86
P. Irato et al.
alimentary tract, where they are desorbed from
the particles and absorbed by the animal during
digestion (Burbidge et al.1994).
Materials and Methods
In May 1998, specimens of M. galloprovincialis. T.
philippinarum and S. inequivalvis were sampled
in two stations with different pollution levels at
Chioggia and Marghera in the Lagoon of Venice.
The shell length of the animals was measured.
Soft tissues were separated from the shell, and
digestive glands and gills were removed. weighed
and frozen at -80°C. Subsequently, glutathione
determination and MT assays were performed
on the samples, dividing the organs into two
parts. For total glutathione determination, the
samples were homogenized (1:5 w/v ratio) in 5%
sulphosalicilic acid and centrifuged at 15,000
rpm for 30 min at 4°C. Total GSH contents were
measured in the resulting supernatant by
Anderson's (1985) method and referred to wet
weights. For MT assays, the samples were
homogenized (1:4 w/v ratio) in 20 mM Tris HCl
pH 7.5, added with 0.006 mM leupeptine, 0.5 mM
PMSF (phenylmethylsulphonylfluoride) as
antiproteolitic agents, and 0.01% p-mercapto ethanol as reducing agent, and centrifuged at
30,000 g for 60 min at 4°C. The MT contents were
measured in the resulting supernatant by the silver saturation method (Scheuhammer and
Cherian 1991) and the values referred to total
protein determined by the method of Lowry et
al. (1951). The digestive glands and gills of other
individual animals were homogenized with the
same buffer as the MT assays and digested with
HN0 3 AristaR in PFA vessels in a CEM MDS-2000
microwave furnace. Metal concentrations (Cr,
Mn, Fe, Co, Ni, Cu, Zn, Cd) were determined with
an inductively coupled plasma-atomic emission
spectrometry (ICP-AES) Spectroflame Modula
sequential and referred to dry weights (identical
amounts of homogenate used for digestion were
dried in an oven at 130°C).
Values are shown as means ± standard error.
Statistical analysis was performed with the
PRIMER statistical program: Student's t-test for
comparison of pairs or ANOVA for different
groups followed by the Student-Newman-Keuls
test for determination of significant differences
(P = O.OS). Linear regression analyses were also
performed.
Results and Discussion
Sediment metal analyses (conducted by Casellato
S. and Degetto S. in 1999; unpublished data)
highlighted the important fact that Marghera has
three times more Cr than Chioggia and that Cd is
only found in Marghera. Results show that MT
contents, in both digestive glands and gills of all
three species, were not significantly different
between the two stations with different levels of
pollution. Instead, digestive gland MT contents
were significantly different in the same station
among the three species: T. philippinarum had
the greatest MT contents, followed by S. inequivalvis andM. galloprovincialis (Fig. la). The greatest amount of MT in the digestive gland of T.
philippinarum is probably due to a higher concentration of eu (Table). Moreover, it is known
that Cu has higher affmity for MT with respect to
Zn, whereas Zn is a good inductor. We therefore
hypothesize that. in T. philippinarum. Cu is
linked to MT induced by Zn, so that Zn can then
induce other MT. T. philippinarum from
Marghera was the species with the greatest
amounts of Mn, Co and Fe (Table). Gill MT contents were not significantly different in the same
station among the three species (Fig. Ib).
GSH digestive gland contents were statistically greater in M. galloprovincialis from
Chioggia than in the same species from
Marghera (p:::: 0.001). In proportion. some metals such as Mn, Co and Fe were also greater in M.
galloprovincialis from Chioggia than in spedmens from Marghera (Table). GSH digestive
gland contents of S. inequivalvis were also
greater in the specimens from Chioggia (P =
0.006).
Comparing GSH contents in the digestive
gland of the three species in the same two stations, T. philippinarum had significant lower
amounts of GSH than M. galloprovincialis and S.
inequivalvis (Fig. 2a). This situation may be due
to the very high induction of MT in Tapes.
Instead, considering gills. the only statistically significant differences were: (1) M. galloprovincialis from Chioggia had a significantly greater
amount of GSH than M. galloprovincialis from
Marghera (P = 0.009); (2) at Marghera, S. inequivalvis had a significantly greater amount of GSH
than M. galloprovincialis (Fig. 2b). The latter
datum is in keeping with the metal contents: the
gills of S. inequivalvis had more metal concentrations (except Cu) than the other two species.
P. Irato et al.
alimentary tract, where they are desorbed from
the particles and absorbed by the animal during
digestion (Burbidge et al.1994).
Materials and Methods
In May 1998, specimens of M. galloprovincialis. T.
philippinarum and S. inequivalvis were sampled
in two stations with different pollution levels at
Chioggia and Marghera in the Lagoon of Venice.
The shell length of the animals was measured.
Soft tissues were separated from the shell, and
digestive glands and gills were removed. weighed
and frozen at -80°C. Subsequently, glutathione
determination and MT assays were performed
on the samples, dividing the organs into two
parts. For total glutathione determination, the
samples were homogenized (1:5 w/v ratio) in 5%
sulphosalicilic acid and centrifuged at 15,000
rpm for 30 min at 4°C. Total GSH contents were
measured in the resulting supernatant by
Anderson's (1985) method and referred to wet
weights. For MT assays, the samples were
homogenized (1:4 w/v ratio) in 20 mM Tris HCl
pH 7.5, added with 0.006 mM leupeptine, 0.5 mM
PMSF (phenylmethylsulphonylfluoride) as
antiproteolitic agents, and 0.01% p-mercapto ethanol as reducing agent, and centrifuged at
30,000 g for 60 min at 4°C. The MT contents were
measured in the resulting supernatant by the silver saturation method (Scheuhammer and
Cherian 1991) and the values referred to total
protein determined by the method of Lowry et
al. (1951). The digestive glands and gills of other
individual animals were homogenized with the
same buffer as the MT assays and digested with
HN0 3 AristaR in PFA vessels in a CEM MDS-2000
microwave furnace. Metal concentrations (Cr,
Mn, Fe, Co, Ni, Cu, Zn, Cd) were determined with
an inductively coupled plasma-atomic emission
spectrometry (ICP-AES) Spectroflame Modula
sequential and referred to dry weights (identical
amounts of homogenate used for digestion were
dried in an oven at 130°C).
Values are shown as means ± standard error.
Statistical analysis was performed with the
PRIMER statistical program: Student's t-test for
comparison of pairs or ANOVA for different
groups followed by the Student-Newman-Keuls
test for determination of significant differences
(P = O.OS). Linear regression analyses were also
performed.
Results and Discussion
Sediment metal analyses (conducted by Casellato
S. and Degetto S. in 1999; unpublished data)
highlighted the important fact that Marghera has
three times more Cr than Chioggia and that Cd is
only found in Marghera. Results show that MT
contents, in both digestive glands and gills of all
three species, were not significantly different
between the two stations with different levels of
pollution. Instead, digestive gland MT contents
were significantly different in the same station
among the three species: T. philippinarum had
the greatest MT contents, followed by S. inequivalvis andM. galloprovincialis (Fig. la). The greatest amount of MT in the digestive gland of T.
philippinarum is probably due to a higher concentration of eu (Table). Moreover, it is known
that Cu has higher affmity for MT with respect to
Zn, whereas Zn is a good inductor. We therefore
hypothesize that. in T. philippinarum. Cu is
linked to MT induced by Zn, so that Zn can then
induce other MT. T. philippinarum from
Marghera was the species with the greatest
amounts of Mn, Co and Fe (Table). Gill MT contents were not significantly different in the same
station among the three species (Fig. Ib).
GSH digestive gland contents were statistically greater in M. galloprovincialis from
Chioggia than in the same species from
Marghera (p:::: 0.001). In proportion. some metals such as Mn, Co and Fe were also greater in M.
galloprovincialis from Chioggia than in spedmens from Marghera (Table). GSH digestive
gland contents of S. inequivalvis were also
greater in the specimens from Chioggia (P =
0.006).
Comparing GSH contents in the digestive
gland of the three species in the same two stations, T. philippinarum had significant lower
amounts of GSH than M. galloprovincialis and S.
inequivalvis (Fig. 2a). This situation may be due
to the very high induction of MT in Tapes.
Instead, considering gills. the only statistically significant differences were: (1) M. galloprovincialis from Chioggia had a significantly greater
amount of GSH than M. galloprovincialis from
Marghera (P = 0.009); (2) at Marghera, S. inequivalvis had a significantly greater amount of GSH
than M. galloprovincialis (Fig. 2b). The latter
datum is in keeping with the metal contents: the
gills of S. inequivalvis had more metal concentrations (except Cu) than the other two species.