Page 3 - AmbioBonacci_etal_2007

MATERIALS AND METHODS
Description of Study Area and Experimental Design
In September 2002, specimens of the three species were collected
in 12 sites in the Egadi MPA, Sicily, Italy (Fig. 1). The study
area measures 53 992 ha and was instituted in 1991. It
comprises the archipelago of the Egadi Islands consisting of
Favignana (33 km2), Marettimo (12 km2), Levanzo (10 km2),
and some smaller islands such as Maraone and Formica (43).
The area is divided into three main protection levels, according
to national criteria applied to all Italian MPAs (44): total
reserve, or Zone A, where fishing and harvesting marine
resources are forbidden, and only authorized personnel can
have access for monitoring, research, and maintenance; general
reserve, or Zone B, where only low-impact tourism is allowed;
and partial reserve, or Zone C, which is usually a buffer area
between unprotected sea and Zones A and B. Recreational
navigation and some sporting and commercial fishing are
allowed in Zone C. In the Egadi MPA, Zone A is in two parts,
one in an inshore area of Marettimo and the other around
Maraone; Zone B is in five parts: two inshore areas of
Marettimo, and one each in Favignana, in Levanzo, and
around Formica; Zone C is in two parts, one around Marettimo
and the other around Favignana, Levanzo, Maraone, and
Formica. The two C zones include the A and B zones already
defined.
The MPA is quite distant from the anthropized Sicilian coast
and is putatively free from significant negative effects of local
industries and agriculture (11). The only significant source of
man-made pollution in the MPA is thought to be the harbor of
Favignana.
Ten specimens of fish and 20 specimens of each invertebrate
species were collected at each of the 12 sites and immediately
humanely killed. Biometric parameters were recorded, and
tissues were immediately dissected and stored in liquid N2.
Rainbow wrasse were 13.9–17.6 cm in length and 13.9–35.8 g in
weight, with a male/female percentage ratio of 79/21. Gastro-
pod limpets were 2.0–3.0 cm long, 1.6–2.4 cm wide, and 0.7–1.2
cm high. Sea urchins were 3.6–4.5 cm in theca diameter.
Samples were shipped to the laboratory and stored at À808C
until analysis.
Determination of P450 Enzyme Activities
P450 enzyme activities were measured in microsomal fractions
of the following organs: liver of C. julis, visceral portion of P. Wilk W test and for homogeneity of variances by the Levine
caerulea, and pyloric caeca of P. lividus. Invertebrate tissues
from the same sampling site were grouped in seven pools of two
or three individuals each; fish samples were analyzed one by
one. Microsomal fractions were prepared by differential
centrifuging (9000 3 g for 20 min and 100 000 3 g for 60 min)
in a Sorvall RC28S ultracentrifuge, after homogenization 1:4
(w/v) with sucrose buffer (50 mM K2HPO4, 0.75 mM sucrose, 1
mM EDTA, 0.5 mM DTT, 400 lM PMSF, 10 lM leupeptin, 1
lM pepstatin, 1 mg LÀ1 aprotinin, pH 7.5) in a glass–Teflon
Potter-Elvenjem homogenizer at 2000 rpm. The resulting
microsomal pellets were separated from the supernatants,
resuspended 1:2.6 (w/v) in Tris-(base) buffer (10 mM Tris
[base], 20% w/v glycerol, 0.5 mM DTT, 400 lM PMSF, 10 lM
leupeptin, 1 lM pepstatin, 1 mg LÀ1 aprotinin, pH 7.5), and
used as enzyme source for the monooxygenase bioassay. All
procedures were carried out at 48C.
Hepatic microsomal EROD and BaPMO activities were
measured in duplicate by fluorometric methods according to
Burke and Mayer (45) and Kurelec et al. (46), respectively, with
a LS50B luminescence spectrofluorometer. EROD assay
conditions in the reaction mixture (final volume 2.25 mL) were
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as follows: pH 7.5, 308C, in a fluorometer cuvette containing 50
mM Tris-HCl, 25 mM MgCl2 6H2O, 10 lL 125 lM NADPH,
and about 25–50 lL of microsomal suspension. 7-Ethoxyresor-
ufin (10 lL 0.1 mg mLÀ1 in DMSO) was used as substrate. The
reaction was started by adding NADPH, and the progressive
increase in fluorescence was recorded for 4 min at kEX ¼ 522 nm
and kEM ¼ 586 nm. The amount of resorufin produced was
calculated from a resorufin standard. EROD activity was
expressed as picomoles of resorufin produced per minute per
milligram of total microsomal protein (pmol minÀ1 mg protÀ1).
BaPMO assay conditions in the reaction mixture (final
volume 1.01– 1.06 mL) were as follows: pH 7.5, 308C, in a
fluorometer cuvette containing 110 mM Tris-HCl, 15 mM
MgCl2 6H2O, 1.8 lM NADPH and about 50–100 lL of
microsomal suspension. Benzo(a)pyrene (B[a]P) (2 mM) was
used as substrate in a 1-h reaction stopped with cool acetone.
The amount of 3 OH-B(a)P produced was read at kEX ¼ 396 nm
and kEM ¼ 522 nm, with 1M H2SO4 and 1 lg mLÀ1 quinine
sulfate as standards. BaPMO activity was compared with a
blank treated with acetone before incubation and expressed as
fluorescence units per hour per milligram of total microsomal
protein (FU minÀ1 mg protÀ1).
NADH–ferry red and NADH–cyt c red activities were
assayed at 258C by following the method of Livingstone and
Farrar (47). The reaction mixture (1 mL final volume)
contained Tris-HCl buffer (100 mM, pH 7.6), 50 lL potassium
cyanide (KCN) (20 mM final concentration), 50 lL of either
potassium ferricyanide (10 mM) or cytochrome c (1.2 mM) as
substrates, and distilled water. The reaction was started by
adding 50 lL NADH (6 mM). Decreasing or increasing
absorbance was recorded for 1 min at 420 nm and 550 nm for
NADH–ferry red and NADH–cyt c red activities, respectively.
The instrument was a Shimadzu UV-160A visible recording
spectrophotometer. Enzyme activities were expressed as nano-
moles of metabolized substrate per minute per milligram of
total microsomal proteins (nmol mg protÀ1 minÀ1). Microsomal
protein concentration was measured as described by Bradford
(48) in a Shimadzu UV-160A visible recording spectrometer
with bovine serum albumin used as a standard.
Statistical Analysis
Results for each sampling site were expressed as mean 6
standard deviation. All determinations were performed in
quadruplicate for each pool aliquot and in duplicate for single
samples. The data were tested for normality by the Shapiro-
test. Statistically significant differences between P450 activities
in sample groups from different areas were determined by one-
way analysis of variance with Newman-Keuls post hoc
comparison for parametric data and the Kruskal-Wallis test
for nonparametric data. Correlations were assessed by the
Pearson correlation coefficient (r). A probability level less than
0.05 was considered significant. Statistical analysis was per-
formed with Statistica 5.1 software (StatSoft).
RESULTS AND DISCUSSION
Coris julis
EROD and BaPMO activities are shown in Figures 2 and 3.
EROD activities ranged from 0.528 to 1.217 nmol mg protÀ1
minÀ1 (Stations 2A and 1N, respectively). Statistically signifi-
cant differences in EROD activity were observed between
Favignana Harbor and Zone A of Marettimo (Station 1A),
Zone A of Maraone (Station 2A), and Zone C of Marettimo
(Station 1C). Similar results were found for BaPMO activities,
which ranged from 8.86 to 48.9 FU mg protÀ1 hÀ1 (Stations 2A
Ambio Vol. 36, No. 4, June 2007