INTRODUCTIONTop

Over the last few decades, the use and development of the biomonitoring approach in environmental studies has increased considerably. Indeed, this type of biological test provides supplementary information that may not be revealed by chemical methods. Biomonitoring programmes based on measuring hazardous pollutants in marine organisms are interesting tools from a human health viewpoint (Contardo-Jara et al. 2009Contardo-Jara V., Galanti L.N., Amé M.V., et al. 2009. Biotransformation and antioxidant enzymes of Limnoperna fortunei detect site impact in water courses of Córdoba, Argentina. Ecotoxicol. Environ. Saf. 72: 1871- 1880.). Bivalves are among the first sentinel organisms used in programmes for monitoring chemical contamination in marine and freshwater ecosystems. They have a high water-filtering activity, good accumulation ability, low excretion rates and a low metabolic capacity for contaminant degradation (Zorita et al. 2006Zorita L., Ortiz-Zarragoitia M., Solo M., et al. 2006. Biomarkers in mussels from a copper site gradient (Visnes, Norway): an integrated biochemical, histochemical and histological study. Aquat. Toxicol. 78: 109-116., Sheehan and McDonagh 2008Sheehan D., McDonagh B. 2008. Oxidative stress and bivalves: a proteomic approach. Invertebr. Surviv. J. 5: 110-123.). Mactra stultorum (Mollusca: Bivalvia) is sedentary and easy to collect and keep alive in the laboratory (Chetoui et al. 2010Chetoui I., Rabeh I., Telahigue K., et al. 2010. Valorisation de l’apportnutritionnel d’un mollusque bivalve Mactra corallina des côtestunisiennes (Kalaât El Andalous). Bull. Inst. Natn. Scien. Tech. Mer de Salmmbô 37: 83-88., Abdallah 2013Abdallah M.A.M. 2013. Bioaccumulation of Heavy Metals in Mollusca Species and Assessment of Potential Risks to Human Health. Bull. Environ. Contam. Toxicol. 90: 552-557.).

Acrylamide (ACR), a ubiquitous chemical, is an industrial compound with a variety of applications, including water treatment, agricultural processes, textile manufacturing and life science research (Friedman 2003Friedman M. 2003. Chemistry, acrylamide: A review. J. Agric. Food. Chem. 51: 4504-4526., Tepe and Çebi 2017Tepe Y., Çebi A. 2017. Acrylamide in environmental water: a review on sources, exposure, and public health risks. Exposure and Health 11: 3-12.). Due to its substantial hydrophilicity, ACR has a high migration capability and may be easily transferred to the environmental soil and groundwater (Tepe 2015Tepe Y. 2015. Acrylamide in surface and drinking water. acrylamide in food: analysis, content and potential health effects. Giresun Univ., Turkey, pp. 275-293.). ACR was first discovered in numerous fried or baked carbohydrate-rich foods such as French fries, potato chips, bread, biscuits and coffee (Mottram et al. 2002Mottram D.S., Wedzicha B.L., Dodson A.T. 2002. Acrylamide is formed in the Maillard reaction. Nature 419: 448-449.). It is considered one of the most dumped contaminants, reaching high concentrations (≈1000 mg L^–1) in several accident cases (Kusnin et al. 2015Kusnin N., Syed M.A., Ahmad S.A. 2015. Toxicity, pollution and biodegradation of acrylamide - a mini review. J. Biochem. Microbiol. Biotechnol. 3: 6-12., Tepe 2015Tepe Y. 2015. Acrylamide in surface and drinking water. acrylamide in food: analysis, content and potential health effects. Giresun Univ., Turkey, pp. 275-293.). High ACR levels found in the environment are especially encountered in workplaces where ACR is used or in the vicinity of industrial facilities (Kusnin et al. 2015Kusnin N., Syed M.A., Ahmad S.A. 2015. Toxicity, pollution and biodegradation of acrylamide - a mini review. J. Biochem. Microbiol. Biotechnol. 3: 6-12.). The stationary point sources of ARC (about 93%) are located in China, USA, Western Europe and Japan (Touzé et al. 2015Touzé S., Guerin V., Guezennec A.G., et al. 2015. Dissemination of acrylamide monomer from polyacrylamide-based flocculant use—sand and gravel quarry case study. Environ. Sci. Pollut. Res. 22: 6423-6430.).

ACR has been classified as a “Group 2A carcinogen” by the International Agency for Research on Cancer (IARC) (IARC 1994International Agency for Research on Cancer (IARC). 1994. IARC working group on the evaluation of carcinogenic risks to humans: some industrial chemicals. IARC Monogr. Eval. Carcinog. Risks. Hum. 60: 1-560.). It is common knowledge that its low molecular weight and high water solubility enable this compound to easily pass through various biological membranes. A study conducted by Larguinho et al. (2014)Larguinho M., Cordeiro A., Diniz M.S., et al. 2014. Metabolic and histopathological alterations in the marine bivalve Mytilus galloprovincialis induced by chronic exposure to acrylamide. Environ. Res. 135: 55-62. showed that the LC50 (96 h) for ACR in water is estimated to be 411 mg L^–1 in bivalves (Mytilus galloprovincialis). Indeed, the chemical structure of ACR is distinctive as a result of an α,β-unsaturated carbonyl group which constitutes its “Michael-type” reactivity towards thiol, hydroxyl or amino groups, and to a lesser extent the nucleophilic centres in DNA (Adamsa et al. 2010Adamsa A., Hamdania S., Van Lanckera F., et al. 2010. Stability of acrylamide in model systems and its reactivity with selected nucleophiles. Food. Res. Int. 43: 1517-1522.). However, recent reports have revealed that ACR exposure is associated with carcinogenesis, genotoxicity, neurotoxicity and reproductive toxicity (Erkekoglu and Baydar 2014Erkekoglu P., Baydar T. 2014. Acrylamide neurotoxicity. Nutr. Neurosci. 17: 49- 57., Duan et al. 2015Duan X., Wang Q.C., Chen K.L., et al. 2015. Acrylamide toxic effects on mouse oocyte quality and fertility in vivo. Sci. Rep. 5: 11562.).In toxicological studies, ACR has been observed to elicit its toxicity through oxidative stress induction associated with the over-generation of reactive oxygen species (ROS) including the superoxide anion radical (O₂^•–), hydrogen peroxide (H₂O₂) and the hydroxyl radical (OH^•) (Yilmaz et al. 2017Yilmaz B., Yildizbayrak N., Aydin Y., et al. 2017. Evidence of acrylamide- and glycidamide-induced oxidative stress and apoptosis in Leydig and Sertoli cells. Hum. Exp. Toxicol. 36: 1225-1235., Trabelsi et al. 2019Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acids composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208.). ROS are able to damage all cellular biomolecules, including proteins, DNA and lipids (Fasulo et al. 2012Fasulo B., Deuring R., Murawska M., et al. 2012. The Drosophila Mi-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo. PLoS. Genet. 8: e1002878., Kim et al. 2015Kim S.M., Beak J.M., Lim S.M., et al. 2015. Modified Lipoproteins by Acrylamide Showed More Atherogenic Properties and Exposure of Acrylamide Induces Acute Hyperlipidemia and Fatty Liver Changes in Zebrafish. Cardiovasc. Toxicol. 15: 300-308.).

Our knowledge of potential toxicity following ACR exposure in bivalves is largely based on very limited data (Larguinho et al. 2014Larguinho M., Cordeiro A., Diniz M.S., et al. 2014. Metabolic and histopathological alterations in the marine bivalve Mytilus galloprovincialis induced by chronic exposure to acrylamide. Environ. Res. 135: 55-62., Trabelsi et al. 2019Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acids composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208.), suggesting that more extensive experimental studies in this field are required. In an effort to improve the mechanisms of ACR toxicity, the present work was performed to examine the effect of different concentrations of ACR on the function of M. stultorum gills.

MATERIALS AND METHODSTop

Sampling and experimental design

Clams of Mactra stultorum (3.5±0.63cm length and 8.03±0.47 g weight) were collected from Bizerta lagoon situated in northern Tunisia. The clams were transferred directly to the laboratory, where they were acclimated for a week in 20-L aquaria renewed daily with fresh seawater. The acclimation arrangement contained a closed-circuit system with continuous aeration, and some physical-chemical parameters such as salinity (34±1) and photoperiod (12/12h) were maintained. After acclimation, clams were randomly divided into four groups and transferred to 50-L experimental aquaria in duplicate (n=8 clams per replicate). They were exposed for five days under controlled conditions, as mentioned above, to graded acrylamide (ACR, powder 99%) concentrations as follows:

1. Control group
2. Group A: 5 mg L^–1
3. Group B: 10 mg L^–1
4. Group C: 20 mg L^–1.

The doses of ACR were chosen according to other studies involving aquatic organisms (Larguinho et al. 2014Larguinho M., Cordeiro A., Diniz M.S., et al. 2014. Metabolic and histopathological alterations in the marine bivalve Mytilus galloprovincialis induced by chronic exposure to acrylamide. Environ. Res. 135: 55-62., Trabelsi et al. 2019Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acids composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208.). The doses were also selected to ensure observable effects in order to investigate the toxic effects of ACR and its mechanism of action in terms of oxidative stress and metabolic alteration. During the experimental test, the clams were not fed to avoid prandial effects. No mortality was detected after five days. Specimens were collected and dissected, and the gills were removed using sterile scissors. Some portions of the tissue were homogenized in 10% of Tris-HCl buffer (100 mM, pH: 7.4) and then centrifuged at 10000 rpm for 25 min at 4°C. The obtained supernatants were stored at –80°C in aliquots for further biomarker and protein assays.

Chemicals

All reagents used in the present study were of the highest quality available and were obtained from Sigma Aldrich (Germany) or Merck (Germany).

Biochemical analyses in Mactra stultorum gills

Protein quantification

Proteins contents were measured according to the method of Lowry et al. (1951)Lowry O.H., Roseborouch N.I., Farrand A.L., et al. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 263-275. using bovine serum albumin as standard. Results were expressed as mg/g of tissue.

Malondialdehyde (MDA)

Lipid peroxidation was estimated by measuring thiobarbituric acid (TBA)–reactive substances and was expressed in terms of malondialdehyde (MDA) content by a colorimetric method according to Draper and Hadley (1990)Draper H.H., Hadley M. 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 86: 421-431.. An aliquot of 0.2 mL of the tissue extract supernatant was mixed with 1 mL of trichloroacetic acid solution and centrifuged at 2500×g for 10 min. The resulting supernatant was then incubated at 90°C for 15 min with 1 mL of a solution containing 0.67% TBA. The absorbance of TBA–MDA complex was measured spectrophotometrically at 532 nm. The MDA amounts were calculated using 1,1,3,3-tetraethoxypropane as standard and expressed as nmol/mg of protein.

Lipid hydroperoxides

Lipid hydroperoxide (LOOH) levels in gills were estimated using the ferrous oxidation in the xylenol orange assay (FOX assay) described by Jiang et al. (1992)Jiang Z.Y., Hunt J.V., Wolff S.P. 1992. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low-density lipoprotein. Anal. Biochem. 202: 384-389.. The amount of hydroperoxides produced was calculated using the molar extinction coefficient of 4.6×9×104/M/cm and the results were expressed as nmol/mg of protein.

Advanced oxidation protein products

The advanced oxidation protein product (AOPP) levels were determined according to the method of Kayali et al. (2006)Kayali R., Cakatay U., Akcay T., et al. 2006. Effect of alphalipoicacid supplementation on markers of protein oxidation in postmitotictissues of ageing rat. Cell. Biochem. Funct. 24: 79-85.. Briefly, 400 µL of digestive gland supernatant was treated with 800 µL Tris-Hcl (100 mM, pH 7.4). After 2 min, 100 µl of potassium iodide (1.16 M) was treated with the previous solution followed by 0.1 mL of acetic acid. The reaction mixture was immediately recorded at 340 nm. The concentration of AOPP was calculated using the extinction coefficient (261/cm/mM) and results were expressed as µmol/mg of protein.

Non-enzymatic antioxidants

Reduced glutathione (GSH): GSH level in M. stultorum digestives glands was determined according to Ellman’s (1959)Ellman G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82: 70-77. method as modified by Jollow et al. (1974)Jollow D.J., Mitchell J.R., Zampaglione N., et al. 1974. Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 11: 151-169.. The method is based on the reductive cleavage of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) by a sulfhydryl (-SH) group to yield a yellow colour. Briefly, a mixture of tissue homogenates in phosphate buffer and sulfosalicylic acid (4%) was centrifuged at 1600×g for 15 min. A volume of the obtained supernatant was taken and added to Ellman’s reagent. Reduced GSHwas measured as the difference in the absorbance values of samples in the presence and the absence of DTNB at 412 nm. GSH value was expressed as μg/mg of protein.

Ascorbic acid (Vit C): Ascorbic acid levels were determined according to Jacques-Silva et al. (2001)Jacques-Silva M.C., Nogueira C.W., Broch L.C. 2001. Diphenyl diselenide and ascorbic acid changes deposition of selenium and ascorbic acid in liver and brain of mice. Pharmacol. Toxicol. 88: 119-125. in clams’ digestive glands. Supernatants were precipitated in a cold trichloroacetic acid solution (5%). After incubation at 85°C for 30 min, 1 mL of sulfuric acid (H₂SO₄) solution (65%) was added to the previous mixture. The reaction product was determined using colour reagent containing dinitrophenyl hydrazine and copper sulphate and measured at 540 nm. Results were expressed as nmol/mg of protein.

Metallothioneins (MTs): MT content was determined according to the method of Viarengo et al. (1997)Viarengo A., Ponzano E., Dondero F., et al. 1997. A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antartic molluscs. Mar. Environ. Res. 44: 69-84. modified by Petrovic et al. (2001)Petrovic S., Ozretic B., Krajnovic-Ozretic M., et al. 2001. Lysosomal membrane stability and metallothioneins in digestive gland of mussels (Mytilus galloprovincialis Lam.) as biomarkers in afield study. Mar. Pollut. Bull. 42: 1373-1378.. An aliquot of the supernatant was mixed with cold absolute ethanol and chloroform and centrifuged at 6000 g for 10 min. The collected supernatant was combined with three volumes of cold ethanol, maintained at −20°C for 1 h and centrifuged at 6000 g for 10 min. The obtained MT-containing pellets were then rinsed with 87% ethanol and1% chloroform and centrifuged at 6000 g for 10 min. MT concentration was quantified by evaluating the SH residue content by a spectrophotometric method, using Ellman’s reagent (DTNB). The absorbance was evaluated at 412 nm. Concentrations were expressed as μmol GSH/mg of protein.

Enzymatic antioxidant activities

Glutathione peroxidase (GPx): GPx was assayedby Flohe and Gunzler’s (1984)Flohe L., Gunzler W.A. 1984. Assays of gluthathione peroxidase. Methods. Enzymol. 105: 114-121. method. GPx oxidizes reduced glutathione in the presence of peroxides. A reaction mixture containing 0.3 mL of phosphate buffer (0.1 M, pH 7.4), 0.2 mL of 2 mM glutathione (GSH), 0.1 mL of sodium acid (10 mM), 0.1 mL of H₂O₂ (1 mM) and 0.3 mL of tissue homogenates was used. After 15 min incubation at 37°C, 0.5 mL 5% trichloroacetic acidwas added to the mixture. The tubes were centrifuged at 1500 g for 10 min and the collected supernatant was assayed for glutathione content using DTNB reagent (10 mM). The absorbance was recorded at 420 nm. Resultswere expressed as nmol of GSH/min/mg of protein.

Superoxide dismutase (SOD): SOD activity was estimated by the riboflavin/nitro blue tetrazolium (NBT) photochemistry analysis method, as described by Beauchamp and Fridovich (1971)Beauchamp C., Fridovich I. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44 : 276-277.. The reaction mixture consisted of tissue homogenates in potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mML-methionine, 2 μM riboflavin and 75 μM NBT. The reduction of NBT by superoxide radicals to blue colour was followed at 560 nm. The SOD activity was expressed as the amount of enzyme required to inhibit the reduction of NBT by 50% and the activity was expressed as U/mg of protein.

Acetylcholinesterase activity

Acetylcholinesterase (AChE) activity was determined in M. stultorum digestive glands according to the method described by Ellman et al. (1961)Ellman G.L., Courtney K.D., Andres V. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88-95., using acetylthiocholine iodide as a substrate. Enzyme activity was recorded over 5 min after addition of the substrate concentration. The enzymatic reaction rate was measured spectrophotometrically at 412 nm against a blank without substrate for each activity. AChE activity was expressed as nmol of substrate/min/mg protein.

Statistical analysis

Multivariate statistical analysis was performed using Statistica software analysis (8.0, USA). The obtained results were assessed for normality and homogeneity via the Shapiro test. A one-way ANOVA (Tukey HSD test) and a non-parametric Kruskal-Wallis test (Student t-test) for analysed parameters were performed in order to determine the significant differences between control and treated groups at p<0.05. The whole data set of the non-enzymatic and enzymatic antioxidants in gills of M. stultorum was statistically treated by principal component analysis (PCA) using R software version 2.15.2 (R Core Team 2017R Development Core Team. 2017. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.). PCA is used to look for directions in space that best represent the correlations between random variables. The aim of PCA is to obtain an approximate representation of the cloud in a low-dimensional subspace “k” by projection on well-chosen axes. The main “k” axes are those that maximize the inertia of the projected cloud, that is, the weighted average of the squares of the distances from the projected points to their centre of gravity.

RESULTSTop

Estimation of MDA, LOOH and AOPP levels in M. stultorum gills

The exposure of groups A, B and C to ACR (5, 10 and 20 mg L^–1, respectively) caused a significant, dose-dependent increase in MDA levels (88%, 150% and 150%, respectively), LOOH levels (134%, 124% and 125%, respectively) and AOPP levels (29%, 74% and 61%, respectively) in gills of M. stultorum in comparison with the control group (Table 1). The AOPP levels showed a positive correlation with MDA (p<0.01) and LOOH (p<0.001) in gills of M. stultorum (Table 2).

Non-enzymatic antioxidant status of M. stultorum gills

Exposure to ACR of groups A, B and C increased the ascorbic acid levels (24%, 44% and 39%, respectively) in M. stultorum gills in comparison with the control group (Table 3). They also significantly increased GSH levels (p<0.001) and MT levels (p<0.001). In addition, MT levels showed a strong positive correlation with GSH content (p<0.001) (Table 2).

Enzymatic antioxidant status in M. stultorum gills

Exposure to ACR of groups B and C led to a significant increase (28% and 21%, respectively) in SOD activity in gills of M. stultorum in comparison with the control group (Table 3). In groups A, B and C it also led to a significant decrease in GPx activity (17%, 120% and 30%, respectively) (Table 3).

AChE activity in M. stultorum gills

Exposure to ACR of groups A, B and C caused a significant decrease in AChE activity (79%, 96% and 94%, respectively) in comparison with the control group (Fig. 1). Levels of AChE in gills were positively correlated with protein content (Table 2).

Principal component analysis

The PCA was applied to our biomarker measurements in order to have a better understanding of their variation in M. stultorum gills exposed to graded doses of ACR after five days of treatment. The explanation of the results is limited to the first two factorial axes, which explain most of the cloud variance of the initial variables. The two principal components (F1, F2) explained 83.4% of the total variance. The first axis, with 73.1% of variance, allowed an almost complete classification of the different experimental groups by separating the controls from those exposed to ACR-treated groups. This axis was negatively correlated with MDA, LOOH, AOPP, SOD, GSH, Vit C and MTs and showed a positive contribution to proteins and AChE. However, the second axis (10.3%) represented the high positive loads with GPx. The projection of individuals (each specimen from each dose) on the same factorial plan 1-2 (Fig. 2) showed that M. stultorum of control and treated groups were clustered into four groups. The group belonging to the controls showed a positive contribution tothe second component (10.3%). This correlation coincided with the high AChE and GPx activities and protein concentration, the low MDA, LOOH, AOPP, GSH, vit C and MT levels, and the low SOD activity. Clams exposed to 5 and 10 mg L^–1 overlap in a group that is explained by the similarity in response to oxidative stress. The group of specimens exposed to 20 mg L^–1 contribute positively tothe axis 1 and negatively to axis 2. MDA, LOOH, AOPP, GSH, vit C, MTs levels and SOD activity increased when those of AChE, GPx and proteins decreased.

DISCUSSIONTop

Acrylamide is considered one of a variety of dumped contaminants that reach high concentrations in marine ecosystems. It is an organic molecule with very high water solubility, which facilitates its rapid absorption by and distribution through aquatic organisms (Andersen et al. 2005Andersen F.A. 2005. Amended final report on the safety assessment of polyacrylamide and acrylamide residues in cosmetics. Int. J. Toxicol. 24: 21-50., Kusnin et al. 2015Kusnin N., Syed M.A., Ahmad S.A. 2015. Toxicity, pollution and biodegradation of acrylamide - a mini review. J. Biochem. Microbiol. Biotechnol. 3: 6-12.). Bivalves’ gill are widely used in monitoring programmes, because that organ perform a multitude of functions in organisms’ body and represent the first point of pollutant access from the surrounding aquatic environment (Della Torre et al. 2015Della Torre C., Balbib T., Grassia G., et al. 2015. Titanium dioxide nanoparticles modulate the toxicological response to cadmium in the gills of Mytilus galloprovincialis. J. Hazard. Mater. 297: 92-100.). Therefore, the present study was designed to examine the impact of ACR on redox status in M. stultorum gills.

Gills are sensitive sensory organs and the first barrier against natural impacts (pathogens and natural toxins) and anthropogenic pollution. This tissue may therefore be the main organ involved in prooxidant and antioxidant processes, resulting in both increased production and accumulation of ROS (Della Torre et al. 2015Della Torre C., Balbib T., Grassia G., et al. 2015. Titanium dioxide nanoparticles modulate the toxicological response to cadmium in the gills of Mytilus galloprovincialis. J. Hazard. Mater. 297: 92-100., Bejaoui et al. 2018Bejaoui S., Telahigue K., Chetoui I., et al. 2018. Integrated Effect of Metal Accumulation, Oxidative Stress Responses and DNA Damage in Venerupisdecussata Gills Collected From Two Coast Tunisian Lagoons. J. Chem. Environ. Biol. Eng. 2: 44-51.). Lipids and protein, the mainfunctional and structural components of cell membranes, are the target of free radical stress (Favier 2003Favier A. 2003. Le Stress oxydant. Intérêt conceptuel et expérimentaldans la compréhension des mécanismes des maladies et potentielthérapeutique. L’actual. Chim. 11: 108-115.). In cell membranes, polyunsaturated fatty acids (PUFAs), fatty acids with two or more double bonds, are the main target of ROS, leading to their peroxidation and to functional and structural disorders (Stanicka et al. 2015Stanicka J., Landry W., Cotter T.G. 2015. Oxidative stress biomarkers and ROS molecular probes. Oxidative Stress: Diagnostics, Prevention. and Therapy. Vol. 2, pp. 353-374.). The result of our present study showed an increase in levels of MDA and LOOH, the products of lipid peroxidation, in the gills of ACR-exposed clams. A conclusive and positive correlation (p<0.001) was found between MDA and LOOH levels in the gill tissues. The results of the current study are corroborated by the findings of Trabelsi et al. (2019)Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acids composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208., who found a rise in the MDA content in M. stultorum’s digestive gland when it was exposed to ACR.

However, increased formation of ROS also leads to enhanced oxidative damage to proteins and polypeptides. Protein oxidation can lead to ROS-induced modifications of amino acid residues/prosthetic groups of enzymes, as well as fragmentation/aggregation of proteins in the cell (Adams 2001Adams S. 2001. Reactive carbonyl formation by oxidative and non-oxidative pathways. Front. Biosci. 6: 17-24.). The most frequently evaluated biomarkers of oxidative protein damage are AOPPs, which are dityrosine-containing cross-linked protein products (Alderman et al. 2002Alderman C.J.J., Shah S., Foreman J.C., et al. 2002. The role of advanced oxidation protein products in regulation of dendritic cell function. Free. Radic. Biol. Med. 32: 377-385.). In this study, the assessed AOPP was significantly higher in the gills of ACR-treated species than in the control group. Hence, it may reflect the prooxidant effect of the ACR active compound, which can easily react with free sulfhydryl groups in proteins (LoPachin and Gavin 2012LoPachin R.M., Gavin T. 2012. Molecular mechanism of acrylamide neurotoxicity: lessons learned from organic chemistry. Environ. Health Perspect. 120: 1650-1657.). Furthermore, a positive correlation (p<0.01) and (p<0.001) was found between the AOPP levels and the lipid peroxydation products (MDA and LOOH, respectively) in the gills of treated groups compared with controls.

The consequences of the generation of free radicals can bring further changes to antioxidant enzyme activities (Schwarz 1996Schwarz K.B. 1996. Oxidative stress during viral infection: a review. Free Radic. Biol. Med. 21: 641-649.). GPx is a glycoprotein comprised of a single seleno-cysteine residue at the active site of each sub-unit. Hence, it participates in the detoxification of LOOHs through its reduction to water and the related alcohols (Jomova and Valko 2011Jomova K., Valko M. 2011. Advances in metal-induced oxidative stress and human disease. Toxicology 283 : 65-87.). In the present study, we found that the activity of GPx decreased in the gills of M. stultorum under ACR exposure, which may imply that GPx was degraded by ROS or over utilized to overcome the excess of lipid hydroperoxides. Moreover, SOD is considered to be the first line of defence against the superoxide anion radical (O₂^•–) by catalysing its conversion to H₂O₂ (Haleng et al. 2007Haleng J., Pincemail J., Defraigne J.O., et al. 2007. Oxidative stress. Rev. Med. Liege 62: 628-638.). Our findings show an increase in SOD activity in the treated animals. The increased SOD activity suggests the protective behaviour of gill cells against O₂^•– radicals generated under a plausible ACR toxicity. Trabelsi et al. (2019)Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acids composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208. reported a disruption of the enzymatic antioxidant status in digestive glands of the ACR-treated M. stultorum. Meanwhile, other indicators of oxidative stress, GSH, Vitamin C and MTs were measured to evaluate the potential oxidative damage induced by ACR exposure. During the test, the gills of ACR-treated organisms had a high content of GSH. It is the abundant non-protein thiol (–SH) that is implicated in cell protection by suppressing toxicants and quenching unstable species such as free radicals (Birben et al. 2012Birben E., Sahiner U.M., Sackesen C., et al. 2012. Oxidative stress and antioxidant defense. World. Allergy. Organ. J. 5: 9-19.). Furthermore, the detoxification pathway of ACR is driven by its conjugation with a GSH molecule (Greim and Snyde 2018Greim H., Snyder R. 2018. Toxicology and Risk Assessment: A Comprehensive Introduction. John Wiley & Sons, Hoboken, USA, 840 pp.). The latter is also directly implicated in recycling ascorbic acid, an important water soluble antioxidant, which protects the cell membrane from oxidative stress (Krishnan et al. 2009Krishnan N., Kodrík D., Kłudkiewicz B., et al. 2009. Glutathione-ascorbic acid redox cycle and thioredoxin reductase activity in the digestive tract of Leptinotarsa decemlineata (Say). Insect. Biochem. Mol. Biol. 39: 180-188.). Under experimental conditions, ascorbic acid levels increased significantly in the gills of ACR-treated M. stultorum. MTs, cysteine-rich proteins with low molecular weight, are believed to protect DNA, lipids and proteins from ROS production and oxidative injury (Cai et al. 1999Cai L., Satoh M., Tohyama C., et al. 1999. Metallothionein in radiation exposure: its induction and protective role. Toxicology 132: 85-98.). In fact, MTs have the potential to directly inactivate the hydroxyl, (OH^•) and superoxide radicals (O₂^•–) (Zhou et al. 2002Zhou Z., Sun X., Kang Y.J. 2002. Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress. Exp. Biol. Med. 227: 214-222.). In our study, the levels of MTs increased in all the treated groups. Indeed, the active compound (α,β-unsaturated carbonyl) in an ACR molecule is likely to be added to the thiol group in MT molecules and forms cysteine-acrylamide adducts (Zamora et al. 2010Zamora R., Delgado R.M., Hidalgo F.J. 2010. Model reactions of acrylamide with selected amino compounds. J. Agric. Food. Chem. 58: 1708-1713.). Recently, high MT levels were found in M. stultorum digestive glands treated with graded concentrations of ACR (Trabelsi et al. 2019Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acids composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208.). This finding is also supported by the Pearson’s correlation results, in which GSH was positively correlated with MT levels (p<0.01). This increase in thiol-rich molecules may be a compensatory adaptive mechanism to neutralize increased levels of ACR molecules binding with the –SH group of GSH and MTs.

Acetylcholine is an important neurotransmitter. Its action is dependent on it being able to metabolize the AChE enzyme, which is essential for the correct transmission of the nerve impulse (Schmatz et al. 2009Schmatz R., Mazzanti C.M., Spanevello R., et al. 2009. Resveratrol prevents memory deficits and the increase in acetylcholinesterase activity in streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 610: 42-48.). In the present study, the AChE activity of M. stultorum gills exposed to graded doses of ACR was significantly inhibited. This enzyme activity can be inhibited by free radicals generated by ACR and its inhibition could disturb the metabolic and nervous activities and induce differential membrane permeability and ionic refluxes. As noted above, the neurotoxic effect of ACR must be related to the reaction of α,β-unsaturated carbonyl with the thiol molecules, either in the presynaptic membranes, with the subsequent reduction of the neurotransmitter releasing AChE, or in the enzyme itself. Recent studies have demonstrated the cholinergic damage of ACR through the inhibition of the AChE activity in the digestive glands of M. stultorum bivalves (Trabelsi et al. 2019Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acids composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208.).

CONCLUSIONTop

Although the ACR concentrations used in our study are unlikely to occur in coastal areas, this study shows that ACR might have a hazardous impact on M. stultorum gills following the installation of oxidative stress. Additionally, our results are particularly important because the observed alterations might induce several types of physiological damage depending on the doses used. We can therefore consider the biomarker responses to be useful tools for evaluating ACR toxicity. Further research using lower concentrations is required to validate the exact signalling pathways targeted by ACR and mediating its toxicity action in cell metabolism.

ACKNOWLEDGEMENTSTop

This study did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. We are also indebted to the editor and the anonymous reviewers for agreeing to review this work.

Conflict of Interest The authors declare that they have no competing interests.

REFERENCESTop

Abdallah M.A.M. 2013. Bioaccumulation of Heavy Metals in Mollusca Species and Assessment of Potential Risks to Human Health. Bull. Environ. Contam. Toxicol. 90: 552-557.
https://doi.org/10.1007/s00128-013-0959-x

Adams S. 2001. Reactive carbonyl formation by oxidative and non-oxidative pathways. Front. Biosci. 6: 17-24.
https://doi.org/10.2741/adams

Adamsa A., Hamdania S., Van Lanckera F., et al. 2010. Stability of acrylamide in model systems and its reactivity with selected nucleophiles. Food. Res. Int. 43: 1517-1522.
https://doi.org/10.1016/j.foodres.2010.04.033

Alderman C.J.J., Shah S., Foreman J.C., et al. 2002. The role of advanced oxidation protein products in regulation of dendritic cell function. Free. Radic. Biol. Med. 32: 377-385.
https://doi.org/10.1016/S0891-5849(01)00735-3

Andersen F.A. 2005. Amended final report on the safety assessment of polyacrylamide and acrylamide residues in cosmetics. Int. J. Toxicol. 24: 21-50.
https://doi.org/10.1080/10915810590953842

Beauchamp C., Fridovich I. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44 : 276-277.
https://doi.org/10.1016/0003-2697(71)90370-8

Bejaoui S., Telahigue K., Chetoui I., et al. 2018. Integrated Effect of Metal Accumulation, Oxidative Stress Responses and DNA Damage in Venerupisdecussata Gills Collected From Two Coast Tunisian Lagoons. J. Chem. Environ. Biol. Eng. 2: 44-51.

Birben E., Sahiner U.M., Sackesen C., et al. 2012. Oxidative stress and antioxidant defense. World. Allergy. Organ. J. 5: 9-19.
https://doi.org/10.1097/WOX.0b013e3182439613

Cai L., Satoh M., Tohyama C., et al. 1999. Metallothionein in radiation exposure: its induction and protective role. Toxicology 132: 85-98.
https://doi.org/10.1016/S0300-483X(98)00150-4

Chetoui I., Rabeh I., Telahigue K., et al. 2010. Valorisation de l’apportnutritionnel d’un mollusque bivalve Mactra corallina des côtestunisiennes (Kalaât El Andalous). Bull. Inst. Natn. Scien. Tech. Mer de Salmmbô 37: 83-88.

Contardo-Jara V., Galanti L.N., Amé M.V., et al. 2009. Biotransformation and antioxidant enzymes of Limnoperna fortunei detect site impact in water courses of Córdoba, Argentina. Ecotoxicol. Environ. Saf. 72: 1871- 1880.
https://doi.org/10.1016/j.ecoenv.2009.07.001

Della Torre C., Balbib T., Grassia G., et al. 2015. Titanium dioxide nanoparticles modulate the toxicological response to cadmium in the gills of Mytilus galloprovincialis. J. Hazard. Mater. 297: 92-100.
https://doi.org/10.1016/j.jhazmat.2015.04.072

Draper H.H., Hadley M. 1990. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 86: 421-431.
https://doi.org/10.1016/0076-6879(90)86135-I

Duan X., Wang Q.C., Chen K.L., et al. 2015. Acrylamide toxic effects on mouse oocyte quality and fertility in vivo. Sci. Rep. 5: 11562.
https://doi.org/10.1038/srep11562

Ellman G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82: 70-77.
https://doi.org/10.1016/0003-9861(59)90090-6

Ellman G.L., Courtney K.D., Andres V. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88-95.
https://doi.org/10.1016/0006-2952(61)90145-9

Erkekoglu P., Baydar T. 2014. Acrylamide neurotoxicity. Nutr. Neurosci. 17: 49- 57.
https://doi.org/10.1179/1476830513Y.0000000065

Fasulo B., Deuring R., Murawska M., et al. 2012. The Drosophila Mi-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo. PLoS. Genet. 8: e1002878.
https://doi.org/10.1371/journal.pgen.1002878

Favier A. 2003. Le Stress oxydant. Intérêt conceptuel et expérimentaldans la compréhension des mécanismes des maladies et potentielthérapeutique. L’actual. Chim. 11: 108-115.

Flohe L., Gunzler W.A. 1984. Assays of gluthathione peroxidase. Methods. Enzymol. 105: 114-121.
https://doi.org/10.1016/S0076-6879(84)05015-1

Friedman M. 2003. Chemistry, acrylamide: A review. J. Agric. Food. Chem. 51: 4504-4526.
https://doi.org/10.1021/jf030204+

Greim H., Snyder R. 2018. Toxicology and Risk Assessment: A Comprehensive Introduction. John Wiley & Sons, Hoboken, USA, 840 pp.
https://doi.org/10.1002/9781119135944

Haleng J., Pincemail J., Defraigne J.O., et al. 2007. Oxidative stress. Rev. Med. Liege 62: 628-638.

International Agency for Research on Cancer (IARC). 1994. IARC working group on the evaluation of carcinogenic risks to humans: some industrial chemicals. IARC Monogr. Eval. Carcinog. Risks. Hum. 60: 1-560.

Jacques-Silva M.C., Nogueira C.W., Broch L.C. 2001. Diphenyl diselenide and ascorbic acid changes deposition of selenium and ascorbic acid in liver and brain of mice. Pharmacol. Toxicol. 88: 119-125.
https://doi.org/10.1034/j.1600-0773.2001.d01-92.x

Jiang Z.Y., Hunt J.V., Wolff S.P. 1992. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low-density lipoprotein. Anal. Biochem. 202: 384-389.
https://doi.org/10.1016/0003-2697(92)90122-N

Jollow D.J., Mitchell J.R., Zampaglione N., et al. 1974. Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 11: 151-169.
https://doi.org/10.1159/000136485

Jomova K., Valko M. 2011. Advances in metal-induced oxidative stress and human disease. Toxicology 283 : 65-87.
https://doi.org/10.1016/j.tox.2011.03.001

Kayali R., Cakatay U., Akcay T., et al. 2006. Effect of alphalipoicacid supplementation on markers of protein oxidation in postmitotictissues of ageing rat. Cell. Biochem. Funct. 24: 79-85.
https://doi.org/10.1002/cbf.1190

Kim S.M., Beak J.M., Lim S.M., et al. 2015. Modified Lipoproteins by Acrylamide Showed More Atherogenic Properties and Exposure of Acrylamide Induces Acute Hyperlipidemia and Fatty Liver Changes in Zebrafish. Cardiovasc. Toxicol. 15: 300-308.
https://doi.org/10.1007/s12012-014-9294-7

Krishnan N., Kodrík D., Kłudkiewicz B., et al. 2009. Glutathione-ascorbic acid redox cycle and thioredoxin reductase activity in the digestive tract of Leptinotarsa decemlineata (Say). Insect. Biochem. Mol. Biol. 39: 180-188.
https://doi.org/10.1016/j.ibmb.2008.11.001

Kusnin N., Syed M.A., Ahmad S.A. 2015. Toxicity, pollution and biodegradation of acrylamide - a mini review. J. Biochem. Microbiol. Biotechnol. 3: 6-12.

Larguinho M., Cordeiro A., Diniz M.S., et al. 2014. Metabolic and histopathological alterations in the marine bivalve Mytilus galloprovincialis induced by chronic exposure to acrylamide. Environ. Res. 135: 55-62.
https://doi.org/10.1016/j.envres.2014.09.004

LoPachin R.M., Gavin T. 2012. Molecular mechanism of acrylamide neurotoxicity: lessons learned from organic chemistry. Environ. Health Perspect. 120: 1650-1657.
https://doi.org/10.1289/ehp.1205432

Lowry O.H., Roseborouch N.I., Farrand A.L., et al. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 263-275.

Mottram D.S., Wedzicha B.L., Dodson A.T. 2002. Acrylamide is formed in the Maillard reaction. Nature 419: 448-449.
https://doi.org/10.1038/419448a

Petrovic S., Ozretic B., Krajnovic-Ozretic M., et al. 2001. Lysosomal membrane stability and metallothioneins in digestive gland of mussels (Mytilus galloprovincialis Lam.) as biomarkers in afield study. Mar. Pollut. Bull. 42: 1373-1378.
https://doi.org/10.1016/S0025-326X(01)00167-9

R Development Core Team. 2017. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.

Schmatz R., Mazzanti C.M., Spanevello R., et al. 2009. Resveratrol prevents memory deficits and the increase in acetylcholinesterase activity in streptozotocin-induced diabetic rats. Eur. J. Pharmacol. 610: 42-48.
https://doi.org/10.1016/j.ejphar.2009.03.032

Schwarz K.B. 1996. Oxidative stress during viral infection: a review. Free Radic. Biol. Med. 21: 641-649.
https://doi.org/10.1016/0891-5849(96)00131-1

Sheehan D., McDonagh B. 2008.Oxidative stress and bivalves: a proteomic approach. Invertebr. Surviv. J. 5: 110-123.

Stanicka J., Landry W., Cotter T.G. 2015. Oxidative stress biomarkers and ROS molecular probes. Oxidative Stress: Diagnostics, Prevention. and Therapy. Vol. 2, pp. 353-374.
https://doi.org/10.1021/bk-2015-1200.ch015

Tepe Y. 2015. Acrylamide in surface and drinking water. acrylamide in food: analysis, content and potential health effects. Giresun Univ., Turkey, pp. 275-293.
https://doi.org/10.1016/B978-0-12-802832-2.00014-0

Tepe Y., Çebi A. 2017. Acrylamide in environmental water: a review on sources, exposure, and public health risks. Exposure and Health 11: 3-12.
https://doi.org/10.1007/s12403-017-0261-y

Touzé S., Guerin V., Guezennec A.G., et al. 2015. Dissemination of acrylamide monomer from polyacrylamide-based flocculant use—sand and gravel quarry case study. Environ. Sci. Pollut. Res. 22: 6423-6430.
https://doi.org/10.1007/s11356-014-3177-0

Trabelsi W., Chetoui I., Fouzai C., et al. 2019. Redox status and fatty acids composition of Mactra corallina digestive gland following exposure to acrylamide. Environ. Sci. Pollut. Res. 26: 22197-22208.
https://doi.org/10.1007/s11356-019-05492-5

Viarengo A., Ponzano E., Dondero F., et al. 1997. A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antartic molluscs. Mar. Environ. Res. 44: 69-84.
https://doi.org/10.1016/S0141-1136(96)00103-1

Yilmaz B., Yildizbayrak N., Aydin Y., et al. 2017. Evidence of acrylamide- and glycidamide-induced oxidative stress and apoptosis in Leydig and Sertoli cells. Hum. Exp. Toxicol. 36: 1225-1235.
https://doi.org/10.1177/0960327116686818

Zamora R., Delgado R.M., Hidalgo F.J. 2010. Model reactions of acrylamide with selected amino compounds. J. Agric. Food. Chem. 58: 1708-1713.
https://doi.org/10.1021/jf903378x

Zhou Z., Sun X., Kang Y.J. 2002. Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress. Exp. Biol. Med. 227: 214-222.
https://doi.org/10.1177/153537020222700310

Zorita L., Ortiz-Zarragoitia M., Solo M., et al. 2006. Biomarkers in mussels from a copper site gradient (Visnes, Norway): an integrated biochemical, histochemical and histological study. Aquat. Toxicol. 78: 109-116.
https://doi.org/10.1016/j.aquatox.2006.02.032