pellets collected from exponentially growing cultures of

A. doliolum were suspended in cell lysis buffer (pH 7)

and subjected to sonication at 4 °C. The cell lysis buffer

contained 1 mM EDTA and 1% polyvinylpyrrolidone

(PVP) with the addition of 1 mM ASA in case of APX

assay. The sonicated samples were centrifuged at

15,000 × g for 30 min at 4 °C and the resulting super-

natant was used for the assay of the antioxidant

enzymes. Total SOD activity was assayed by monitoring

the inhibition of reduction of nitro blue tetrazolium

(NBT) according to the method of Giannopolitis and

Ries [21]. A 3 ml reaction mixture contained 50 mM

potassium phosphate buffer (pH 7.8), 13 mM methion-

ine, 75 µM NBT, 2 µM riboflavin, 0.1 mM EDTA and

100 µl of enzyme extract. The reaction mixture was

illuminated for 20 min at a light intensity of 5000 µmol

photon m–2 s–1. One unit of SOD activity was defined as

the amount of enzyme required to cause 50% inhibi-

tion of NBT reduction monitored at 560 nm. CAT activ-

ity was determined by measuring the consumption of

H2O2 (extinction coefficient 39.4 mM–1 cm–1) at 240 nm

for 3 min [22]. The reaction mixture contained 50 mM

potassium phosphate buffer (pH 7), 10 mM H2O2 and



200 µl of the enzyme extract in a 3 ml volume. APX

activity was determined by measuring the decrease

in absorbance at 290 nm (A290) (extinction coefficient

2.8 mM–1 cm–1) for 1 min in 1 ml reaction mixture

containing 50 mM potassium phosphate buffer (pH 7),

0.5 mM ascorbic acid, 0.1 mM H2O2 and 200 µl of the

enzyme extract. The reaction was started by adding

enzyme extract. Corrections were made for low, nonen-

zymatic oxidation of H2O2 [23]. GR activity was deter-

mined by measuring the oxidation of NADPH at 340 nm

(extinction coefficient 6.2 mM–1 cm–1) for 3 min in

1 ml of assay mixture containing 50 mM potassium

phosphate buffer (pH 7.8), 2 mM Na2EDTA, 0.15 mM

NADPH, 0.5 mM GSSG (glutathione oxidized) and 200 µl

of enzyme extract. The reaction was initiated by adding

NADPH. Corrections were made for the background

absorbance at 340 nm without NADPH [24].

Assay of non-enzymatic antioxidants Ascorbate was measured as per the method of Keller

and Schawger [25]. A. doliolum pellet was sonicated in ice

cold extracting buffer containing 0.25 M oxalic acid and

1 mM EDTA. This was centrifuged at 6000 × g for

15 min. The supernatant was mixed with 5 ml of 20 µg

DCPIP ml–1. The absorbance was measured at 520 nm.

Ascorbic acid content was calculated with the help of a

standard curve.

Glutathione was estimated by 5,5′-dithiobis-(2-nitro-

benzoic acid) (DTNB)-glutathione reductase coupled

assay as described in Anderson [26]. Cells were har-

vested and resuspended in 5% sulphosalicylic acid and

vortexed vigorously for 5 min. The extract was then

centrifuged and the supernatant was assayed to deter-

mine the glutathione content.

α-Tocopherol was extracted as per Munne-Bosch et al.

[27]. The pellet was sonicated in 5 ml ice-cold methanol

containing 1% ascorbate. α-tocopherol was extracted in

4 ml hexane by vigorous mixing for 2 min. After centri-

fuging the samples at 1500 × g for 20 min, the upper

hexane layer was carefully removed and evaporated to

dryness under vacuum. The dried hexane extract was

dissolved in 2 ml methanol and injected into a 10 µM

HPLC column (300 × 3.9 mm, C-18 column, Waters

Chromatography Division CAT No. 27324, USA) and

detected at 295 nm. Pure ± α-tocopherol was used as a

standard.

Proline was measured according to the method of

Bates et al. [28]. Control and treated cyanobacterial cells

were harvested and the pellet was resuspended in 10 ml

of 3% (v/v) sulphosalicylic acid and subjected to ultra-

sonic disruption for 5 min. The resulting cell extract

was centrifuged (9000 × g, 15 min) to remove cell debris

and the supernatant with free proline was treated with

freshly prepared acidic ninhydrin for 1 h at 80 °C. The

samples were put on ice to terminate the reaction, di-

luted with toluene, vortexed vigorously to extract the

colour into the organic phase and swirled to ensure

uniform mixing of the organic phase and the absorb-

ance read at 520 nm. A proline standard was prepared

by dissolving proline in 3% (v/v) sulphosalicylic acid.

The proline level was expressed as nmol mg–1 protein.

Statistical analysis Results were firstly analyzed by a two way ANOVA,

followed by a post hoc Duncan’s Multiple Range Test

(DMRT) to compare changes within a group during the

study period and between groups at the same selected

time. This was followed by Pearson correlation coeffi-

cient analysis. All the statistical analyses were per-

formed using SPSS ver. 12 software. When a time

course is followed, these data are included at time 0 to

indicate the onset of the time course in our experimen-

tal design. The number of independent variables for

each experiment was three.

Results

The EC25, EC50, EC75 and lethal doses were found to be 9,

11, 13.5 and 15 mM for As(III) and 35, 58, 77.5 and

110 mM for As(V) respectively in A. doliolum. Approxi-

mately 32.5, 67.5 and 31.25 and 65% reduction in final

growth yield was observed after 72 h exposure of the

test organism to 11 and 15 and 58 and 110 mM of

As(III) and As(V) respectively (data not shown). These

doses were selected for further study.

Photosynthetic pigments As(III) and As(V) produced dose dependent inhibition of

chlorophyll and phycocyanin in A. doliolum. The data of

these parameters showed insignificant change in

stressed cells as compared to the control after 0 h. Con-

trary to this, a similar pattern of significant inhibition

was observed after As(III) and As(V) treatment, how-

ever, this being more pronounced for As(V) (68.5 and

69.8%) than As(III) (30.3 and 31.5%) for chlorophyll

and phycocyanin respectively after 72 h of treatment

(Fig. 1a and c) as compared to their respective controls.

Further, the selected doses of As(III) and As(V) depicted

a more apparent inhibition of phycocyanin than chl-a.

Approximately 1.2 and 1.8 fold increase in carotenoid

content was noticed in cells treated with 15 mM As(III)

and 110 mM As(V) for 72 h, respectively over their con-

trols (Fig. 1b). Further, CAR content showed significant



Figure 1. Effects of arsenite and arsenate on the (A) chlorophyll content (B) carotenoid content and (C) phycocyanin content of Anabaena doliolum. Error bars indicate SD of three replicates. Different letters on the bars represents significant change (P < 0.05) in the treatments as compared to the control of 0 h as revealed from Duncan multiple range test.

change when compared within the group (P = 0.008)

(separately for As(III) and As(V) treatment) after dif-

ferent time interval as well as between the groups

(P < 0.001) (two way ANOVA and DMRT). Interestingly,

chl content showed a positive with phycocyanin con-

tent, and negative correlation with CAR content.

Lipid peroxidation, electrolyte leakage and peroxide content All the three parameters depicted significant increase

(DMRT) in their contents following exposure to both

As(III) and As(V) except after 0 h, the increase being

greater in case of As(V) as compared to As(III) (Fig. 2a–c).



Figure 2. Effects of arsenite and arsenate on the (A) malondialdehyde content (µMol mg–1 protein) (B) electrolyte leakage (%) and (C) peroxide content (µMol mg–1 protein) of Anabaena doliolum. Different letters on the bars represents significant change (P < 0.05) in the treatments as compared to the control of 0 h as revealed from Duncan multiple range test.

However, MDA content emerged more sensitive and

depicted significant alteration (P < 0.05) to both the

stressor as well as after different time period for each

treatment as compared to their respective controls.

Peroxide content increased maximally by 4.2 fold over

respective control following 110 mM As(V) after 72 h.

MDA and electrolyte leakage registered a 5.8 and 1.1

fold increase respectively on exposing the cells to

110 mM As(V) as compared to respective control after

72 h. Further, Pearson correlation coefficient revealed a

significant positive correlation (P < 0.01) between all

three parameters used for the estimation of membrane

damage.

Antioxidative enzyme activities Data pertaining to the effect of both concentrations of

As(III) and As(V) on SOD, CAT, APX and GR activities are

compiled in Fig. 3a–d. The activities of these enzymes

except GR were found to be insignificantly high in

stressed cells after 0 h. Of the antioxidative enzymes



Figure 3. Effects of arsenite and arsenate on the (A) SOD (U SOD mg protein–1) (B) CAT (µM min–1 mg protein–1) (C) APX (µM min–1 mg protein–1) (D) GR (µM min–1 mg protein–1) of Anabaena doliolum. Different letters on the bars represents significant change (P < 0.05) in the treatments as compared to the control of 0 h as revealed from Duncan multiple range test.

SOD and CAT showed an increase with increase in the

As(V) concentration; this being 4.3 and 2.1 fold respec-

tively after 110 mM As(V) as compared to their respec-

tive control. Interestingly APX gave a mixed response,

decline on exposure to As(III) and increase with As(V).

A 1.9 fold increase in APX activity was observed at

110 mM As(V) after 72 h of exposure. In contrast to

above, both As(III) and As(V) inhibited the GR activity

continuously up to 72 h as compared to the control.

Approximately 62.6% inhibition in GR activity was

observed with 110 mM of As(V) over the respective

control after 72 h. The statistical analysis showed that

the change in GR activity was insignificant between

group and within group except after As(V) treatment as

compared to control. The activities of these enzymes

were positively correlated (for SOD and CAT: P < 0.01,

SOD and APX: P < 0.05 and CAT and APX: P < 0.05) ex-

cept GR, which showed negative correlation. However,

GR and GSH were positively correlated (P < 0.01) as

revealed by Pearson correlation coefficient.

Nonenzymatic antioxidant content Fig. 4a–d depicts significant changes in ASA, GSH,

α-TOC and proline contents of A. doliolum subjected to

different concentrations of As(III) and As(V). All the

non-enzymatic antioxidants except GSH registered an

increase in a dose and time dependent manner except

0 h. 15 mM As(III) and 110 mM As(V) produced 2 and

4.2 fold and 2.5 and 4.4 fold increase in ASA content

over their controls after 48 and 72 h treatment respec-

tively. About 6.0 and 7.0 fold increase in α-TOC con-

tent was observed at 15 and 110 mM concentrations of

As(III) and As(V) respectively after 72 h of exposure as

compared to their respective controls. 110 mM of As(V)

significantly enhanced the proline accumulation in the

cells. About 3.6 and 4.4 and 2.2 and 3.1 fold increase in

proline content over their controls was reported at

selected doses of As(V) and As(III) respectively after 72 h

exposure. The two way ANOVA and DMRT showed a

significant difference (P < 0.001, between the groups

and P < 0.027, within the group) in proline content as

compared to control. All studied non-enzymatic anti-

oxidants including CAR content showed significant

positive correlation (P < 0.01) except GSH content.

Discussion

Requirement of high dosage of As(III) and As(V) to pro-

duce 50 and 100% growth inhibition can be attributed

to the ability of cyanobacteria including Anabaena vari-

abilis [11] to withstand and accumulate high concentra-

tions of these metalloids. The results of the require-

ment of a comparatively low amount of As(III) than



Figure 4. Effects of arsenite and arsenate on the (A) ASA (mg mg protein–1) (B) GSH (nM mg protein–1) (C) α-TOC (mg mg protein–1) (D) proline (nM mg protein–1) of Anabaena doliolum. Different letters on the bars represents significant change (P < 0.05) in the treatments as compared to the control of 0 h as revealed from Duncan multiple range test.

As(V) to produce 50% reduction in specific growth rate

(EC50) of A. doliolum offer direct support to our first hy-

pothesis that As(III) is more toxic than As(V) to the test

cyanobacterium. Cyanobacterial species are able to

grow in the presence of high concentrations of As(V)

(up to 100 mM) and low-millimolar concentrations of

As(III). This is also due to the fact that arsenate is a

phosphate analogue and competes with phosphate to

enter the cell. Since the affinity of the phosphate up-

take system for As(V) is very low as compared to phos-

phate hence a much higher concentration of As(V) will

be required to enter the cell. Arsenite on the other

hand can enter through aquaglyceroporins freely and

thus even a very little concentration may exert toxicity

to the cell.

Both chlorophyll and phycocyanin contents were

inhibited by As(V) as well as As(III). However, a more

pronounced inhibition of phycocyanin as compared to

chlorophyll (Fig. 1) may be attributed to its location on

the exterior side of the thylakoid membrane and thus a

prolonged exposure to the toxicant. However, the re-

duction in chl-a content may be assigned to the inhibi-

tion of chlorophyll biosynthesis brought about by

arsenic mediated inhibition of δ-aminolevulinic acid

dehydrogenase. The α-amino levulinic acid, required for

porphyrin synthesis, can be synthesized by two differ-

ent routes (i) by condensation of glycine with succinyl

Co-A, and (ii) from intact glutamic acid. In view of the

amelioration of As-induced inhibition of chlorophyll

biosynthesis following supplementation of precursors

of the pathways such as 2-oxoglutarate, glutamine,

glycine and succinate [7], arsenic induced inhibition of

the two pathways of chlorophyll biosynthesis becomes

evident.

Notwithstanding above, the data of lipid peroxida-

tion, electrolyte leakage and peroxide content which

indicate the level of oxidative damage in a cell offer

testimony to our second hypothesis that As(V) causes

more oxidative damage than As(III) (Fig. 1). As(III) is a

known inducer of oxidative stress [6] as well as of

NADH oxidase to produce superoxide. Arsenate induced

oxidative damage may be attributed to its known role

in uncoupling the oxidative phosphorylation thereby

inhibiting ATP synthesis and disrupting the ΔpH thus

leading to leakage of electrons and generation of reac-

tive oxygen species [29]. Further, As(V) metabolism

involves the formation of As(III) via cytochrome/cyto-

chrome oxidase, using oxygen as a final electron accep-

tor [41] and in the process generates superoxide radi-

cals. Presuming that the oxidative damage caused by

As(III), whether coming from diffusion entry or by con-

version of As(V) in the cell, would be equal, As(V) could

have an edge over As(III) in causing oxidative stress.

This may be due to the conversion of As(V) to As(III)



which is an extra step to cause oxidative damage in

comparison to As(III) alone. However, the conversion

depends on redox state and pH of the medium. Since

cyanobacteria are responsible for creating oxidizing

atmosphere, As(V) may become stable and dominate

and thus the severity of oxidative stress in cyanobacte-

ria supplemented with As(V) may also be attributed to

this.

A comparison of the oxidative damage caused by

As(III) and As(V) was further examined by measuring

the induction of enzymatic and non-enzymatic antioxi-

dants. SOD and CAT activities were found to be aug-

mented in response to both As(III) and As(V), but extent

of increase was more for As(V) (Fig. 3). Superoxide dis-

mutase, is a prominent biomarker of defence against

oxidative stress. A pronounced increase in SOD activity

as observed in the present study is quite in tune with

red clover [10]. Enhanced CAT may be a requirement

for the cell to scavenge the peroxide generated as a

result of superoxide scavenging via SOD. The other

peroxide-scavenging enzyme APX was enhanced on

As(V) treatment but declined after As(III) treatment.

Although the exact mechanism of As(III) induced inhi-

bition of APX is not known, As(III) might react with the

hydroxyl groups (2 OH and 3 OH) which are responsible

for H bonding with Arg 172 of APX of ascorbate [30],

thereby rendering them unfit for further reaction.

Further, GR activity showed a greater inhibition follow-

ing As(V) than As(III) treatment. As(III) directly inacti-

vates GR by reacting with the thiol group [3], while

As(V) reduces GSH pool (Fig. 4) which is a substrate for

GR. A decline in the GSH pool of the cell can be attrib-

uted to the utilization of GSH in phytochelatin synthe-

sis [3].

Except GSH the other non-enzymatic antioxidants

ASA, α-TOC and CAR showed similar response as ob-

served for enzymatic antioxidants. Ascorbate functions

as an antioxidant through removal of H2O2 via ascor-

bate-glutathione cycle. It not only acts as a substrate

for APX but also participates in the regeneration of

membrane-bound carotenoid. A significant increase in

α-tocopherol content in arsenic-stressed cells can be

attributed to its multiple functions viz. (i) chain break-

ing ability within membranes and lipoproteins, (ii)

regulation of H2O2 production, (iii) protection of poly-

unsaturated fatty acids from lipid peroxidation, and (iv)

increase in the activity of HPT enzyme involved in

α-TOC biosynthesis during abiotic stresses [31]. Not-

withstanding above, an increase in carotenoid content

is a necessity of the cell because it is associated in pho-

tosynthetic process and serves as antioxidant. It is also

known to quench singlet oxygen and scavenge free

radicals. Further, increased accumulation of proline

provides osmotic adjustment as well as protection by

chelating heavy metals in cytoplasm, scavenging hy-

droxyl radicals and maintaining water balance, which

is often disturbed by heavy metals [32, 33]. It also offers

protection by detoxifying free radicals and maintaining

NAD(P)+/NAD(P)H ratio [34]. Our results find support

from the work of Mishra and Dubey [35] who observed

significant accumulation of proline content in Oryza

sativa seedlings following exposure to arsenic.

This study clearly reveals that As(V), though less

toxic, caused more oxidative damage in A. doliolum than

As(III) as evident by a greater lipid peroxidation, perox-

ide content, and electrolyte leakage. Our results of a

minor increase in CAT and inhibition of APX on As(III)

treatment suggests that As(III) fails to stimulate the

peroxide scavenging enzymes, rendering the organism

susceptible to ROS damage. However, a two fold in-

crease in both CAT and APX activities following As(V)

treatment provides a kind of initial armory for the

survival of the test organism. In view of the greater

stimulation of antioxidative defense system in As(V)

than As(III), cyanobacteria appear to have a better po-

tential to survive under As(V) than As(III) rich envi-

ronments.

Acknowledgements

L. C. Rai is thankful to CSIR for financial support in the

form of projects. Poonam Bhargava is thankful to UGC

for the award of SRF. We are thankful to Prof. A.P.

Singh, Institute of Agricultural Sciences, B.H.U. for

help in measuring electrolyte leakage and to Prof.

K.P. Joy, Department of Zoology, B.H.U. for measuring

the α- tocopherol.

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differential response of antioxidative defense system of anabaena doliolum under arsenite and...

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