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]. Highlights in BioScience Page 1 of 8 January 2021|Volume 4 https://doi.org/10.36462/H.BioSci.202101 https://creativecommons.org/licenses/by/4.0/ http://highlightsin.org/index.php/bioscience 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 Highlights in BioScience Page 2 of 8 January 2021|Volume 4 http://highlightsin.org/index.php/bioscience 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 Highlights in BioScience Page 3 of 8 January 2021|Volume 4 http://highlightsin.org/index.php/bioscience 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, Highlights in BioScience Page 4 of 8 January 2021|Volume 4 http://highlightsin.org/index.php/bioscience 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 Highlights in BioScience Page 5 of 8 January 2021|Volume 4 http://highlightsin.org/index.php/bioscience 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 Highlights in BioScience Page 6 of 8 January 2021|Volume 4 http://highlightsin.org/index.php/bioscience 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. References 1. Di Salvatore P, Calcagno JA, Ortiz N et al. Effect of season- ality on oxidative stress responses and metal accumulation in soft tissues of Aulacomya atra, a mussel from the South Atlantic Patagonian coast. Mar. Environ. Res.2013; 92:244-252. 2. Velez C, Figueira E, Soares AMVM et al. Accumulation and cellular partitioning of metals and As in the clam venerupis cor- rugata: Different strategies towards different elements. Chemo- sphere. 2016; 156:128-134. 3. Company R, Serafim A et al. Source and impact of lead contam- ination on d- aminolevulinic acid dehydratase activity in several marine bivalve species along the Gulf of Cadiz. Aqua. Toxicol. 2011; 10:146-154. 4. 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Highlights in BioScience Page 8 of 8 January 2021|Volume 4 http://highlightsin.org/index.php/bioscience 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