Comparative Biochemistry and Physiology, Part C 146 (2007) 118 – 123

www.elsevier.com/locate/cbpc

Does starvation influence the antioxidant status of the digestive gland

of Nacella concinna in experimental conditions? ☆
Martín Ansaldo a,⁎, Hernan Sacristán b , Eva Wider c
a
Instituto Antártico Argentino, Dirección Nacional del Antártico, Cerrito 1248, (1010) Buenos Aires, Argentina
b
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Ciudad Universitaria (1428), Buenos Aires, Argentina
c
Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, Rivadavia 1917 (1033), Buenos Aires, Argentina
Received 13 March 2006; received in revised form 20 November 2006; accepted 20 November 2006
Available online 24 November 2006

Abstract

In a previous study we analysed the effect of diesel seawater contamination in the digestive gland of the Antarctic limpet Nacella concinna. We
observed that antioxidant enzyme activities decreased after one-week starvation prior to the experiment, and this was considered in the analysis of
the obtained results. To know whether the digestive gland oxidant–antioxidant status may be altered by starvation and experimental conditions, we
evaluated the food deprivation effect in limpets from the nearshore shallow waters of Potter Cove, Antarctica. Organisms were acclimated to
laboratory conditions and were divided in fed and starved groups, and maintained in these conditions during one month. Every week 20 limpets
were sampled from each group. Digestive glands were dissected and kept frozen until they were processed. Superoxide dismutase (SOD), catalase
(CAT) and glutathione S-transferase (GST) activities, as well as lipid peroxidation (LPO) measured as thiobarbituric reactive substances
(TBARS), protein oxidation (PO) and reduced glutathione (GSH) were measured. For both groups of limpets, SOD increased its activity in the
first week of the exposure period, with a maximum in the second week. CAT activity increased significantly in the second week, only for the
starved group. Similarly, GST activity also increased for starved group in the second week; but maintained this tendency for both groups until the
fourth week. In fed and starved limpets, TBARS values increased significantly, during the first week and then returned to normal values. The PO
levels in the starved group increased only during the first week. The GSH content, for the fed group, increased significantly after the third week.
The obtained results indicate that biochemical or physiological studies conducted with N. concinna should consider the effects of food deprivation
and time spent under experimental conditions.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Lipid-peroxidation; Protein oxidation; GSH; SOD; CAT; GST; Molluscs; Nacella concinna

1. Introduction compared with tropical and temperate marine environments

(Clarke, 1988; Clarke and Leakey, 1996). In general, the slow
The Antarctic marine environment is characterised by low growth of marine Antarctic organisms is influenced by both
but very stable temperatures and a highly seasonal food supply. food availability and temperature (Clarke, 1983). Many benthic
Food availability and temperature are effectively uncoupled species cease feeding in winter for periods ranging from a few
weeks to many months (Brockington and Clarke, 2001). In
☆
This paper is part of the 4th special issue of CBP dedicated to The Face of
contrast, the limpet Nacella concinna (Mollusca, Patellidae) is
Latin American Comparative Biochemistry and Physiology organized by unusual for Antarctic marine ectotherms because it feeds
Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene throughout the year, although feeding rates decrease signifi-
Beleboni (Brazil), Rodrigo Stabeli (Brazil), Tania Zenteno-Savín (Mexico) and cantly in winter (Fraser et al., 2002; Clarke et al., 2004).
the editors of CBP. This issue is dedicated to the memory of two exceptional N. concinna is a common invertebrate of the Antarctic and
men, Peter L. Lutz, one of the pioneers of comparative and integrative
physiology, and Cicero Lima, journalist, science lover and Hermes-Lima's dad.
Sub-Antarctic nearshore fauna. It has a circumpolar distribution
⁎ Corresponding author. Tel./fax: +54 011 4813 7807. and lives between the intertidal zones and depths around 100 m
E-mail address: tincho@qb.fcen.uba.ar (M. Ansaldo). (Cadée, 1999). The littoral features of N. concinna make it a
1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2006.11.004
 M. Ansaldo et al. / Comparative Biochemistry and Physiology, Part C 146 (2007) 118–123 119

suitable model for studies involving: histology, physiology, granulatus hepatopancreas after one week of starvation, without
biochemistry and pollution (Kennicutt et al., 1992; Cripps and any measurable modification in SOD activity. Ansaldo et al.
Shears, 1997; Abele et al., 1998; Najle et al., 2000; Peck and (2005) reported that, during the acclimation week previous to the
Veal, 2001; Fraser et al., 2002; Ahn et al., 2002; Clarke et al., start of the experiments, SOD and CAT activities decreased
2004; Ansaldo et al., 2005). significantly in the digestive gland of N. concinna. After this
Starvation exerts a wide range of effects in molluscs: e.g., period, SOD activity of control limpets remained unchanged,
decline in heartbeat frequency of Patella caerulea and Patella while CAT activity continued decreasing. Hence, starvation must
rustica (Santini et al., 2002), alterations in lysosomal membrane be considered as a pro-oxidant condition that could synergize
integrity (Moore, 2004), depletion of glycogen in the nervous external factors such as pollution and/or seasonality.
ganglia of Helix aspersa (Borges et al., 2004), decrease in the The aim of the present work was to determine whether food
RNA/DNA ratio from muscle, digestive gland and gills in green deprivation under experimental conditions could change the
mussels (Bracho et al., 2000), increased aminotransferases pro-oxidant/antioxidant balance, altering the normal oxidative
activity in the hemolymph of Bradybaena similaris (Pinheiro status of the Antarctic limpet N. concinna.
et al., 2001), and increased D-lactate dehydrogenase together with
decreased D-lactate in the mantle of Octopus ocellatus (Fujisawa 2. Materials and methods
et al., 2005). Moreover, starvation has also been reported to have
pro-oxidant effects in mammals, and has been considered Specimens of Nacella concinna with a mean shell length of
responsible for most of the harmful effects derived from food 28.3 ± 3.0 mm (wet mass 3.04 ± 0.60 g), were collected by
deprivation, e.g. increased generation of reactive oxygen species SCUBA diving at a depths ranging from 2 to 4 m near Jubany
(ROS) that is not effectively neutralized by antioxidant systems Station (Argentina), 25 de Mayo (King George) Island, South
(Robinson et al., 1997; Domenicali et al., 2001). Shetland Islands, during the summer of 2003. Two groups of 80
Oxidative stress can be understood as a situation derived limpets were randomly assigned to two treatments: “fed” and
either from an enhanced rate of ROS generation and a reduced “starved”. Twenty animals were kept in 20L Plexiglas aquaria
level of antioxidant defences (Sies, 1985). It may be estimated filled with filtered seawater at 1 ± 0.5 °C under natural
by the physicochemical condition in which an increase in the photoperiodic conditions. Seawater was continuously aerated,
steady-state levels of oxidative species (i.e., O2−, H2O2, HO˙, R˙ and completely changed every 48 h.
and ROO.) occurs. This increased steady-state level of oxidants Organisms in the fed group were fed fresh macroalgae
may consequently lead to reversible or irreversible cell damage, (mainly: crustose red algae and the brown alga Ascoseira
and eventually to cell death. ROS are produced in a series of mirabilis; secondly: the red algae Iridaea cordata, Gigartina
biochemical reactions that will normally occur within the skottsbergii and the brown alga Geminocarpus germinatus)
cellular compartments (i.e., mitochondrium and the endoplasmic which constitute their natural diet, and grow on the intertidal
reticulum are the most important ROS sources; Halliwell and rocks present in the shallow waters ecosystem of Potter Cove
Gutteridge, 1999). It is well known that a number of different (Iken et al., 1998). Every 48 h, the rocks covered with algae were
enzymes and non-enzymatic compounds participate in the replaced in each fed group in order to maintain the natural source
antioxidant chain in biological systems. Among the enzymes, of food. The replaced rocks were those without limpets on their
superoxide dismutase (SOD) converts superoxide anion (O2−) to surface avoiding any possible disturbance caused by handling.
hydrogen peroxide (H2O2), catalase (CAT) reduces H2O2 to Twenty animals from each group were sacrificed (cooling on
water, and glutathione S-transferases (GSTs), which constitute a ice, 6–8 min) at days 7, 14, 21 and 30 of the experiment (weeks
large family of multifunctional enzymes, conjugate the reduced 1–4 hereafter). The shell was carefully removed and the whole
glutathione (GSH) to xenobiotics and aldehydic products of lipid body washed in saline solution, placed on filter paper to drain
peroxidation such as 4-hydroxialkenals. As a non-enzymatic extra fluids, and weighed. Then, the digestive gland was
compound, GSH is the principal nonprotein thiol involved in the dissected, immediately immersed in liquid nitrogen and kept
antioxidant cellular defence; it provides reducing capacity for frozen until they were analysed in the laboratory. The “wild or
several reactions, and plays an important role in detoxification of natural” condition of antioxidant variables was studied on an
hydrogen peroxide, other peroxides and free radicals (Halliwell additional group of 20 animals (week 0), randomly labelled fed
and Gutteridge, 1999; Hermes-Lima, 2004). or starved, by measuring the same parameters (see below)
The effect of oxidative stress elicited by starvation was studied immediately after collection at the Jubany Station laboratory.
in several mammal models (see references in Pascual et al., 2003), All procedures were performed at 0–2 °C. Digestive glands
but only a few studies were performed in non-mammal were homogenised (1:9 w/v) in a cold (4 °C) buffer solution
vertebrates like fish (Pascual et al., 2003; Morales et al., 2004), containing Tris-base (125 mM), 2-mercaptoethanol (1 mM),
and aquatic invertebrates, like molluscs and crabs (Abele et al., and PMSF (0.1 mM) (Vijayavel et al., 2004) with pH adjusted to
1998; Pinho et al., 2003; Ansaldo et al., 2005). Abele et al. (1998) 6.8. Homogenates were centrifuged at 10,000 ×g for 10 min at
have recorded reduced levels of SOD in the digestive gland and 4 °C, and the supernatant used for enzyme activity, LPO and PO
CAT in the gills of N. concinna after one month starvation. They studies.
also observed that gill CAT activity of starved animals decreased SOD, CAT and GST activities were determined using the
under pro-oxidant treatments as H2O2 exposure. Pinho et al. spectrophotometric methods described by Misra and Fridovich
(2003) observed a decrease in CAT activity from Chasmagnathus (1972), Aebi (1984) and Habig et al. (1974) respectively.
120 M. Ansaldo et al. / Comparative Biochemistry and Physiology, Part C 146 (2007) 118–123

Specific enzyme activity was calculated considering the total compared to wild groups (week 0) (Fig. 1a). From the second
protein content of the supernatant; results were expressed as week up to the end of the assay, the TBARS levels decreased to
enzyme units/min.mg of protein. One SOD unit is the amount of the control values, remaining unchanged. No differences in
enzyme necessary to inhibit 50% the rate of autocatalytic TBARS levels were observed between fed and starved limpets
adrenochrome formation measured at 480 nm. One CAT unit is throughout the experiment ( p N 0.05).
the amount of enzyme necessary to degrade 1 μmol of H2O2, Protein oxidation, measured as carbonyl content level, only
measured at 240 nm. One GST unit represents the amount of increased significantly ( p b 0.05) in the first week for the
enzyme required to conjugate 1 μmol of 1-chloro-2.4-dinitro- starved animals (Fig. 1b). From the second week and until the
benzene, determined at 340 nm. end of the assay, values decreased back to those measured in the
The LPO level was measured according to Buege and Aust control group. The fed group remained unchanged throughout
(1978), by the formation of thiobarbituric acid reactive all the experiment ( p N 0.05).
substances (TBARS). Fresh homogenates were added to the The GSH level of the starved group showed no significantly
reaction mixture (trichloroacetic acid 15% (w/v), 2-thiobarbi- changes during the time of the assay ( p N 0.05). In the fed group,
turic acid 0.375% (w/v), and butylhydroxytoluene 0.147 mM) the level of GSH began to increase in the first week, and was
in a ratio of 1:5 (v/v). The mixture was vigorously shaken, significantly different in the third week ( p b 0.01), remaining
maintained in boiling water for 60 min, and immediately cooled high during the fourth week (Fig. 1c).
at 5 °C for 5 min (Ohkawa et al., 1979). Then it was centrifuged
at 5000×g for 10 min, and the supernatant was measured
spectrophotometrically at 535 nm.
The PO level was evaluated according to Reznick and Packer
(1994), by detecting the formation of protein hydrazones as a
result of the reaction of dinitrophenyl hydrazine (DNPH) with
protein carbonyls. Some minor modifications were performed
to the original protocol; briefly, after the protein hydrazone
formation, they were precipitated using TCA 30% (Fagan et al.,
1999), and then washed 3 times with ethanol: ethyl acetate
(1:1). After the final wash, the protein was solubilized in 1 mL
of urea (6 M in 20 mM potassium phosphate, pH 2.5) instead of
guanidine hydrochloride. To speed up the solubilization
process, the samples were incubated at 37 °C in a water bath
for 60 min. The final solution was centrifuged to remove any
insoluble material. The carbonyl content was calculated from
the absorbance measurement at 375 nm, using an absorption
coefficient ε = 22,000 M− 1 cm− 1. It was verified that urea
blanks showed the same absorbance peak at 375 nm as
guanidine hydrochloride blanks.
The reduced glutathione (GSH) level was determined by the
method of Moron et al. (1979). Digestive glands were
homogenized (1:10) in EDTA 0.02 M. After deproteinization
with trichloroacetic acid (TCA 50%), free endogenous GSH
was determined using 5,5-dithio-bis-2-nitrobenzoic acid
(DTNB) 0.5 mM. The absorbance was read at 412 nm. GSH
was used as standard to calculate nmol/mg wet tissue.
The total protein quantity of the homogenates was measured
by the method of Lowry et al. (1951) using bovine serum
albumin as standard.
Data were analysed through an ANOVA followed by a
posteriori mean comparisons (Tukey test) among wild (week 0)
and experimental groups as well as between treatments (fed vs.
starved). Previously, ANOVA assumptions were verified and
transformations applied if lack of normality and/or variance
heterogeneity were detected (Sokal and Rohlf, 1997).

3. Results
Fig. 1. The TBARS, PO and GSH levels in the digestive gland of N. concinna in
fed and starved groups at different time in aquaculture conditions. Data are
The TBARS levels for both groups augmented in the first expressed as mean ± standard error. Different letters indicate significant
week and this increment were significantly different ( p b 0.001) differences (P b 0.05) among wild (week 0), fed and starved groups.
 M. Ansaldo et al. / Comparative Biochemistry and Physiology, Part C 146 (2007) 118–123 121

The SOD activity increased significantly ( p b 0.05) for fed and the assay (Fig. 2b). During the third week, a decrease in CAT
starved groups in the end of the first week (Fig. 2a). Both groups activity was observed, and this value was not significantly
of limpets registered the highest values during the second week different ( p N 0.05) from the week 0 groups. The fed group only
( p b 0.001). The SOD activity of the starved group remained registered a significant increment ( p b 0.05) in the fourth week.
significantly different ( p b 0.05) with similar levels to those Starved limpets showed an increment of GST activity during
observed in the first week of the assay ( p N 0.05). During the third the second week, which was not statistically different from week
week, the SOD activity of the fed group returned to the week 0 0 group ( p N 0.05; Fig. 2c), whereas the fed limpets did not show
values ( p N 0.05). Fed and starved groups showed increased any change in the enzyme activity. From the third week and until
values in the end of the experimental period, although there was the end of the assay, both groups showed increased values which
no significant difference between them ( p N 0.05), but they were were statistically different ( p b 0.05) from the week 0 groups.
significantly different ( p b 0.01) from the week 0 groups.
The CAT activity showed significantly ( p b 0.05) increased 4. Discussion
values for the starved group in the second and fourth weeks of
It has been reported that most of the negative effects of
starvation could be mainly attributed to the participation of ROS
generated under food deprivation (Pascual et al., 2003, Morales
et al., 2004). However, taking into account the present results,
we should consider that the antioxidant status of the Antarctic
limpets may be influenced by starvation as well as by exper-
imental conditions.
Our results showed a marked increment in the TBARS levels
in the digestive gland of N. concinna during the first week of
aquaria acclimation, probably due to the stress produced by
experimental conditions, because this increment was observed
both in fed and starved groups. Simultaneously, an increase was
also observed for protein oxidation levels of the starved group.
The SOD, CAT and GST activities increased in a synchronized
way in the second week trying to counterbalance the oxidative
damage. This fact was mainly evident in the starved group, and
was reflected in the decrease of lipid and protein oxidation
levels, which diminished to control values (week 0) after the
second week. Throughout the experimental period, the CAT
activity seems to be slightly higher in the starved group than in
the fed one. Guderley et al. (2003) also reported increased CAT
and GST activities in the liver of the starved fish Gadus
morhua. Starvation decreases tissue metabolic capacities but, on
the other hand, the food deprivation causes degradation of
endogenous sources of energy (lipids, glycogen, and proteins)
in order to maintain the fish physiological homeostasis. They
found that the activity of certain lysosomal enzymes and
antioxidant defences were maintained or enhanced even after
the starvation period. These findings on fish antioxidant status
addressed the interpretation that when facing energetic limita-
tions such as food deprivation and/or severe hypoxia, aerobic
organisms maintain relatively high levels of their antioxidant
defences to cope with oxidative stress as metabolic priorities
compared to other important functions such as weight gain and
reproduction (Wilhelm Filho et al., 2005). Changes observed in
the antioxidant status during the reproductive cycle of molluscs
may be a determinant for the expression of the antioxidant
enzymes, as well as for the synthesis of endogenous anti-
oxidants such as GSH (Wilhelm Filho et al., 2001). In the
present study, limpets were collected in the summer, and
therefore no effect on the antioxidant status could be attributed
Fig. 2. The SOD, CAT and GST activities in the digestive gland of N. concinna
in fed and starved groups at different time in aquaculture conditions. Data are to emission of gametes by the organism because this event only
expressed as mean ± standard error. Different letters indicate significant happens in spring, in a very narrow time-frame (Stanwell-Smith
differences (P b 0.05) among wild (week 0), fed and starved groups. and Clarke, 1998).
122 M. Ansaldo et al. / Comparative Biochemistry and Physiology, Part C 146 (2007) 118–123

The effect of starvation enhancing oxidative stress could “cold” period corresponding to the summer of 2003, showed
also be attributed to the deficiency of the diet with low mole- the opposite condition: permanent snow and ice covering the
cular weight components such as ascorbic acid, α-tocopherol coast, with high amounts of ice debris in the intertidal zone
and carotenoids, which are well known antioxidants in marine turning it inaccessible for benthic fauna. These environmental
algae (Dummermuth et al., 2003). A deficit of these com- characteristics determined by the extreme difference in tem-
pounds could become a critical factor with regard to the anti- perature, might be influencing the antioxidant enzyme acti-
oxidant status of starved animals (Abele et al., 1998). However, vities. Fraser et al. (2002) reported the seasonal decrease in the
in the present study, the GSH level for the starved group Antarctic limpet metabolism due to lowering temperatures.
remained unchanged, probably due to the peptidases activities Hence, we conclude that differences observed in the antiox-
on peptides from the intracellular pool (Hermes-Lima, 2004) idant enzyme activities registered between the Ansaldo et al.
that kept the GSH level similar to the control group (0 week). (2005) and the present study, were also due to the temperature
By contrast, the fed group showed increasing values, probably fluctuation registered in both sampling years.
due to the food intake. In the present study, we observed that the starved and fed
The NADPH supply is critical because it is involved in the limpets maintained in laboratory conditions had relatively high
reduction of GSSG to GSH. The glucose-6-phosphate dehy- levels of their antioxidant defences to cope with oxidative stress
drogenase (G6PDH), a key enzyme from the pentose phosphate but the additional effect of experimental conditions probably
pathway is involved in the production of NADPH. The masked the food deprivation effect per se.
inhibition or reduction of the G6PDH activity was reported In conclusion, and according to the present findings, we
during food deprivation (Barroso et al., 1998; Morales et al., suggest that to avoid misinterpretation of experimental results,
2004), or hypometabolism due to estivation (Ramnanan and any assay involving N. concinna in aquarium conditions must
Story, 2006). Thus, the generation of oxidative stress might be take into account the acclimation period to this “artificial new
related to the inhibition of G6PDH activity in the digestive environment”. Climatic and environmental conditions during
gland metabolism in the food-deprived limpets. animal sampling should also be considered due to the fact that
In a previous study, values of SOD and CAT activities 10- they might affect the limpet's physiological status.
folded those registered in the present study for the 0 week group
(Ansaldo et al., 2005). Similarly, comparing their studies, Fraser Acknowledgements
et al. (2002) and Peck and Veal (2001) observed differences in
the oxygen consumption rates of N. concinna. Fraser et al. This study was supported by the Instituto Antártico
(2002) speculated that higher respiration rates reported by Peck Argentino (project number 57, 2002–2004) and the Universidad
and Veal (2001) had been the result of the starvation and feeding de Buenos Aires. We thank Dr. Javier Calcagno for his
protocol used. Hence, the lower respiration rates reported by invaluable help in statistical analysis. We also thank Dr. Gustavo
Fraser et al. (2002) were probably related to experimental Lovrich, Dr. Norberto Lemcoff and Lic. Patricia Pérez Barros for
protocols designed to minimise handling and experimental revising translation and for their helpful comments.
stress. In the present work, to avoid excessive handling stress,
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