































Highlights in BioScience
ISSN:2682-4043
DOI:10.36462/H.BioSci.202101

Research Article

Open Access

1 Faculty of Sciences of Tunis, Biology Depart-

ment, Research Unit of Physiology and Aquatic

Environment, University of Tunis El Manar,

2092 Tunis, Tunisia.
2 Aquatic Environment Exploitation Resources

Unit, Higher institute fishing and fish farming

of Bizerte, Tunisia.

Contacts of authors

* To whom correspondence should be
addressed: Imene Chetoui

Received: September 24, 2020

Accepted: January 12, 2021

Published: January 20, 2021

Citation: Chetoui I, Ghribi F, Bejaoui S, Ghal-
ghaa M, El Cafsi M, Soudani N . Assessment of
stress biomarkers responses in mantle and adduc-
tor muscles of Mactra stultorum following lead
exposure. 2021 Jan 20;4:bs202101

Copyright: © 2021 Chetoui et al.. This is an
open access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.

Data Availability Statement: All relevant data are
within the paper and supplementary materials.

Funding: The authors have no support or
funding to report.

Competing interests: The authors declare
that they have no competing interests.

Assessment of stress biomarkers responses in mantle and adductor
muscles of Mactra stultorum following lead exposure

Imene Chetoui*1, Feriel Ghribi1, Safa Bejaoui1, Mohamed Ghalghaa2, M'hamed El Cafsi
1, Nejla Soudani1

Abstract
The objective of the present work is to evaluate the possible toxic effect engendered

by graded doses of lead chloride (PbCl2) on Mactra stultorum mantle and adductor mus-
cles through a battery of biomarkers responses. M. stultorum were divided into 4 groups
and exposed to three concentrations of PbCl2 (D1:1mg/L, D2: 2.5 mg/L and D3: 5 mg/L)
with control during five days. Our findings showed decreases of lipid contents in both
organs following PbCl2 exposure, while, proteins declined only in the adductor muscles
of the treated M. stultorum. During our experiment, the PbCl2 exposure induced the levels
of metallothionein (MTs), malondialdehyde (MDA) and advanced oxidation protein prod-
ucts (AOPP) in both organs as compared to the control. These biomarkers responses are
distinctly different between mantle and adductor muscles.

Keywords: Lead chloride, Mactra stultorum, Mantle, Adductor muscles, Biomarkers responses.

Introduction
The contamination of aquatic ecosystems by several environmental pollutants has become a

worldwide problem in the last years [1]. The presence of heavy metals in those environments and

their accumulation in marine organisms has been largely investigated during the last decades because

of their harmful effects and persistence [2]. For the global environmental health, lead (Pb) is consid-

ered to be a major hazard. This non-essential and toxic heavy metal is the most abundant metal in the

aquatic system. In nature, it is present as a divalent cation and principally forming stable complexes

with sulfur. It has a natural origin or it is realized from many industrials discharges such as lead ore

mining and smelting, refining, alkyl-lead petroleum combustion, batteries and cement manufacture

[3]. At the national level, lead is one of metals contaminating the Tunisian coasts because of its

highest concentrations and has been considered a major source of pollution in Tunisian waters [4,5].

Thus, high Pb levels which are exceeding the permissible limit (1 mg/kg), [6] have been recorded in

Tunisian bivalves tissues (values comprised between 5 and 9 mg/kg DW) [5,7]. Moreover, their accu-

mulation in aquatic ecosystems can become dangerous to all kinds of organisms including bivalves,

fishes, aquatic plants and human life, causing many toxic effects [8].

Mactra stultorum is considered as ecologically important components of marine environments

and an important edible marine bivalve due to their richness of protein and essentials fatty acids [9,

10]. It has a wide distribution along the Mediterranean and Atlantic coasts and estuaries [11]. So, it

is abundant specie in the subtidal area and shallow seas along the coast of Tunisia [12]. Moreover,

due to their benthic and sedentary mode of life; suspension-feeding mode and high filtration rate, it

is easily exposed to environmental pollution [13]. M. stultorum can accrued a enormous number and

high concentration of heavy metals in their tissues [14,15,16]. Therefore, similar to other species

of Mactra, Mussels and Oysters, M stultorum can be used as a good bioindicator of heavy metal

pollution in marine environments [17].

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Chetoui et al., 2021 Assessment of stress biomarkers responses in Mactra stultorum following lead exposure

The excessive accumulation of lead in the bivalve tissues
can induce oxidative stress through the overproduction of re-
active oxygen species (ROS) in the cells which affect cellular
functions [18,19]. So, when these increases of ROS levels get
over the scavenging capacity of organisms, the superfluous free
radicals may advantage to oxidative damage in basic biologi-
cal molecules, such as lipid peroxidation, protein oxidation and
DNA damage [18,19,20].

Thus, the lipid peroxidation constitutes an involvement of
self-propagating sequence of chemical reactions that occurred
in the bulk phase of cell membrane lipid bilayers. Malondialde-
hyde (MDA) and 4-hydroxyalkenals are the degradation prod-
ucts of lipid peroxidation and their levels reflect the degree of
oxidative damage and constitute a biomarker specific to environ-
mental stresses [21].

Moreover, lipid peroxidation and other damages resulting
from metal toxicity are modulated by antioxidant systems and
stress proteins such as metallothioneins (MT) [22]. These sys-
tems of defense play a key role in the alive organisms which
can provide for the different cells the protection against environ-
mental toxicity control metabolism homeostasis [22]. Metalloth-
ionein (MT) is a low-molecular-weight and cysteine-rich protein,
identified for the first time in the kidney of the horse [23]. MT’s
plays a crucial role in metal metabolism and principally in the
detoxification mechanisms as a metal-chelating agent for the ex-
cess of metals in the cells [24]. Further, it ensure an essential role
in immune response [25], antioxidant processes [26,27], and re-
sponse to estrogenic compounds [28]. In aquatic environments,
MT has been implied to be used as a bioindicator for metal con-
tamination because of its possibility to bind to particular metals.

Even though there are investigations about the impact of
PbCl2 on M. stultorum gills and digestive gland [15,16], still
information are lacking about the impact of this metal on the
metabolism and redox status of other organs. Thus, in this study,
we focused on identifying the metabolic and redox strategies de-
veloped by M. stultorum adductor muscles and mantle to cope
with graded PbCl2 concentrations.

Materials and Methods
Experimental protocol

Mature clams individuals (Shell length (SL): 3.5 ± 0.63 cm
and Total weight (TW) 8.03±0.47 g) were collected at 1 m depth
by scuba divers from the Bizerte lagoon. After sampling, clams
were acclimated for 7 days in aquaria (20 L). The water was
daily renewed and physicochemical parameters were controlled
(temperature (18°C), salinity (30 psu), pH (7.4 ± 0.2), and pho-
toperiod (12h/12h)). During the acclimation period, green mi-
croalgae Isochrysis affinis galbana (t-ISO, 2 million cells per
ml) was fed regularly to M. stultorum. At the end of the acclima-
tion period, clams were divided in 4 groups of 18 clams. Each
group was placed in 8 L plastic aquaria and was represented
in triplicate (n=6 clams per replicate). Clams first group was
kept in aquaria1 containing filtered natural seawater (control),

while other groups were exposed for 5 days to different concen-
trations of unmixed PbCl2 metal (Lead chloride; PbCl2; Sigma-
Aldrich; powder 98%) which was dissolved in pure water. Dur-
ing metal exposure, clams were exposed to graded concentra-
tions of PbCl2 as follows: aquaria1: control; aquaria2: 1mg/L;
aquaria3: 2.5mg/L and aquaria4: 5mg/L with controlled condi-
tions as mentioned above (Figure 1) and no added food. Half
(50%) aquaria water volume was replaced every 24 h in order
to maintain the water quality, and concentrations of PbCl2 were
reestablished. PbCl2 concentrations were selected based on pre-
vious trials achieved on other bivalves [29,30]. During the ex-
perimental period, no mortality has been reported.

Preparation of the samples for biochemical analyses
After PbCl2 exposure, clams were sacrificed and the man-

tle and adductor muscle were quickly removed and rinsed with
cold distilled water in order to remove the externally bound Pb.
6 replicate of each group tissues (mantle and adductor muscle;
n=3 for each replicate) were homogenized in a Tris-HCl buffer
(20mM; pH=7.4) in cold conditions, then centrifuged at 10.000
× g for 20 min (4°C). Tissues supernatants were stored at -80°C
for oxidative stress parameters analysis.

Biochemical analyses
For biochemical analysis, chemicals were purchased from lo-

cal commercial suppliers. Except for 5,5-dithio-bis-(2- nitroben-
zoic acid) (DTNB) and 2-thiobarbituric acid) (TBA) they were
purchased from Sigma chemical Co (Saint Louis, MO 63103,
USA).

Protein quantification
Based on Lowry et al. [31] method, protein content was es-

timated using Folin Reagent and bovine serum albumin (BSA)
as a standard. 2 ml of a solution mixture (sodium carbonate hy-
drate dissolved in a solution of NaOH (0.1N), copper sulfate and
sodium hydrate dissolved in water) were added to 10 µl super-
natants. Then, 200 µl of Folin Reagen was added to the formed
mixture for reaction activation. After 30 min incubation, protein
content was determined at 540nm using the spectrophotometric
method.

Lipid quantification
According to the method of Goldsworthy et al. [32], lipids

were determined and the extraction was carried out following
Shibko et al. [33] method. 0.5 g of tissues are removed, cut and
macerated in 10 ml of trichloroacetic acid (TCA, 20%). After
grinding and filtration and a first centrifugation 5000× g for 10
min, the pellet is kept in the same tube which we added 1 ml
of the mixture Ether / Chloroform (1 v/ 1v). Subsequently, the
last volume is submitted to second centrifugation 5000× g for
10 min and 100 μl of the supernatant is taken which we added 1
ml of sulfuric acid. And after stirring, the tubes were placed in
a hot water bath (100 °C) for 10 min. Then, we added 2.5 ml of
the sulfophospho-vanillin mixture (85%) to 200 μl of the extract

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Chetoui et al., 2021 Assessment of stress biomarkers responses in Mactra stultorum following lead exposure

Figure 1. Experimental conception of Mactra stultorum exposed to graded doses of lead chloride (PbCl2).

which was incubated for 30 minutes in a darkroom. Lipid quan-
tities are determined by the spectrophotometric method at 530
nm. A calibration range was carried out from a stock solution
prepared from sunflower oil. The lipid contents are expressed in
mg/g wet weight (mg/gWW)

Malondialdehyde (MDA) measurement
MDA level was determined according to Draper and Hadley

[34]. An aliquot of 0.5ml of each tissue supernatant was incu-
bated for 1 hour in heated water (37°C) and mixed with 0.5 ml
of trichloroacetic acid (TCA 30%). After centrifugation for 10
min (3500× g/4 °C), we added 500µl of thiobarbituric acid (TBA
0.67%) to 0.5 ml of supernatant. After incubation for 10min,
MDA levels were determined by spectrophotometric method at
532 nm and expressed as nmol /mg protein.

Advanced oxidation protein products level (AOPP) measurement
The advanced oxidation protein products (AOPP) levels were

determined following the method of Kayali et al. [35]. After pro-
tein precipitation in double volumes of phosphate buffer (0.1M;

pH = 7.4). Then, potassium iodide (1.16M) and absolute acetic
acid (200µl) were added to clams supernatants. We used the ex-
tinction coefficient of 261 cm-1 mM-1 for AOPP quantification.
AOPP levels were determined at 340 nm and expressed as μmol/
mg of protein.

Metallothionein (MTs) content
According to the method of Viarengo et al. [36] modified

by Petrovic et al. (2001), MTs were determined. The super-
natant of each tissue from each group (500µl) was mixed with
ethanol/chloroform solution (95%; 1%). After centrifugation for
6000 x g during 10 min in cold, EDTA (1mM) and NaCl (0.25M)
were added to the pellets and MTs absorbance was measured at
412nm. MTs were expressed as nmol GSH/mg protein.

Statistical analysis
Statistica software version 5.0 was used for statistical anal-

ysis. The homogeneity and normality of variables were tested
using the Shapiro-Wilcoxon test. Differences between variables

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Chetoui et al., 2021 Assessment of stress biomarkers responses in Mactra stultorum following lead exposure

were assessed by One-way ANOVA and deemed significant at p
< 0.05. For each parameter, results were expressed as means ±
standard deviation (SD). Pearson correlation matrix and princi-
pal component analysis (PCA) were used to discriminate signif-
icant correlations between biochemical parameters.

Results
The PbCl2 effects on general behavior of M. stultorum

No mortality was noticed in treated clams by PbCl2 differ-
ent doses. During the experimental period, clams behavior was
regularly monitored (e.g. filtration activity, reduced respiration,
siphon retraction. . . ). Those parameters remained stable even in
clams exposed to the high dose of PbCl2 (D3).

Estimation of total protein contents
Results showed no significant variation in protein content of

clam's mantle tissues after PbCl2 treatment. However, signifi-
cant decreases in the amount of total protein were revealed in
the adductor muscles of M. stultorum following lead exposure
(-23%, -24% and -20% in clams exposed to doses D1; D2 and
D3 respectively compared to controls (Table 1).

Estimation of lipid contents
Lipids contents decreased significantly in clams mantle and

adductor muscle tissues after 120 hours of exposition to PbCl2.
Compared to the control, this decline was recorded for the man-
tle by -37%, -60% and -67% in clams exposed to 1mg/L; 2.5mg/L
and 5mg/L respectively) and for the adductor muscles by -32 %
and -60% in clams treated by D2 and D3 respectively (Table 1).

Estimation of malondialdehyde (MDA) levels
Results showed that the progressive accumulation of Pb in

both M. stultorum tissues induced lipid peroxidation in all treated
clams by PbCl2 which was revealed by MDA levels enhance-
ment. In the mantle tissues and compared to the control, MDA
levels increased with graded doses (D1, D2 and D3, respectively)
by 57%, 150% and 575%. We also noted that MDA levels in the
adductor muscles increased significantly by 58% and 74%, in
clams treated with the highest doses (D1 and D2) (Figure 2).

Estimation of advanced protein oxidation products (AOPP) levels
Significant increases in AOPP levels were recorded in the

mantle of all PbCl2 treated groups (1, 2.5 and 5 mg/L PbCl2)
with a dose dependent manner (+60, +65and +205% respec-
tively). While in the adductor muscles tissue and compared
to control, the increase of AOPP levels was observed only for
clams exposed to 2.5mg /L; (+102%) and 5mg / L; (+132%)
(Figure 3).

Estimation of metallothionein (MT’s) levels
MT’s levels increased significantly only in clams mantle tis-

sues exposed to the highest dose of PbCl2 (5mg / L; 163%) dur-
ing 5 days. Furthermore, the MT’s levels increased significantly
in the adductor muscles with a dose dependent manner (+30,
+45 and +268%) with D1, D2 and D3 PbCl2 doses compared to
control (Figure 4).

Figure 2. The MDA levels in the control and treated M. stultorum mantle
and adductor muscles with Pb Cl2 graded doses (D1, D2, D3) during 5 days.
Values are expressed as means ± SD, 6 replicate in each group and tissues (n=3
clams). Pb Cl2 graded doses: D1 (1mg/L Pb Cl2); D2 (2.5mg/L Pb Cl2); D3
(5mg/L Pb Cl2). *** P <0.001: Pb Cl2 groups VS controls for each tissue. ++
<0.01; +++ P <0.001: mantle VS adductor muscles for each condition.

Figure 3. The AOPP levels in the control and treated M. stultorum mantle
and adductor muscles with Pb Cl2 graded doses (D1, D2, D3) during 5 days.
Values are expressed as means ± SD, 6 replicate in each group and tissues (n=3
clams). Pb Cl2 graded doses: D1 (1mg/L Pb Cl2); D2 (2.5mg/L Pb Cl2); D3
(5mg/L Pb Cl2). ** P <0.01;*** P <0.001: Pb Cl2 groups VS controls for
each tissue. ++P <0.01; +++ P <0.001: mantle VS adductor muscles for each
condition.

Principal component analysis (PCA) and correlation matrix
Correlation matrix and PCA were established in order to un-

derstand in the first time the effect of lead graded doses on stress
biomarkers in mantle and adductor muscles tissues of Mactra
stultorum and in second time to compare the response of these
biomarkers between these tissues (Figure 5 and Table 2). The
first two factorial axes that explain 90.96% of the total variance
(Figure 5). Factor 1 (56.38%) was characterized by high MDA
and MT’s levels (Figure 5). Factor 2 (33.37 %) was charac-
terized by AOPP levels. Results showed that protein and lipid
contents are intermediates compounds for F1 and F2. PCA re-
sults showed that there were two significant separations, the first
one between controls of mantle and adductor muscles and the
treated groups and the second one between both tissues from all
treated groups (Figure 5). Control mantle and adductor mus-
cles were projected in the positive sides of two factorials axes,

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Chetoui et al., 2021 Assessment of stress biomarkers responses in Mactra stultorum following lead exposure

Table 1. Protein and Lipid contents in the control and treated M. stultorun mantle and adductor muscle with PbCl2 graded Doses (D1. D2. D3) during 5 days.
Values are expressed as means ± SD, 6 replicate in each group and each tissues (n=3 clams.). a: nmol/mg protein. b: mg/mg protein. Pb Cl2 graded doses: D1
(1mg/L Pb Cl2); D2 (2.5mg/L Pb Cl2); D3 (5mg/L Pb Cl2). ** P <0.01;*** P <0.001: Pb Cl2 groups VS controls for each tissue. +++P <0.01; +++ P <0.001:
mantle VS adductor muscles for each condition.

CT D1 D2 D3

Proteina Mantle 10.4± 0.67+++ 8.37± 0.76+++ 9.84± 0.64+++ 7.84± 0.69+++

Adductor muscle 43.4± 4.91 33.4± 2.02* 32.8± 1.94* 34.7± 5.14*

Mantle 4.2± 0.92++ 2.62± 0.71*
+++ 1.67± 50.27*

+++ 1.42± 0.32*
++

Lipidb Adductor muscle 5.92± 0.35 5.89± 0.26 4.02± 0.28* 2.37± 0.83*

Figure 4. The MT’s levels in the control and treated M. stultorum mantle
and adductor muscles with Pb Cl2 graded doses (D1, D2, D3) during 5 days.
Values are expressed as means ± SD, 6 replicate in each group and tissues (n=3
clams). Pb Cl2 graded doses: D1 (1mg/L Pb Cl2); D2 (2.5mg/L Pb Cl2); D3
(5mg/L Pb Cl2). ** P <0.01;*** P <0.001: Pb Cl2 groups VS controls for each
tissue. +++ P <0.001: mantle VS adductor muscles for each condition.

explaining by the high contents of lipids and protein and minor
levels of lipid peroxidation and MT’s. Second group was dom-
inated by treated mantle clams by PbCl2 which represented the
negative side of F1 and the positive side of F2; revealing im-
portant lipid and protein oxidation. The third one including the
adductor muscles from M. stultorum from all treated groups was
characterized by a minor response of stress biomarkers compar-
ing to mantle tissues and by a remarkable decrease in protein and
lipid contents especially for clams exposed to high dose. Clearly,
biomarkers responses involved in oxidative stress were signifi-
cantly enhanced in both tissues clams treated with high PbCl2
dose when compared to control groups.

Table 2. Correlation analysis (Pearson correlation) between the biochemical
parameters in the control and treated M. stultorun mantle and adductor muscle
with Pb Cl2 graded doses (D11.D2. D33 during 5 days. Correlation coefficients
statistically significant (p <0.05) . ns: not significant (p <0.05).

Mantle Adductor muscles

Protein Lipid MDA AOPP Protein Lipid MDA AOPP

Lipid ns ns

MDA ns -0.98 0.98 ns

AOPP ns ns 0.98 -0.95 -0.96 ns

MT ns ns 0.99 0.99 0.97 ns 0.99 ns

Figure 5. Principal analysis component (PCA) represented by two factors
F1 and F2 and produced by biochemical variables in control and stultorum
mantle and adductor muscles with Pb Cl2 graded doses (D1, D2, D3) during
5 days. Projection of the variables and the cases on the factor-plane (1×2);
D1:1mg/L PbCl2; D2:2.5mg/L PbCl2; D3:5mg/L PbCl2; MT: mantle; AM:
adductor muscles.

Pearson correlation matrix showed that protein contents in
adductor muscles is significantly negatively correlated (p<0.05)
with AOPP levels. While, lipid contents in mantle showed a neg-
ative correlation with MDA levels (p<0.05). Positive correlation
was recorded between AOPP, MDA and MTs levels for mantle
tissue. While, negative correlation was observed between AOPP
levels and lipid contents for adductor muscles tissue. Thus, MT’s
presents in this tissue positive correlation only with MDA and
protein levels (Table 2).

Discussion
Biological responses in native indicator species can give to-

tal and pertinent information on the potential impact of metal
toxicity on ecosystem dynamics. Heavy metals constitute a group
among environmental pollutants because of their bioaccumula-
tion and non-degradable property. Lead is a non-essential metal,
abundant in the oceans and exhibits widely regarded toxic effects
associated with the stimulation of radical processes [37].

Lead is accumulated by different organisms in aquatic sys-
tems and became dangerous to all kinds of organisms, including

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Chetoui et al., 2021 Assessment of stress biomarkers responses in Mactra stultorum following lead exposure

bivalves, fishes, and aquatic plants and finally is transferred to
human life [3]. Lead in the divalent cation form Pb2+ can be
transported into the intracellular part through the Ca2+ transport
systems thereby might altering the calcium homeostasis [38], af-
fecting different cellular functions and enzymatic activities and
causing DNA damage [39]. To our knowledge, our study is
the first to assess the redox status in the mantle and adductor
muscles of M. stultorum after Pb Cl2 exposure. Furthermore,
among the established mechanisms of lead toxicity is its ability
to induce oxidative stress following an overproduction of ROS.
An imbalance between antioxidants and pro-oxidants is a result
of this toxicity and causing an alteration in redox status, lipids
peroxidation and protein oxidations [39-40]. Malondialdehyde
(MDA) constitutes the final products of lipid peroxidation indi-
cating the degradation of lipids; it is therefore used as a good
biomarker of lipid damage in aquatic organisms [4]. The oxida-
tion of lipids during our experiment was confirmed by significant
increases in MDA levels in the mantle and adductor muscles in
all animals treated groups. The lipid membranes are the primary
targets of oxidative damage [42]. So, the membrane integrity
and fluidity in the bivalve’s cells is altered by the pro-oxidant
effects of PbCl2 which was proved by these raise of MDA levels.
As well, these high MDA levels are accompanied by high and
significant decreases of lipid quantities in both tissues mainly
for the clams exposed to D2 and D3 which also demonstrated
a significant and negative correlation with the lipid contents in
both tissues.. Moreover, the hypothesis that the inorganic cations
as Pb2+ can stimulate the lipid peroxidation processes through
the oxidation of polyunsatured fatty acids (PUFA) is generally
suggested [37]. Indeed, these PUFA are extremely sensitive to
oxidation via ROS’s attack due to their high number of double
bonds per fatty acid molecule [43]. In this context, the richness
of mantle and adductor muscles by PUFA recorded by Chetoui
et al. [9]can explain these low levels of lipids content and high
MDA levels which are necessary the results of the toxic effects
of lead [29,44].

Additionally, the generation of ROS is the main consequence
of protein damage [45]. In our experiment, increases of AOPP
levels are recorded in all M. stultorun treated groups for mantle
and in M. stultorun treated with D2 and D3 for adductor mus-
cle suggested that the harmful effects of Pb accumulation are
leading to excessive protein oxidation. This oxidation of pro-
tein in the adductor muscles was associated with a remarkable
and significant decrease in the amount of total protein during the
treatment which probably due to their richness of total proteins
compared to mantle [10]. These declines of protein quantities
in AM are negatively correlated with the protein contents in this
tissue. However, in the mantle, the total protein has not changed
during the treatment. Previous research has shown that the alter-
ation of protein in other tissues of M. corallina and Venus ver-
rcosa are observed following the lead toxicity [16,46]. Similar
decreases of protein content have been demonstrated in oyster
gills and mussel digestive glands after metals exposure [43].

It is widely reported that the induction of MT’s in marine
organisms by metals is the result of their use as metal-pollution
biomarkers [47]. These sulfhydryl groups (-SH) are involved in
the detoxification processes [26]. And by capturing free radi-
cals, these metalloproteins were able to acquire protective activ-
ity [48]. Our data showed that treatment with high Pb concentra-
tions (5mg/L) leads to an increase of MT’s content in the mantle
tissue which is highly and positively correlated MDA and AOPP
levels. However, we revealed that total Metallothionein (MTs)
contents in adductor muscle enhanced in a dose-dependent man-
ner which demonstrated a high and positive correlation with pro-
teins and MDA levels. These MT‘s inductions reflected the im-
pairment of both tissues functions in M. stultorum. Moreover, by
comparing to the adductor muscles, this protein (MT's) is more
active (or more expressed) in the mantle for each treated group.
According to Kumari [49], the increase in MT concentrations
seems to be a result of the increased transcriptions. And this sug-
gestion has been demonstrated in M. veneriformis following mer-
cury exposure which the basal MvMT mRNA expression was ac-
cording to the ranking of the tissues like the following: digestive
gland>mantle> gill>adductor muscle>foot [50]. Besides, these
high MT’s induction in the mantle than in the adductor muscles
in our study can probably be firstly by the possibility of that
tissue to accumulate more quantity of metals than the adductor
muscle [29,51,52]and secondly by the activation of their main
role in metal detoxification when the lipid peroxidation and pro-
tein oxidation reached the maximum. Our results are therefore
in agreement with previous studies carried out on Mytilus gallo-
provincialis mantle and M. corallina tissues after lead exposure
[15,16,29].

In the literature, the metals accumulated (as lead) in mantle
and adductor muscles tissues seem to be lower than in the di-
gestive gland and gills of Venus veruscosa, Mytilus galloprovin-
cialis, Callista chione, Perna viridis and Modiolus metcalfei [29,
51,52]. Moreover, in our study, the stress biomarker response
is clearly and significantly different between the two organs and
appears to be more accentuated in the mantle of M. stultorum
treated groups. These results can be explained that similar to
gills, the mantle is also located in the mantle cavity which can
directly interact with marine pollution and consequently can ac-
cumulate metals more than adductor muscle [29,51,52].

Conclusions
In conclusion, the present results demonstrate that PbCl2 ex-

posure (1mg / L; 2.5mg / L and 5mg / L) alters similarly the
redox status of M. stultorum mantle and adductor muscles. The
toxic effect of lead induces similarly the lipids peroxidation con-
firmed by the increases of MDA levels which they were asso-
ciated with decreases in lipids contents in both tissues. The al-
teration of proteins expressed by the elevation of AOPP levels
in both tissues confirms the harmful effects of lead. They were
correlated with a decrease in protein content only in adductor
muscles. The capacity of M. stultorum to increase their MT’s

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Chetoui et al., 2021 Assessment of stress biomarkers responses in Mactra stultorum following lead exposure

concentrations in both tissues seems to be an essential cellular
adaptive system defending the animal against the lead-induced
toxicity. These biomarkers responses in mantle and adductor
muscle tissues elucidate the installation of oxidative stress by
their increases in PbCl2 treatment as compared to controls. How-
ever, they are distinctly different between mantle and adductor
muscles. We can then deduce that the toxic effects of lead are
greater in the mantle than in adductor muscles.

Acknowledgments
The Tunis University of Sciences and the research Unit of

Physiology and Aquatic Environment supported this work.

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Highlights in BioScience Page 8 of 8 January 2021|Volume 4

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	Abstract
	Introduction
	Materials and Methods
	Experimental protocol
	Preparation of the samples for biochemical analyses
	Biochemical analyses 
	Statistical analysis

	Results
	The PbCl2 effects on general behavior of M. stultorum 
	Estimation of total protein contents
	Estimation of lipid contents
	Estimation of malondialdehyde (MDA) levels
	Estimation of advanced protein oxidation products (AOPP) levels
	Estimation of metallothionein (MT’s) levels
	Principal component analysis (PCA) and correlation matrix

	Discussion
	Conclusions
	Acknowledgments
	References

