Total oxidant scavenging capacity (TOSC) of microsomal and cytosolic fractions from Antarctic, Arctic and Mediterranean scallops: Differentiation between three potent oxidants

Article abstract of DOI:10.1016/S0166-445X(99)00070-3

The enhanced formation of reactive oxygen species (ROS) is a common pathway of toxicity induced by stressful environmental conditions. In polar environments, characterization of antioxidant defences in key sentinel species may be of particular value as early detection biomarkers of unforeseen effects of human activities which are progressively increasing in these remote areas. The complexities associated with predicting the consequences at the 'organism level' of variations of specific antioxidant defences have been recently overcome by the ability to quantify an index of specific biological resistance to various kinds of ROS. The total oxyradical scavenging capacity (TOSC) assay has been used in three species of scallops for quantifying their ability to neutralize peroxyl (ROO·) and hydroxyl (·OH) radicals and peroxynitrite (HOONO). Adamussium colbecki and Chlamys islandicus represent key organisms for monitoring Antarctic and Arctic regions while Pecten jacobaeus was chosen for a comparison with a related temperate species. TOSC values for ROO· were significantly higher in A. colbecki indicating this species as the most efficient scavenger of ROO·. Mediterranean scallops had the lowest TOSC for ROO·. A. colbecki also exhibited the highest scavenging capacity for ·OH with values more than 2- fold greater than for C. islandicus and P. jacobaeus. TOSC for HOONO was lower for all scallops as compared to those for ROO· or ·OH. TOSC for microsomes was not significantly different among the species for any ROS studied, and the percentage contribution to the specific TOSC for the various oxidants of microsomes of all scallops accounted for 1-3% of the total TOSC of the post-mitochondrial fraction. The specific TOSC of scallop microsomes for ·OH was approximately ten times lower than that for ROO· or HOONO. The higher basal capability of the Antarctic scallop to neutralize different reactive oxygen species is discussed in terms of a possible adaptation to this extreme environment and TOSC is validated as a quantifiable measure of susceptibility to oxidative stress in marine organisms. (C) 2000 Elsevier Science B.V.

Full text of DOI:10.1016/S0166-445X(99)00070-3

Aquatic Toxicology 49 (2000) 13–25
www.elsevier.com/locate/aquatox
Total oxidant scavenging capacity (TOSC) of microsomal
and cytosolic fractions from Antarctic, Arctic and
Mediterranean scallops: differentiation between three
potent oxidants
Francesco Regoli a,*, Marco Nigro b, Stefano Bompadre c, Gary W. Winston d
a Istituto di Biologia e Genetica, Uni6ersita` di Ancona, Via Ranieri Monte d’Ago, 60100 Ancona, Italy
b Dipartimento di Morfologia Umana e Biologia Applicata, Uni6ersita` di Pisa, Pisa, Italy
c Istituto di Scienze Biomediche, Uni6ersita` di Ancona, Ancona, Italy
d Department of Toxicology, North Carolina State Uni6ersity, Raleigh, NC, USA
Received 28 April 1999; received in revised form 10 August 1999; accepted 17 August 1999
Abstract
The enhanced formation of reactive oxygen species (ROS) is a common pathway of toxicity induced by stressful
environmental conditions. In polar environments, characterization of antioxidant defences in key sentinel species may
be of particular value as early detection biomarkers of unforeseen effects of human activities which are progressively
increasing in these remote areas.
The complexities associated with predicting the consequences at the ‘organism level’ of variations of specific
antioxidant defences have been recently overcome by the ability to quantify an index of specific biological resistance
to various kinds of ROS.
The total oxyradical scavenging capacity (TOSC) assay has been used in three species of scallops for quantifying
’
’
their ability to neutralize peroxyl (ROO ) and hydroxyl ( OH) radicals and peroxynitrite (HOONO). Adamussium
colbecki and Chlamys islandicus represent key organisms for monitoring Antarctic and Arctic regions while Pecten
’
jacobaeus was chosen for a comparison with a related temperate species. TOSC values for ROO were significantly
’
higher in A. colbecki indicating this species as the most efficient scavenger of ROO . Mediterranean scallops had the
lowest TOSC for ROO . A. colbecki also exhibited the highest scavenging capacity for OH with values more than
2-fold greater than for C. islandicus and P. jacobaeus. TOSC for HOONO was lower for all scallops as compared to
those for ROO or OH. TOSC for microsomes was not significantly different among the species for any ROS studied,
and the percentage contribution to the specific TOSC for the various oxidants of microsomes of all scallops accounted
for 1–3% of the total TOSC of the post-mitochondrial fraction. The specific TOSC of scallop microsomes for OH
’
’
’
’
’
’
was approximately ten times lower than that for ROO or HOONO.
The higher basal capability of the Antarctic scallop to neutralize different reactive oxygen species is discussed in
* Corresponding author. Tel.: +39-071-2204613; fax: +39-071-2204609.
E-mail address: regoli@popcsi.unian.it (F. Regoli)
0166-445X/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S0166-445X(99)00070-3

14
F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
terms of a possible adaptation to this extreme environment and TOSC is validated as a quantifiable measure of
susceptibility to oxidative stress in marine organisms. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Adamussium colbecki; Antioxidant defences; Biomarkers; Total antioxidant capacity; Oxidative stress; Reactive oxygen
species
Porte et al., 1991; Ribera et al., 1991; Winston
and Di Giulio, 1991; Stein et al., 1992; Di Giulio
et al., 1993; Livingstone, 1993; Regoli and Princi-
pato, 1995; Regoli et al., 1998a). Investigations of
the susceptibility to oxidative stress in marine
organisms have been classically based on analysis
of specific, single antioxidants, their biosynthetic
pathways, intracellular localization, mode of ac-
tion and responses to different stressors (Di
Giulio et al., 1989; Viarengo et al., 1989; Winston
et al., 1990, 1991; Livingstone, 1991a,b; Viarengo
et al., 1991a,b; Livingstone et al., 1992; Winston
et al., 1996; Regoli, 1998; Regoli et al., 1998b).
These approaches have been of significant benefit
in understanding environmentally-induced oxida-
tive stress. Nevertheless, in light of the complexi-
ties associated with predicting the consequences of
environmental alteration on adaptation to prooxi-
dant challenge and antioxidant defences, such
study of specific responses does not provide a
holistic understanding of the antioxidant capacity
of organisms in quantifiable terms with respect to
oxidant scavenging potential.
We have recently developed an analytical
method for measuring and quantifying the capa-
bility of biological samples to neutralize ROS;
thus, providing an index of specific biological
resistance to various kinds of ROS (Winston et
al., 1998; Regoli and Winston, 1999). In the total
oxyradical scavenging capacity (TOSC) assay dif-
ferent ROS can be generated at a constant rate. In
this manner, ROS can oxidize the substrate a-
keto-g-methiolbutyric acid (KMBA), to ethylene
gas, and the course of ethylene formation is mea-
sured. The efficiency of cellular antioxidants as
scavengers of the generated ROS is a function of
their ability to inhibit the reaction between ROS
and KMBA. The degree of inhibition of ethylene
formation relative to controls yields a parameter
termed the TOSC value, which is an index of the
antioxidant capacity of a sample for a defined
1. Introduction
Aerobic organisms are normally exposed to re-
active oxygen species (ROS), which are formed in
several metabolic pathways. These include the
four-electron reduction of molecular oxygen to
water coupled with oxidative phosphorylation,
other microsomal or photosynthetic electron-
transport chains, active phagocytosis and the ac-
tivities of several enzymes which produce ROS as
intermediates (Asada et al., 1974; Fridovich, 1978;
Halliwell, 1978; Winston and Cederbaum, 1983;
Babior, 1984; Cadenas et al., 1984; Halliwell and
Gutteridge, 1984). The main ROS produced in
these cellular processes include superoxide anion
(O−2 ), hydrogen peroxide (H2O2), hydroxyl radi-
’
’
cals (HO ) peroxyl radicals (ROO ), alkoxyl radi-
’
cals (RO ) and peroxynitrite (HOONO). All of
these species are strong oxidants, however, their
reactivity/toxicity towards biological macro-
molecules varies greatly; hydroxyl radicals and
superoxide anion, respectively are generally re-
garded as the strongest and weakest oxidants
(Halliwell and Aruoma, 1991). Intracellular for-
mation of ROS does not preclude toxicity since
damaging effects of ROS to biological tissues are
normally counteracted by a complex array of low
molecular weight antioxidants (both water- and
lipid-soluble) and specially adapted enzymes. Oxi-
dative stress is suppressed if the balance between
prooxidant challenge and antioxidant defences is
not exceeded (Winston, 1991).
The areas of free radical biology and oxidative
stress are of increasing interest to the field of
ecotoxicology for their applications in biomoni-
toring programs. Since environmental pollutants
can enhance intracellular formation of ROS, vari-
ations in the levels or activities of the main an-
tioxidants have been proposed as biomarkers of
contaminant-mediated oxidative stress in marine
organisms (Lee, 1988; Collier and Varanasi, 1991;

F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
15
ROS. Since the relative efficiency of antioxidants
may considerably vary towards different ROS, the
TOSC assay has been standardized for measuring
scavenging capacity with respect to various ROS
(Regoli and Winston, 1999) including peroxyl rad-
icals, hydroxyl radicals and peroxynitrite or oxi-
dants generated from the decomposition of
peroxynitrite (Squadrito et al., 1996). The TOSC
assay has been used recently by our laboratory to
evaluate the scavenging capacity of tissues from
different marine invertebrates towards peroxyl
radicals (Regoli et al., 1998a,b; Regoli and Win-
ston, 1998).
somal fractions of A. colbecki, C. islandicus and
P. jacobaeus towards peroxyl radical, hydroxyl
radical and peroxynitrite. These data provide the
first baseline data sets for resistance to toxicity of
reactive oxygen species in three important sentinel
organisms and validate the use of TOSC assay as
a tool for ecotoxicologists dealing with oxidative
stress in marine organisms.
2. Materials and methods
2.1. Sampling
Knowledge of the susceptibility of organisms to
oxidative stress is of particular importance in
polar environments in which characterization of
the responses of antioxidant defences in key sen-
tinel species may be of value as early detection
biomarkers of unforseen effects of human activi-
ties, which are progressively increasing in these
remote areas. Previous investigations on the
Antarctic scallop Adamussium colbecki, revealed a
general enhancement of several antioxidant de-
fences in comparison to the Mediterranean species
Pecten jacobaeus, suggesting some influence of the
extreme environmental conditions such as the low
temperature of seawater with higher levels of dis-
solved oxygen (Regoli et al., 1997b).
Specimes of C. islandicus were collected in Au-
gust 1995 at Solovetsky Island (White Sea, Rus-
sia) during an international cruise under the aegis
of an INTAS (International Association for the
Promotion and Cooperation with Scientists from
the Independent States of the Former Soviet
Union) project on biodiversity and adaptation
strategies of Arctic coastal marine benthos (Fig.
1). A. colbecki was sampled in January at Terra
Nova Bay (Ross Sea) during the XIII Italian
Antarctic Expedition (1997–1998) and specimens
of P. jacobaeus were obtained in June from a local
farm in Chioggia, Venice (Italy). Digestive glands
from each of the three species were dissected from
30 individuals, grouped into six pools and main-
tained in liquid nitrogen until processed for
analyses.
Herein, we report on the antioxidant defence
capabilities of three species of scallops, which live
in uniquely different environments, namely, A.
colbecki, Chlamys islandicus and P. jacobaeus, re-
spectively from the Antarctic, Arctic and the
Mediterranean sea.
2.2. Enzymatic analyses
The levels of total glutathione and the activity
of several antioxidant enzymes (catalase, superox-
ide dismutase, glutathione peroxidases, glu-
tathione reductase, glutathione S-transferases,
glyoxalase I and glyoxalase II) have been analysed
in the Arctic scallop C. islandicus and compared
with values measured in A. colbecki and P. ja-
cobaeus. Although Arctic organisms live under
extreme conditions during the winter, they experi-
ence much higher temperatures during the sum-
mer; thus, the Arctic environment may be
considered as ‘intermediate’ to that of the Antarc-
tic and Mediterranean environments. Finally, we
report the TOSC values for cytosolic and micro-
Total glutathione was analysed in samples ho-
mogenized (1:5 w/v ratio) in 5% sulphosalicilic
acid with 4 mM EDTA, maintained for 45 min on
ice and centrifuged at 37 000×g for 15 min. The
resulting supernatants were assayed by the enzy-
matic method of Akerboom and Sies (1981).
For enzymatic activities of cytosolic fractions,
samples were homogenized (1:4 w/v ratio) in 100
mM Tris–HCl buffer pH 8.0, 0.1 mM phenyl-
methylsulphonyl fluoride (PMSF), 0.1 mg/ml bac-
itracin, 0.008 TIU/ml aprotinin, NaCl 3%, and
centrifuged at 110 000×g for 1 h at 4°C. Mea-
surements were made with a Varian (model Cary
5) spectrophotometer at a constant temperature

16
F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
Glyoxalase II (EC 3.1.2.6) activity was mea-
sured at 412 nm from the reaction between 5,5%-
dithio-bis-2-nitrobenzoic acid (DTNB) with GSH
of 15°C; all of the assay conditions have been
detailed elsewhere (Regoli et al., 1997a,b, 1998b).
Glutathione reductase (EC 1.6.4.2) activity was
measured by following the oxidation of NADPH
at 340 nm during the reduction of GSSG (extinc-
tion coefficient, m=6.22/mM/cm). The assay con-
ditions were 100 mM Na-phosphate buffer pH
7.0, 1 mM GSSG and 60 mM NADPH.
formed from S-D-lactoylglutathione (LSG). The
assay conditions were 100 mM Na-phosphate
buffer pH 7.2 with 0.2 mM DTNB, 0.9 mM LSG
(m=13.6/mM/cm).
Glutathione peroxidases (GPx) activities were
assayed in a coupled enzyme system where the
formed GSSG is reduced to GSH from excess
glutathione reductase. The decrease in absorbance
at 340 nm (m= −6.22/mM/cm) due to NADPH
oxidation was followed in 100 mM Na-phosphate
buffer pH 7.5, 1 mM EDTA, 1 mM dithiothreitol
(DTT), 1 mM NaN3 (only for hydrogen peroxide
Glyoxalase I (EC 4.4.1.5) activity was deter-
mined by monitoring the formation of S-D-lac-
toylglutathione from the hemimercaptal adduct of
methylglyoxal (MG) and reduced glutathione at
240 nm (m=3.37/mM/cm). The working buffer
contained 1.5 mM GSH and 1.5 mM MG in 100
mM Na-phosphate buffer pH 6.8.
Fig. 1. Sampling sites in the Arctic region (Solovetsky Island, White Sea), in the Mediterranean (Chioggia, Venice) and in Antarctica
(Terra Nova Bay, Ross Sea).

F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
17
assays), 2 mM GSH, 1 unit glutathione reductase,
0.24 mM NADPH and 0.6 mM hydrogen perox-
ide or 0.8 mM cumene hydroperoxide, respec-
tively, were used to differentiate between the
Se-dependent (EC 1.11.1.9) and the sum of Se-de-
pendent and Se-independent (EC 2.5.1.18)
activities.
Catalase (EC 1.11.1.6) was measured by the
decrease in absorbance at 240 nm (m=0.04/mM/
cm) due to H2O2 consumption (12 mM H2O2 in
100 mM Na-phosphate buffer pH 7.0).
tions were finally resuspended in 100 mM potas-
sium phosphate buffer, pH 7.5, with 2.5% NaCl,
aliquoted and stored at −80°C.
The time-course of ethylene formation from
KMBA was followed by gas chromatographic
analysis of aliquots removed from the head-space
of the reaction vessels. The capability of a sample
to scavenge oxyradicals is quantified from its abil-
ity to inhibit ethylene formation relative to a
control reaction, which contained no biological
sample (Winston et al., 1998).
Glutathione S-transferases (GST) (EC 2.5.1.18)
were determined at 340 nm using 1-chloro-2,4-
dinitrobenzene (CDNB) as substrate (m=9.6/mM/
cm). The assay was carried out in 100 mM
Na-phosphate buffer pH 6.5, 1.5 mM CDNB, 1
mM GSH.
Three oxidant generating systems designed to
generate independently peroxyl radicals, hydroxyl
radicals and peroxynitrite were used to compare
the antioxidant scavenging capacity of subcellular
fractions isolated from tissues of scallops towards
representative oxidants that may be produced in
the cell. Holding the oxidizable substrate KMBA
at a constant concentration appropriate assay
conditions were developed in which each oxidant
produced an equivalent yield of ethylene produc-
tion throughout the reaction time-course. Thus,
the relative efficiency of cellular antioxidants has
been compared for their ability to counteract
different oxidants under quantitatively similar
conditions of KMBA oxidation. Peroxyl radicals
were produced by the thermal homolysis of 2-2%-
azo-bis-(2 methylpropionamidine)-dihydrochlo-
ride (ABAP), hydroxyl radicals were generated by
an iron-ascorbate Fenton reaction and peroxyni-
trite was generated from 3-morpholinosydnon-
Superoxide dismutase (SOD) (EC 1.15.1.1) was
quantified by measuring its ability to inhibit the
reduction of cytochrome c in the presence of the
O−2 -generating system hypoxanthine/xanthine oxi-
dase. The reduction of cytochrome c was moni-
tored in 50 mM Na-phosphate buffer pH 7.8, 0.1
mM EDTA, 50 mM hypoxanthine, 10 mM cy-
tochrome c, 6 mU xanthine oxidase and different
volumes of sample were used to determine the
50% inhibition of reaction rate. One unit of SOD
is defined as the amount of enzyme required to
inhibit by 50% the reduction of cytochrome c.
The protein concentrations in the 110 000×g
supernatants were determined according to the
method of Lowry et al. (1951) with bovine serum
albumin (BSA) used as standard.
imine (SIN-1),
a
molecule that releases
concomitantly nitric oxide and superoxide anion,
which rapidly combine to form HOONO. Final
assay conditions were: (a) 0.2 mM KMBA, 20
mM ABAP in 100 mM potassium phosphate
buffer, pH 7.4 for peroxyl radicals; (b) 1.8 mM
Fe3+, 3.6 mM EDTA, 0.2 mM KMBA, 180 mM
ascorbic acid in 100 mM potassium phosphate
buffer, pH 7.4 for hydroxyl radicals; (c) 0.2 mM
KMBA and 80 mM SIN-1 in 100 mM potassium
phosphate buffer, pH 7.4 with 0.1 mM diethylene-
triaminepentaacetic acid (DTPA) for peroxyni-
trite. Reactions were conducted at 35°C in 10 ml
vials sealed with gas-tight Mininert® valves (Su-
pelco, Bellefonte, PA) in a final volume of 1 ml.
Aliquots of 200 ml were taken from the head space
of reaction vessels with a gas-tight syringe at
2.3. Total oxyradical sca6enging capacity (TOSC)
assay
Digestive glands were homogenized in four vol-
umes of 100 mM potassium phosphate buffer, pH
7.5, containing 2.5% NaCl, aprotinin (1 mg/ml),
leupeptin (1 mg/ml) and pepstatin (0.5 mg/ml).
After initial centrifugation at 37 000×g (4°C) for
20 min, the supernatants were further centrifuged
at 110 000×g for 1 h. Cytosolic fractions were
aliquoted and stored at −80°C, while the result-
ing pellets were washed three times in the homog-
enizaton buffer and centrifuged again at
110 000×g (4°C) for 1 h. The microsomal frac-

18
F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
Table 1
Comparison of glutathione content and enzymatic activities measured in cytosolic fractions of C. islandicus (mean values9S.D.,
n=6) with values previously reported for A. colbecki and P. jacobaeus (Regoli et al., 1997a,b)a
C. islandicus
A. colbecki
P. jacobaeus
Glutathione (nmol/mg total protein)
Glutathione reductase (nmol/min/mg protein)
Glyoxalase I (nmol/min/mg protein)
12.992.17 n.s.
36.495.12 a
324941.5 b
25.194.92 a
9.7292.12 ab
12.293.71 ab
6709140 c
12.292.33 n.s.
35.294.77 a
437962.1 a
29.395.17 a
11.593.60 a
14.995.78 a
62609830 a
510981.2 a
10.793.12 n.s.
13.191.52 n.s.
16.293.13 b
135912.9 c
14.294.44 b
6.0791.97 b
7.2293.78 b
39209580 b
51.497.92 c
9.9092.41 n.s.
Glyoxalase II (nmol/min/mg protein)
Glutathione peroxidases (H2O2) (nmol/min/mg protein)
Glutathione peroxidases (CHP) (nmol/min/mg protein)
Glutathione S-transferases (nmol/min/mg protein)
Catalase (mmol/min/mg protein)
117912.2 b
11.593.22 n.s.
Superoxide dismutase (SOD units/mg protein)
a Different letters (a, b, c) indicate significant differences between means of values.
ical tissues were diluted to obtain experimental
TOSC within this range of values, which generally
corresponded to 55910 mg of protein in the
assay.
10–12 min intervals during the time-course of the
reaction and ethylene production was measured
with a Hewlett Packard (HP 4890 series) gas
chromatograph equipped with a Supelco SPB-1
capillary column (30 m×0.32 mm×0.25 mm)
and a flame ionization detector (FID). The oven,
injection and FID temperatures were, respectively,
35, 160 and 220°C; helium was the carrier gas (1
ml/min flow rate) and a split ratio 20:1 was used.
For the various oxidant-generating systems,
TOSC values are quantified from the equation:
TOSC=100−(SA/CA×100), where SA and CA
are the integrated areas calculated under the least
squares kinetic curve produced during the reac-
tion course for sample (SA) and control (CA)
reactions, respectively (Winston et al., 1998).
Thus, a TOSC value of 0 (SA/CA=1) indicates a
sample with no scavenging capacity (i.e. no inhibi-
tion of ethylene formation), while a maximum
theoretical TOSC value of 100 would correspond
2.4. Statistics
To determine differences in biochemical re-
sponses between the investigated species, one-way
analysis of variance (ANOVA) was used. The
homogeneity of variance was analysed by
Cochram C, and the post hoc tests (Newman–
Keuls) were used to discriminate between means
of values.
3. Results
The levels of glutathione and the activities of
antioxidant enzymes measured in C. islandicus are
reported in Table 1. The values are shown in
comparison with those previously measured in A.
colbecki and P. jacobaeus (Regoli et al., 1997a,b).
While similar levels of glutathione were obtained
in the three species of scallops, the activity of
glutathione reductase was significantly higher in
C. islandicus and A. colbecki than in P. jacobaeus.
Compared to the Mediterranean scallop (P. ja-
cobaeus) higher activities of antioxidant enzymes
were observed in polar scallops (A. colbecki and
C. islandicus) for glyoxalase I, glyoxalase II and
glutathione peroxidase (both Se-dependent and
to
a total inhibition of ethylene formation
throughout the assay (SA=0). For all the sam-
ples, a specific TOSC (referred to 1 mg of protein)
was calculated dividing the experimental TOSC
values by the relative protein concentration in mg
contained in the assay. Previous investigations on
both pure antioxidants and biological tissues (Re-
goli and Winston, 1998, 1999; Regoli et al., 1998c;
Winston et al., 1998) revealed that sample dilu-
tions giving experimental TOSC values ranging
from 20 to 40 are in the maximum linearity
interval with a slope value approaching 1. Biolog-

F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
19
-independent forms). Values in C. islandicus were
generally intermediate between those of A. col-
becki and P. jacobaeus. The specific activity of
catalase was also intermediate in C. islandicus,
albeit closer to that of the Mediterranean than of
the Antarctic scallop. The Arctic scallop ex-
pressed considerably lower activities of glu-
tathione S-transferases as compared to the other
species. The differences in the activities of SOD
Fig. 3. Total oxyradical scavenging capacity towards peroxyl
radicals, hydroxyl radicals and peroxynitrite of cytosols from
A. colbecki, C. islandicus and P. jacobaeus. Values are ex-
pressed as TOSC units/mg protein (mean values9S.D., n=
6).
between the three species were not significant.
Fig. 2 shows comparative time-courses of
ethylene production over a 96-min incubation of
0.2 mM KMBA in the ABAP, iron-ascorbate and
SIN-1 oxidant-generating systems. In the presence
of different amounts of biological sample the
ethylene formation proportionally varied (Fig.
2B) indicating a linear relationship between TOSC
values and the concentration of biological mate-
rial (Fig. 2C). The TOSC values towards peroxyl
radicals of cytosols from the three species are
shown in Fig. 3A where it is noted that values
were significantly higher in A. colbecki, indicating
this species as the most efficient scavenger of
Fig. 2. Panel A: Comparative time-courses of ethylene produc-
tion from a 96-min incubation of KMBA in the ABAP, SIN-1
and iron-ascorbate oxidant-generating systems. Panel B: Typi-
cal time-course of KMBA oxidation upon thermal homolysis
of ABAP for a control reaction and reactions with different
amounts of a cytosolic fraction. Panel C: Dose-response of
TOSC values vs. different concentrations of cytosolic frac-
tions.

20
F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
peroxyl radicals. The Mediterranean scallop
showed the lowest capability to scavenge peroxyl
radicals, and consistent with antioxidant enzyme
activities, the TOSC value of the Arctic scallop C.
islandicus was intermediate to those of A. colbecki
and P. jacobaeus. A. colbecki also exhibited the
highest scavenging capacity for hydroxyl radicals
(Fig. 3B); the TOSC value was approximately
2-fold greater than those obtained with C. islandi-
cus and P. jacobaeus. For all the scallops a signifi-
cant contribution in preventing the oxidation of
KMBA by hydroxyl radical appears to be due to
catalase since the addition of 1 mM sodium azide
to the cytosolic fractions lowered their TOSC
values towards these radicals by 50, 40 and 30%,
respectively, in A. colbecki, C. islandicus and P.
jacobaeus. This concentration of sodium azide had
no effect on the TOSC value in reactions devoid
of cytosolic fractions or on TOSC values of
known soluble antioxidants.
strong oxidant with the oxidizing power of a
hydroxyl radical. TOSC values towards peroxyni-
trite or its putative secondary decomposition
products were lower for all the scallops as com-
pared to their TOSC measured with peroxyl or
hydroxyl radicals (Fig. 3C). Differences in TOSC
values between the species were not statistically
significant.
TOSC values for microsomal fractions were
also determined, however for this fraction, analy-
sis of variance revealed no significant differences
among the species. Specific TOSC values (TOSC
standardized to 1 mg of microsomal protein) to-
wards peroxyl radicals and peroxynitrite were
slightly lower than cytosols (Fig. 4). However,
when the percentage contribution to the specific
TOSC towards the various oxidants was calcu-
lated for the microsomal fractions of all the spe-
cies, it accounted for only 1–3% of the total
TOSC of the post-mitochondrial fraction (S9).
Microsomal fractions from the three scallops were
very inefficient scavengers of hydroxyl radicals;
the specific TOSC value was about an order of
magnitude lower than those towards peroxyl radi-
cals or peroxynitrite.
SIN-1 simultaneously releases nitric oxide and
superoxide anion, which spontaneously react to
form peroxynitrite. Peroxynitrite, in turn, may
decompose rapidly in aqueous solution to yield a
4. Discussion
Because of their wide-spread distribution
throughout the Arctic and Antarctic regions, scal-
lops may have selective advantages as key sen-
tinels for biological monitoring programs of
environmental pollution. These advantages in-
clude their sedentary habit and a profound capa-
bility to concentrate most pollutants in their
tissues (Mauri et al., 1990; Regoli et al., 1998b).
Considering the increase of human activities in
these remote areas and the effects that pollutants
may have on the biochemistry and physiology of
various organisms, including their endogenous re-
dox status, it was of interest to compare the
susceptibility to oxidative stress of three species of
scallops, A. colbecki, C. islandicus and P. ja-
cobaeus, respectively, from Antarctic, Arctic and
Mediterranean regions.
Fig. 4. Total oxyradical scavenging capacity towards peroxyl
radicals and peroxynitrite of microsomal fractions from A.
colbecki, C. islandicus and P. jacobaeus. Values are expressed
as TOSC units/mg protein (mean values9S.D., n=6).
The present study revealed numerous interspe-
cific differences in antioxidant and related systems

F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
21
Higher antioxidant activities in polar scallops
were not confirmed for glutathione S-transferases
with values which in A. colbecki (62609830) and
P. jacobaeus (39209580) were approximately an
order of magnitude higher than in C. islandicus
(6709140). The high levels of glutathione S-
transferases are typical for scallops and probably
related to some natural component of their diet
(Regoli et al., 1997b); thus, the lower levels in C.
islandicus probably reflect differences in the qual-
ity and/or quantity of organic matter available as
food resource in the Arctic area.
When the three species of scallops are com-
pared there appears to be a general enhancement
of the individual antioxidants under study in the
Antarctic scallop A. colbecki, whereas the Arctic
scallop C. islandicus tended to exhibit values inter-
mediate to those of A. colbecki and the Mediter-
ranean scallop P. jacobaeus. In this regard, Arctic
environmental conditions can be considered ‘in-
termediate’ with respect to the Antarctic and
Mediterranean environment since the organisms
experience marked seasonal fluctuations of
chemico-physical factors, with extreme conditions
occurring during the winter.
The characterization of antioxidant defences
has increasing ecotoxicological application be-
cause knowledge of basal levels in appropriate key
species is the basis for biomarker studies of pollu-
tant exposure. Exposure to chemicals may result
in several responses including induction of some
antioxidant defences and/or the depletion of oth-
ers. The literature indicates a high degree of vari-
ability in the responsiveness of antioxidant
defences as a function of the species being investi-
gated, the kinds of exposure, the class of chemi-
cals, and the time of year of exposure (Suteau et
al., 1988; Wenning et al., 1988; Ribera et al., 1989;
Viarengo et al., 1989; Livingstone, 1991a,b, 1993;
Viarengo et al., 1991a,b; Stein et al., 1992; Regoli,
1998). Despite the fact that specific responses are
difficult to predict, variations in the levels or
activities of antioxidant mechanisms reveal a bio-
logical effect, and a number of studies have confi-
rmed their utility in identifying environmental
stress (Collier and Varanasi, 1991; Porte et al.,
1991; Di Giulio et al., 1993). However, when the
same organisms exhibit an increase of certain
of defence against the toxic ramifications poten-
tially caused by reactive oxygen species. The levels
of total glutathione in cytosol were quite com-
parable in the three species, but polar scallops
exhibited significantly higher activities of glu-
tathione reductase. Despite the absence of data on
the relative Km of this enzyme, this result may
suggest that under conditions of oxidative chal-
lenge, both A. colbecki and C. islandicus are more
capable of maintaining the redox status of glu-
tathione, which not only is important as a cofac-
tor of several enzymes but also as a direct
scavenger of oxyradicals (Winston et al., 1998;
Regoli and Winston, 1999). The greater capacity
to reconvert the oxidized disulfide form of glu-
tathione to its reduced thiol may render these
species more tolerant to glutathione depletion,
which is a common response to metal exposure in
marine bivalves (Suteau et al., 1988; Viarengo et
al., 1990; Regoli and Principato, 1995; Regoli et
al., 1997a, 1998b).
The activities of glyoxalase system enzymes and
glutathione peroxidases were significantly higher
in A. colbecki and C. islandicus as compared to
the Mediterranean scallop. Glyoxalases I and II
are involved in the detoxification of methylglyoxal
and other toxic a-ketoaldehydes which may be
formed during cellular peroxidative processes (Re-
goli et al., 1996). Glyoxalase enzymes are elevated
in mussels exposed to high levels of heavy metals,
probably in response to enhanced metal-mediated
peroxidative processes (Regoli, 1992; Regoli and
Principato, 1995). However, the magnitude of this
enzymatic induction is characterized by marked
seasonal fluctuations (Regoli, 1998).
The effects of stress conditions are generally
more difficult to predict with respect to glu-
tathione peroxidases since both induction and de-
pletion of this enzymatic activity have been
reported in marine invertebrates exposed to pollu-
tants (Winston and Di Giulio, 1991). The catalase
activity of C. islandicus was approximately twice
that of P. jacobaeus but much lower than in A.
colbecki. The higher basal GPx and catalase activ-
ities observed in A. colbecki and C. islandicus
suggests greater protection from peroxide toxicity
in these species.

22
F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
antioxidants concomitant to a decrease of others,
it can be difficult to relate in quantifiable terms
the real biological impact of such variability with
respect to health outcomes of organisms. This is
a key point, since biomarkers that have predic-
tive validity at the organism level have greater
ecological relevance in the assessment of the im-
pact of pollutants (Depledge, 1994). The
difficulty in relating variations of antioxidant de-
fences (biochemical level) to the organism level
has, to an extent, been allayed in our laborato-
ries by the development of the TOSC assay,
which measures the capability of a tissue to neu-
tralize ROS in quantifiable terms; thus, the
TOSC value is a measure or index of resistance
or susceptibility to oxidative stress (Winston et
al., 1998; Regoli and Winston, 1998; Regoli et
al., 1998a,b). The assay, firstly developed for
measuring the scavenging capacity of individual
isolated antioxidants and certain biological tis-
sues towards peroxyl radicals, has recently been
extended for use in measuring scavenging capac-
ity of antioxidants with respect to hydroxyl radi-
cals and peroxynitrite (Regoli and Winston,
1999). Thus, an important feature of the TOSC
assay is its ability to distinguish the reactivity of
various ROS and the relative capacity of antioxi-
dants to scavenge these oxidants.
sol from the Antarctic scallop A. colbecki were
more than 2-fold greater than those obtained in
both C. islandicus and P. jacobaeus.
Shick and Dykens (1985) first hypothesized
that the basal efficiency of oxygen detoxification
systems are influenced by the natural prooxidant
pressure to which organisms are exposed. Their
study of several algal–invertebrate symbioses
from the Great Barrier Reef revealed an hyper-
oxic challenge in zooxanthellae-containing species
where the activity of antioxidant enzymes (super-
oxide dismutase and catalase) were directly re-
lated to the chlorophyll content, an index of the
capacity for O2 production by algae. Similarly,
the higher capability of A. colbecki to counteract
different ROS could be consistent with greater
oxidative pressure in this extreme environment
characterized by constant low temperature of
seawater with higher levels of dissolved oxygen.
The particularly high hydroxyl radical scavenging
capacity shown for A. colbecki may also suggest
the effect of UV radiation which can generate
’
both H2O2 and HO directly in seawater. Both of
these ROS are produced in Antarctic waters
from the UV-induced photolysis of nitrate, dis-
solved organic matter (DOM) and, to a lesser
extent, nitrite (Qian et al., 1995). The rates of
’
HO production have been shown to decrease
The TOSC has been measured in the three
species of scallops towards peroxyl radicals, hy-
droxyl radicals and peroxynitrite, all oxidants
with significant potential to damage biological
tissues, despite their different reactivities and tox-
icities, towards biological macromolecules, in-
cluding DNA, lipids and proteins.
When the TOSC values of cytosolic fractions
are compared in the three species, A. colbecki
reflected the results obtained with measurements
made of individual antioxidants by indicating a
greater capability of this species to counteract
the toxicity of oxyradicals in comparison to C.
islandicus and P. jacobaeus. While the TOSC val-
ues towards peroxyl radicals progressively de-
creased from A. colbecki to P. jacobaeus with
intermediate values in C. islandicus, more marked
interspecific differences for the capability to neu-
tralize hydroxyl radicals were observed. The
TOSC values towards hydroxyl radicals of cyto-
with depth corresponding to the decline in UV-
flux (Qian et al., 1995) while a decrease in ozone
from 336 to 151 Dobson units resulted in a 42%
increase in H2O2 production at Crystal Sound
(66°11%S, 66°28%W; Yocis et al., 1995). Kieber
and Mopper (1995) also calculated that the dark
decay of H2O2 in Antarctic waters is slow (1.4
nM/day) compared to its photochemical produc-
tion rate (4 nM/h) indicating a turnover time of
H2O2 of the order of weeks. The effect of UV
radiation in Antarctica has been demonstrated
also at the biological level. Karentz et al. (1991)
analysed the presence of mycosporine-like amino
acids (MAAs) in 57 Antarctic species (including
algae, invertebrates and fish) and showed a wide-
spread occurrence of these UV-absorbing com-
pounds suggesting that these species have some
degree of natural biochemical protection from
UV exposure. It is recognized that the presence
of MAAs in A. colbecki can be only hypothe-

F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
23
sized at this time and a direct contribution of
these compounds to the high TOSC values and
their ability to act as scavengers of hydroxyl
radicals is unknown. However, our data clearly
demonstrate a role for catalase in protecting or-
environmental conditions, or that these organ-
isms are more susceptible to peroxynitrite-medi-
ated toxicity.
Our TOSC assay was suitable also for measur-
ing the scavenging capacity of the microsomal
fraction of scallops; no interspecific differences
were obtained suggesting a similar protective ef-
fect afforded by lipid-soluble antioxidants to-
wards the various oxidants. In comparing
between A. colbecki and P. jacobaeus, Viarengo et
al. (1994) showed that whereas the membrane
composition of the Antarctic scallop was not
qualitatively or quantitatively different with re-
spect to the various classes of phospholipids,
there was a tendency toward shorter and, to a
greater extent, more saturated fatty acid groups.
The more saturated the fatty acids of lipids the
less susceptible they are to peroxidation by ROS.
Similar TOSC values were obtained towards per-
oxyl radicals and peroxynitrite while the capabil-
ity of microsomal fraction to neutralize hydroxyl
radicals was an order of magnitude less efficient.
These data are consistent with the relatively lower
’
ganisms from HO toxicity as indicated by the
greatly reduced TOSC values upon addition of
sodium azide to the reaction medium in all of the
investigated species. This has credence in light of
’
the observation that the highest HO scavenging
capacity was obtained in A. colbecki, in which the
activity of catalase was much greater than in C.
islandicus and P. jacobaeus. Previous investiga-
tions of the classic antioxidants GSH, ascorbic
acid and Trolox (Regoli and Winston, 1999) re-
vealed relatively poorer efficiency of these
molecules as scavengers of hydroxyl radicals sug-
gesting relatively lower protection afforded in the
cellular environment against the potential toxicity
of these oxidants. Because hydroxyl radicals are
so difficult to neutralize once they are formed, it
seems reasonable to suggest that the most effi-
cient strategy for organisms to cope with the
toxicity of hydroxyl radical would be to reduce
their cellular formation by removing H2O2 which
is the main cellular precursor of these highly
reactive species.
Each of the three species of scallops studied
showed some capability to neutralize oxidants
generated by SIN-1. It has been suggested that
peroxynitrite rapidly decomposes to a strong oxi-
dant with chemical reactivity towards biological
antioxidants different from those of hydroxyl and
peroxyl radicals (Hogg et al., 1992; Squadrito et
al., 1996).
There is recent increasing interest in the role of
peroxynitrite (and putative related decomposition
products) in causing toxicity in mammals (Radi,
1996; Squadrito et al., 1996), while virtually noth-
ing is known for marine invertebrates. The TOSC
values of A. colbecki, C. islandicus and P. ja-
cobaeus cytosolic fractions were lower for perox-
ynitrite as compared to hydroxyl and peroxyl
radicals, although the interspecific differences
were not statistically significant. This apparent
lower capacity to protect against the potential
toxicity of peroxynitrite suggests either that this
oxidant does not play a critical role in mediating
cellular toxicity and is not influenced by different
’
HO scavenging capacity of classic lipid-soluble
antioxidants as vitamin E (Regoli and Winston,
1999) and corroborate our hypothesis that toxic-
ity of these reactive oxygen species are probably
best counteracted by reducing their cellular for-
mation.
In conclusion, the present comparative investi-
gation reports the first baseline datasets for the
TOSC with respect to different oxidants in three
potentially important sentinel organisms. The
analysis of TOSC could be proposed as a poten-
tial biomarker for monitoring aquatic pollution,
since a quantifiable value of susceptibility to oxi-
dative stress may provide important information
for the assessment of biological disturbance and
long-term effects of anthropogenic activities of
use to regulatory agencies and environmental
managers.
Acknowledgements
This research was funded by EC-INTAS (Pro-
ject 94-391) and by the Programma Nazionale
Ricerche in Antartide (PNRA).

24
F. Regoli et al. / Aquatic Toxicology 49 (2000) 13–25
Livingstone, D.R., 1991b. Towards a specific index of impact
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