Evaluation of Metallothionein Expression as a Biomarker of Mercury Exposure in Scatophagus argus
The effect of mercury exposure to total Metallothionein
(MT) response and bioaccumulation under control and acute mercury exposure
were investigated in scats (Scatophagus argus). Scats were exposed
to different mercury concentrations (10, 20 and 30 μg Hg L-1)
for 24, 48 and 72 h. Total MT levels were determined by Enzyme-Linked
Immunosorbent Assay (ELISA) method. Mercury contents were determined through
cold Vapour atomic Absorption spectrometry. Induction of MT during exposure
was tissue specific, displaying different response pattern in gill and
liver. Mercury accumulated much stronger in liver than gill and the latter
also showed lower MT level. MT biosynthesis in liver showed a significant
increase after exposure to different mercury concentration during different
times. This increase was significantly correlated with mercury bioaccumulation.
In contrast, presence of different mercury concentration during different
times did not significantly modify total MT except for 72 h exposure at
30 μg L-1 in gills. The results suggest that this form
of MT existing in S. argus was Hg-inducible and could be extended
the as a biomarker of mercury pollution in ecosystems.
Marine ecosystems are contaminated by different pollutants especially
metals due to human activities. Mercury pollution is one of the world,s
most serious environmental problems (Pilon-Smith and Pilon, 2000). Mercury
exposure is the second most common cause of toxic metal poisoning. It
exists in both organic and inorganic forms and is among the few pollutants
that exhibit biomagnification in aquatic food chain (Ribeiro et al.,
1996). Public health concern over mercury exposure due to contamination
of fish with methyl mercury has long been a topic of debate. Existing
situation in the environment has prompted numerous investigations to consider
effect of mercury on the biological functions of marine organisms, particularly
defense mechanism in fish. Different fish species show differences in
metal accumulation and metallothionein biosynthesis as a detoxification
mechanism. Tissue also accumulates metals to a different extend due to
the differences in physiological and biochemical functions.
The distributions of scats in the pacific region are in harbors, natural
embayments, estuaries and lower parts of freshwater streams, frequently
among mangroves. This species often exist in small aggregation.
Metallothionein among a class of proteins with relatively low molecular
weight 6-8 (kDa) characterized by the intrinsic presence of 20 cysteine
group in their structure which confers unique metal binding properties
to the molecules (Dabrio et al., 2002; Chan et al., 2002).
MT occurs mainly in the cytosol and is also present in the nucleus (Decataldo
et al., 2004). Metallothioneins involves in sequestration of toxic
(Cd, Hg) and essential (Cu, Zn) metals have been proposed as sensitive
biomarker in assessing metal exposure and prediction of potential detrimental
effects induced by metal contamination (Ivankovic et al., 2005
). The induction of MT or similar metal binding proteins in fish was demonstrated
either under experimental or environmental conditions (De Boeck et
al., 2003; Hamza-Chaffai et al., 2000).
In the present study, S. argus were exposed to different levels
of mercury for different times. Bioaccumulation of Hg and MT biosynthesis
in tissues (gill and liver) were measured in such experimental series
at the end of each exposure period. The aim of this study was to assess
Hg-binding capacity of two organs (liver and gill) to biosynthesis metallothionein,
to examining the relationship between MT and Hg concentrations for each
tissue and to determining effects of time and dose on MT response in these
MATERIALS AND METHODS
Fish holding condition: Fishes (S. argus) were collected
from west coast of Persain gulf during summer 2007. Male and Female scats
(with a mean weight of 143±7 g and total length 14±1 cm)
were transferred to laboratory of Khoramshahr Marine Science and Technology
University (KMSU, Iran). They were maintained in two 200 L aquaria for
2 weeks before initiation of the exposure study. They were acclimatized
to aerated and standard OECD water (Organization for Economic Cooperation
and Development, 1993) at temperature of 26±1°C under normal
photoperiod of 12-14 h. The water Hardness was 250 mg L-1 as
CaCO3 and pH 7.4±0.02. The medium used was filtered
and the levels of NH4, NO2 and NO3 in
the water recorded to be within 0.1, 0.1 and 20 mg L-1, respectively.
During acclimatization, scats were being fed once a day with Biomar Co.
fish food at a rate of 1% of fish biomass.
Waterborne mercury exposure experiment: Test scats (n = 5 for
each concentration) were exposed to three contamination levels 0, 10,
20 and 30 μg L-1 for 24, 48 and 72 h at 26±1°C
exposure duration by addition of mercury from a mercury stock solution
prepared in deionised water (HgCl2, 20, extra pure, merck).
No mortality were observed during the experiment . Water replacement was
carried out every day and the mercury concentrations in the water were
determined by CVAAS. The ambient hardness was 250 mg L-1 as
CaCO3 and the water pH varied between 7.37 to 7.68. Scats were
not fed during the experiment and were kept starved for 24 h before the
experiment. After each exposure period Scats were anesthetized by dry
extract of clove pink, then liver and gill samples were dissected on ice.
The samples were divided into two parts, weighed and stored at -80°C
for further processing.
Mercury analysis: Total mercury level were determined using cold
vapour analysis technique. After thawing, 1 g of tissue was digested in
20 mL of 3:1 concentrated redistilled HNO3 and concentrated
H2SO4, and then oxidized with 10 mL of saturated
solution of KMnO4. Excess oxidizing agents and mercury ions
were reduced by 10 mL of reducing solution (3% NaBH4 in 1%
NaOH) in a hydride generator apparatus, and thereafter mercury was vaporized
and measured in the atomic absorption spectrophotometer (Unicam 919).
The instrument was pre-calibrated using standard solutions prepared from
commercial Hg chemical of analytical grade. A blank (n = 3) was run in
the same manner as that of samples and mercury was determined using standard
prepared in the same acid matrix which did not show significant metal
MT analysis: After thawing, the gill and liver samples were prepared
by individually homogenizing in homogenization buffer (10 mM cold Tris-HCl
pH 7.0) containing 5 m M2-mercaptoethanol to prevent oxidation with phenylmethanesulfonylfluoride
(PMSF, protease inhibitor) in a 1:2.5-3.0 (w/v) volume using a Teflon
homogenizer at 1000-1200 rpm. The homogenates were centrifuged at 12000
x g for 40 min at 4°C. The supernatant was heated at 80°C for
10 min in order to denature thermolabile proteins, and then centrifuged
again at 12000 x g for 40 min at 4°C.
Ninety six well palates were coated with 100 μL of the different
samples for 12 h at 4°C. The saturation was realized for 2 h at ambient
temperature with 200 μL of a 3% Bovine Serum Albumin (BSA) in 0.01
mol L-1 Phosphate Buffered Serum (PBS) albumin at pH 7.4. After
4 rinses with 0.01% BSA, 0.05% Tween 20 in PBS, 10 μL of polyclonal
antibody (Rabbit Anti-cod metallothionein diluted 1:1000) were added to
each well and incubated for 2 h at 37°C . After four rinses with previous
buffer, 100 μL of HRP (Peroxidase labeled goat anti-Rabbit IgG) diluted
1:3000 in TBS-Tween was added and incubated for 2 h. After 4 washes, 100
μL well-1 of the ABTS peroxidase substrate (Kirkegaard
and Perry Lab, USA) was added followed by incubation at room temperature
for 20-30 min. Colour development was measured at 405 nm with an automatic
micro-titer plate ELISA reader. The liner regression coefficient (Microsoft
Excel 97 SR-1, 1997, Microsoft Corp, Seattle WA.USA) for the logarithm
for the MT standard concentrations was -0.99 and the slope was -0.2.
Statistical analysis: Statistical analysis of data was carried
out using SPSS Statistical Package Programs (version 13). Data were tested
for homogeneity of variance and normal distribution. ANOVA was calculated.
A post host comparison was made using Tukey,s tests. Differences
between means were testes at 5% probability level. Diagrams were drawn
using Microsoft Excel.
RESULTS AND DISCUSSION
Mercury bioaccumulation: Mercury concentration in the liver was
significantly higher than gills. Mercury bioaccumulation appeared to be
strongly correlated with contamination level of water and exposure duration
(p>0.05). However no interaction was found between these two factors.
Mercury bioaccumulation was rapid during 24 h exposure in the liver then
a plateau tendency was seen between 24 to 48 h in 10 μg Hg L-1
level and thereafter gradual and slow in 20 and 30 μg Hg L-1
level at 48 h exposure. Mercury bioaccumulation at 30 μg Hg L-1
24 h exposure was on the other hand lower than exposure in 20 μg
Hg L-1 even after 72 h exposure (Fig. 1a).
||Mercury bioaccumulation at the (a) liver and (b) gill
of S. argus as a function of three levels of contamination
in the water column (Cw) and of the exposure duration (Ed)
||Mean concentration (±SD) of MTs in liver and
gill of scats; unexposed and exposed to Hg
The values followed by the same letter
are not statistically different among the treatment
In contrast, the accumulation rate showed a significant increases in
gills with time close to linearity but bioaccumulation at 24 h exposure
in 20 and 30 μg Hg L-1 levels was lower than 72 h exposure
in 10 and 20 μg Hg L-1 (Fig. 1b).
MT biosynthesis: MT biosynthesis strongly occurred in the tissues
showing significantly higher level in liver than gill (Fig.
2). The results further indicated significant differences between
control and treatments in the liver. A two way ANOVA (contamination level
and exposure duration) performed on MT concentrations was also significant
(p<0.05), indicating the effect of contamination levels of mercury
and effect of exposure duration on MT biosynthesis. The interaction between
contamination and Exposure duration was also significant (p<0.05).
||MT biosynthesis in two organ level (liver and gill)
of S. argus exposed to Hg. *Significant at p<0.05
||Correlation between MT concentration and Hg bioaccumulation
in the (a) liver and (B) gill of S. argus for 72 h to Hg. R2
= 0.46 are significant
The data envisage no significant differences between 24 and 48 h exposure
time at 10 μg Hg L-1 and also the two higher contamination
level for 24 h exposure duration in liver (Table 1).
It is manifested from the data that the effect of exposure duration and
contamination level was significant but no inter-relationship was found
in the gill. On the other hand no significant differences was established
between control and treatment excepting at the higher level and long exposure
duration in the gill (30 μg Hg L-1 72 h). Among treatments
10 μg Hg L-1 for 24 and 48 h and 20 μg Hg L-1
for 24 h differ significantly with 30 μg Hg L-1 for 72
h in gill (Table 1) while no differences was found in
other treatments irrespective of exposure duration.
Correlation between metal accumulation and the corresponding MT level
in S. argus: Increase in MT biosynthesis in the liver of scats
for 72 h exposure to mercury levels were highly correlated (R2
= 0.79, p = 0.001) with the increases mercury concentration (Fig.
3a). Although no significant difference was found in the gills between
control and exposed scat except for 30 μg L-1 after 72
h, high correlation (R2 = 0.83, p = 0.001) between MT and mercury
was found (Fig. 3b).
Short term exposure of scat to different mercury concentrations ranging
from 0 to 30 μg L-1 during different period of exposure
resulted in increase in mercury accumulation in the gill and liver. Mercury
was accumulated significantly in the liver compared to gill. This accumulation
order was also supported by several authors and might be attributed to
the lower metal-binding capacity of the gills as a consequence of the
low gill MT concentrations present (Cattani et al., 1996; De Smet
and Blust, 2001; Lange et al., 2002). Olsvik et al. (2001)
suggested that the cadmium present in the gills of trout is rapidly cleaned
to the circulation system to the liver and kidney where it could be retained
for a longer time.
MT contents of these tissues appeared to be exposure and level of metal
dependent during 24-72 h period. Elsewhere it has been shown that the
MT content appears to have time and dose dependent in vivo and
in vitro studies. Wu and Hwang (2005) also corroborated this findings
in the liver and gill of Tilapia (Oreochromis mosambicus) after
exposure to cadmium during 24-72 h. This result implies that dose-related
response of MT expression only occur with doses of heavy metals that do
not cause detrimental effects to the physiological functioning of the
fish (Wu et al., 2002). Correlation among MT expression, heavy
metal accumulation, and tolerance of fish to heavy metals are complicated,
specially in in vivo systems (Wu et al., 2002) similar changed
appeared in this in vitro study.
The present results clearly indicate tissue -specific differences of
MT induction in response to mercury exposure. Liver can induce MT biosynthesis
much higher than gill. Gill MT level showed only a moderate increase during
the exposure experiment. Indeed no significant mercury related induction
of gill MT could be detected in the scat even at the highest exposure
level and duration reflecting apparently low capacity of the gill for
Hg-induction after a short water-borne mercury. Chaffai et al.
(1997) and Olsvik et al. (2001) have suggested that the gill do
not constitute a good organ for MT quantification perhaps because MT induction
is dependent on the cell type and occur primarily in the chloride cell
(Burkhardt-Holm et al., 1999; Dang et al., 2000). But this
result shows that a clear correlation exists between mercury and MT level
in the gill. It is remarkable that scat which show the best survival rate
under mercury exposure has the fastest and target organ during exposure.
Mercury toxicity in fish shows a typical shock phase with the extensive
damage in the first hours or days of exposure and repair thereafter (McDonald
and Wood, 1993) and a fast protective response is thus a clear advantage.
This data also indicated a good correlation between Hg and MT levels
in the liver of scat. This positive correlation have also been observed
in roach liver exposed to Cd (Bonwick et al., 1991) and in common
carp and gibel carp (De Boeck et al., 2003), as well as gudgeon
exposed to increased Zn concentrations in the field (Bervoets et al.,
2002). Filipovic and Raspor (2003) have interpreted the positive correlations
between metal and metallothionein content in fish tissue as the metal
sequestration by MT and a poor correlation as metal exceeding the binding
capacity of MT or the involvement of non-MT proteins. According to this
interpretation metal binding capacity of liver (Table 1).
Mean concentration (±SD) of MTs in liver and gill of scats is unexposed
and exposed to Hg. The values followed by the same letter are not statistically
different among the treatment is not exceeded at any mercury concentration
and exposure duration measured in this study and provides the mechanism
for the high mercury bioaccumulation capacity of this organ.
The results from this study show that liver is more efficient organ than
gill to induce MT biosynthesis. There is a significant and early increase
of MT biosynthesis in S. argus after exposure to Hg in liver but
in gill only after higher contamination level occurs. Results also show
significant effect of contamination level and exposure duration on MT
concentration in S. argus. The increase was significantly correlated
to Hg bioaccumulation. The advantage in using scat is the rapid response
within 24 h. These parameter could be extend for use of MT as a biomarker
of mercury pollution in marine ecosystems in S. argus.
Thanks are due to Dr. A.N Mohanty (CIFA) Bhubaneswer India for critically
going through the manuscript and suggesting improvement.
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