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Received: 12 January 2012 / Revised and accepted: 20 June 2012 / Published online: 11 September 2012
# Springer Science+Business Media B.V. 2012
used for circumventing the negative effects arising from ILsinduced toxicity in marine ecosystem.
Keywords Ionic liquid . AMPEP . Marine algae .
DNA damage . Ulva lactuca . Ascophyllum nodosum
Introduction
Room temperature ionic liquids (ILs) have emerged as a
new class of solvents and are widely accepted as green
solvents and potential green replacements for conventional organic solvents due to their unique array of physicochemical properties (Pham et al. 2010). These include
excellent solvation ability for a wide range of compounds,
nonflammability, high chemical/thermal stability, favourable conductivity and negligible vapour pressure (Earle
and Seddon 2000). The low volatility of ILs limits their
release in the atmosphere and thus reduces the risk of air
pollution. Nevertheless, the higher solubility in water coupled with non-biodegradable nature of ILs is a great concern
of aquatic pollution arising from accidental spills (Romero
et al. 2008; Coleman and Gathergood 2010). Therefore, the
green credentials of these ILs have been questioned for their
use in aquatic environments (Pham et al. 2010). In view of
the growing industrial interest in ILs, concerns are rising for
their eventual release in the aquatic environment through
industrial effluents and accidental leakage, and its consequent contamination which may negatively impact the structure and function of aquatic ecosystem.
The 1-alkyl-3-methylimidazolium salts are some of the
most common ILs used in industrial application and are
reported to have poor biodegradability resulting to retain
their properties intact in water (Coleman and Gathergood
2010). Recently, there has been considerable effort to determine the toxicity of ILs on both terrestrial and aquatic
371
Following IL and AMPEP treatments, protoplasts were isolated according to Kumar et al. (2011a) and suspended in
saline phosphate buffer at pH 7.4 and kept on ice at 4 C.
The protoplast yields were estimated by counting the cells
using a haemocytometer under an inverted microscope.
For comet assay the protoplast solution (50 L) containing approximately 1103 protoplasts was mixed with 50 L
of 1 % low-melting temperature agarose (LMPA) dissolved
in phosphate-buffered saline. From this mixture, an aliquot
of 80 L was layered on to a base slide, which was precoated with 1 % agarose, and then covered with a coverslip.
When the agarose gel solidified, the coverslip was gently
slide off and another agarose layer (90 L, 0.5 % LMPA)
was layered while covering it with a new coverslip and then
left for 10 min on a chilled metal plate to solidify the
agarose layer. Following this, the coverslip was removed
and the slides were submerged in alkali lysis solution
(2.5 MNaCl, 10 mM Trizma and 100 mM EDTA) at pH>
13 overnight at 4 C. Thereafter, the glass slide was incubated in fresh, cold electrophoresis buffer (i.e. 0.3 M NaOH
and 1 mM EDTA at pH 13) in a horizontal electrophoresis
tank (Model POWER PAC 200, Bio-Rad Co., USA) for
30 min at room temperature to allow for DNA unwinding.
Electrophoresis was performed for 10 min at 25 V and
300 mA in a chamber cooled in an ice bath. After electrophoresis, the glass slides were neutralised in 0.4 M TrisHCl
(pH 7.5) buffer, washed twice in distilled water and left
overnight for drying at room temperature. The slides were
stained following the silver staining method of Nadin et al.
(2011). Stained slides were examined using an Olympus
Microscope model-BX60 fitted with an Olympus-DP72
camera. The classification of comet category and their tail
measurement was carried out according to Garcia et al.
(2007).
Determination of the enzymatic antioxidants
Extracts for determining the activities of antioxidant
enzymes namely superoxide dismutase (SOD), ascorbate
peroxidase (APX), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) and lipoxygenase (LOX) were
prepared under ice-cooled conditions in the extraction buffer
at a proportion of 1:2 (w/v). For SOD, thalli were homogenised in buffer [0.05 M potassium phosphate buffer (PPB,
pH 7.8), 1 % (w/v) polyvinylpyrrolidone (PVP), 0.1 mM
Na2EDTA and 1 % (v/v) Triton X-100], for APX [0.1 M
PPB (pH 6.8), 1 mM Na2EDTA, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 0.5 mM L-AsA (freshly prepared)], for GR [0.1 M PPB (pH 6.8), 3 mM Na2EDTA, 1 %
(w/v) PVP and 1 % (v/v) Triton X-100] and for GSH-Px
[100 mM Tris HCl (pH 7.9), 1 % ascorbate and 1 % (w/v)
Table 1 Effect of ionic liquid [C12mim]Br exposure on cell viability (in %) in U. lactuca in various treatments, viz. control, 0.5LC50, LC50 and 2
LC50, with and without Acadian marine plant extract powder (AMPEP)
ILs concentration
Control
0.5 LC50
LC50
2xLC50
Without AMPEP
100 a
73.292.6b
48.802.8 c
10.331.7 d
100
96.771.9 a
71.443.5 b
52.965.7 c
14.051.1 d
97.873.6
77.823.8
62.852.8
14.722.4
a
b
c
c
150
200
250
102.223.4a
81.242.7 b
83.183.6 b
16.450.97 c
98.985.6 a
82.904.6 b
81.335.1 b
16.732.2 c
97.044.6
83.834.2
82.975.3
16.270.6
a
b
b
c
Different lower case letters in a columns indicate significant difference at p<0.01 analysed by one way ANOVA (meanSD, n03)
Results
Cell viability
Cell viability of U. lactuca was influenced significantly by
IL exposure and decreased with the increase of IL concentration (Table 1). However, the supplementation of AMPEP
to the cultures during IL exposure (LC50) resulted in a
considerable linear increase in the cell viability. Application
of AMPEP (150 g mL1) led to an increase in cell viability
of U. lactuca to 83.18 % (LC50) when compared to 48.80 %
during IL exposure (LC50). A further increase in the concentration of AMPEP however did not result in
corresponding increase in cell viability. Nevertheless,
AMPEP supplementation (at all the studied concentration)
was inefficient in protecting the algal cells from IL-induced
toxicity (2LC50) and exhibited minimum cell viability of
10.3316.73 % when compared to control. Based on these
findings, the potential of AMPEP to detoxifying the ROS,
protection from DNA damage and the effects on the
373
Fig. 3 Histochemical localization of reactive oxygen species generation: a O2 and b H2O2 in U. lactuca following exposure to the ionic
liquid [C12mim]Br for 4 d. Reactive radicals were detected with nitroblue tetrazolium (NBT) and 3,3-diaminobenzidine (DAB) staining,
respectively. Characters (ad) inside figures represent the various treatments, viz. control, LC50, 2LC50, LC50 +AMPEP, respectively. The
AMPEP was used at a concentration of 150 g mL1 in the culture
medium (Kumar et al. 2011a)
antioxidant system of the alga were studied using the concentration of LC50 [C12mim]Br together with AMPEP at
150 g mL1.
IC50 (g mL-1)
MeanSD, n03
57.962.65
127.237.84
177.787.89
67.654.59
85.014.15
93.981.90
62.065.94
1.550.12
DNA damage
Enzymatic antioxidants
Comet assay was used as a sensitive biological marker for
measuring DNA damage in cells exposed to oxidative stress
representing the disproportion between free radical production and functions of the antioxidant system. Results
obtained from comet assay clearly evident the IL-induced
DNA damage in Ulva. Following exposure to IL, the number of comets belonging to category 3 (high damage, 45
0
1
2
3
4
% DNA
in tail
05
525
2545
4570
>70
Tail length
(M)
Number of comets
Control
LC50
LC50 +
AMPEP
4.28.4
8.524.8
25.038.7
39.055.8
48
9
3
10
4
3
37
14
24
21
1
56.096.3
375
Discussion
The earlier investigations aimed at studying the toxicity of
ILS have indicated that the contamination of marine ecosystem with ILs cause a serious threat to the survival of seaweeds (Kumar et al. 2011a). The present findings revealed
significant changes in biochemical processes in U. lactuca
following exposure to IL (LC50). In general, IL exposure
resulted in reduced cell viability, inhibition of enzymatic
antioxidant defence system, increase in biomarkers level
(ROS and MDA level) and DNA damage. These findings
suggest that ionic liquids may have negative impact on the
structure and function of aquatic ecosystem by accumulating in higher trophic levels through the food chain. Further,
ILs contamination may alter population demographic rates,
species interaction within communities and biogeochemical
processes (Bernot et al. 2005). Our results are in agreement
with the previous findings of Yu et al. (2009) who reported
the production of ROS coupled with the disturbance in
antioxidant system of Daphnia magna exposed to 1-alkyl-
References
APPA (2010) The Asian network for using algae as a CO2 sink. The
Asia Pacific Phycological Association. News letter (6), 1 December 2010.
377
Bernot RJ, Kennedy EE, Lamberti GA (2005) Effects of ionic liquids
on the survival, movement, and feeding behavior of the freshwater snail, Physa acuta. Environ Toxicol Chem 24:17591765
Bonhote P, Dias AP, Papageorgiou N, Kalyanasundaram K, Gratzel M
(1996) Hydrophobic, highly conductive ambient temperature
molten salts. Ing Chem 35:11681178
Cho CW, Jeon YC, Pham TPT, Vijayaraghavan K, Yun YS (2008) The
ecotoxicity of ionic liquids and traditional organic solvents on
microalga Selenastrum capricornutum. Ecotoxicol Environ Safe
71:166171
Coleman D, Gathergood N (2010) Biodegradation studies of ionic
liquids. Chem Soc Rev 39:600637
Couling DJ, Bernot RJ, Docherty KM, Dixon JK, Maginn EJ (2006)
Assessing the factors responsible for ionic liquid toxicity to
aquatic organisms via quantitative structure-property relationship
modelling. Green Chem 8:8290
Earle MJ, Seddon KR (2000) Ionic liquids: green solvents for the
future. Pure Appl Chem 72:13911398
Fan D, Hodges DM, Zhang J, Kirby CW, Ji X, Locke SJ, Critchley AT,
Prithiviraj B (2011) Commercial extract of the brown seaweed
Ascophyllum nodosum enhances phenolic antioxidant content of
spinach (Spinacia oleracea L.) which protects Caenorhabditis
elegans against oxidative and thermal stress. Food Chem
124:195202
Garcia O, Romerob I, Gonzalez J, Mandina ET (2007) Measurements
of DNA damage on silver stained comets using free Internet
software. Mutat Res 627:186190
Hazra B, Biswas S, Mandal N (2008) Antioxidant and free radical
scavenging activity of Spondias pinnata. BMC Complement
Altern Med 8:6372
Holdt SL, Kraan S (2010) Bioactive compounds in seaweed: functional
food applications and legislation. J Appl Phycol 23:543597
Hurtado AQ, Yunque DA, Tibubos K, Critchley AT (2009) Uses of
Acadian marine plant extract powder from Ascophyllum nodosum
in tissue culture of Kappaphycus varieties. J Appl Phycol 21:633
639
Jayaraj J, Norrie J, Punja ZK (2011) Commercial extract from the
brown seaweed Ascophyllum nodosum reduces fungal diseases
in greenhouse cucumber. J Appl Phycol 23:353361
Khan W, Palanisamy R, Hankins SD, Critchley AT, Smith DL,
Papadopoulos Y, Prithiviraj B (2008) Ascophyllum nodosum (L.)
Le Jolis extract improves root nodulation in alfalfa. Can J Plant
Sci 88:728728
Khan W, Hiltz D, Critchley AT, Prithiviraj B (2010) Bioassay to detect
Ascophyllum nodosum extract-induced cytokinin-like activity in
Arabidopsis thaliana. J Appl Phycol 23:409414
Kumar M, Kumari P, Gupta V, Anisha PA, Reddy CRK, Jha B (2010a)
Differential responses to cadmium induced oxidative stress in
marine macroalga Ulva lactuca (Ulvales, Chlorophyta). Biometals 23:315325
Kumar M, Kumari P, Gupta V, Reddy CRK, Jha B (2010b) Biochemical responses of red alga Gracilaria corticata (Gracilariales,
Rhodophyta) to salinity induced oxidative stress. J Exp Mar Biol
Ecol 391:2734
Kumar M, Trivedi N, Reddy CRK, Jha B (2011a) Toxic effects of
imidazolium ionic liquids on the green seaweeds Ulva lactuca:
oxidative stress and DNA damage. Chem Res Toxicol 24:1882
1890
Kumar M, Gupta V, Trivedi N, Kumari P, Bijo AJ, Reddy CRK, Jha B
(2011b) Desiccation induced oxidative stress and its biochemical
responses in intertidal red alga Gracilaria corticata (Gracilariales,
Rhodophyta). Environ Exp Bot 72:194201
Kumar M, Bijo AJ, Baghel RS, Reddy CRK, Jha B (2012) Selenium and
spermine alleviate cadmium induced toxicity in the red seaweed
Gracilaria dura by regulating antioxidants and DNA methylation.
Plant Physiol Biochem 51:129138