1. INTRODUCTION
⌅Over the last centuries, the environmental impacts of heavy metal pollution have increased in the coastal areas, causing a major threat to the ecosystems and the biota they support (Ruiz et al., 2014Ruiz F, González-Regalado ML, Muñoz JM, Abad M, Toscano A, Prudencio MI, Dias MI. 2014. Distribution of heavy metals and pollution pathways in a shallow marine shelf: assessment for a future management. J. Environ. Sci. Technol. 11, 1249. https://doi.org/10.1007/s13762-014-0576-1 ). Among these xenobiotics, mercury (Hg) is listed as one of the most toxic elements due to its tendency to bioaccumulate and biomagnify through the food chain (Balshaw et al., 2007Balshaw S, Edwards JW, Ross KE, Daughtry BJ. 2008. Mercury distribution in the muscular tissue of farmed southern bluefin tuna (Thunnus maccoyii) is inversely related to the lipid content of tissues. Food Chem. 111, 616- 621. https://doi.org/10.1016/j.foodchem.2008.04.041 ). The sources of mercury contamination in aquatic ecosystems include natural and anthropogenic emissions (Verlecar et al., 2008Verlecar XN, Jena KB, Chainy GBN. 2008. Modulation of antioxidant defences in digestive gland of Pernaviridis (L.), on mercury exposures. Chemosphere 71, 1977-1985. https://doi.org/10.1016/j.Chemosphere.2007.12.014 ). The later generally come from incineration, fungicides, paints and industrial processes. One of the established mechanisms of Hg toxicity is its ability to induce cellular oxidative stress through the generation of reactive oxygen species (ROS) which react with macromolecules such as lipids, proteins and DNA (Uttara et al., 2009Uttara B, Singh AV, Zamboni P, Mahajan RT. 2009. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr. Neuropharmacol. 7, 65-74. https://doi.org/10.2174/157015909787602823 ). As the cell membrane is tightly linked to cellular physiology, several authors have reported that the membrane is the primary detector of stress stimuli and activator of the cellular stress response (Dindia et al., 2013Dindia LA, Faught EL, Leonenko Z, Thomas RH, Vijayan MM. 2013. Rapid cortisol signaling in response to acute stress involves changes in plasma membrane order in rainbow trout liver. Am. J. Physiol. Endoc. M. 304, E1157-E1166; Vígh et al., 2007Vigh L, Nakamoto H, Landry J, Gomez-Muñoz A, Harwood DJL, Horvath I. 2007. Membrane regulation of the stress response from prokaryotic models to mammalian cells. Ann. N. Y. Acad. Sci. 1113, 40-51. https://doi.org/10.1196/annals.1391.027 ). According to several authors, changes in the membrane biophysical properties as a primary response to stress, mainly trigger changes in lipid and fatty acid compositions (Los et al., 2004Los DA, Murata N. 2004. Membrane fluidity and its roles in the perception of environmental signals. Biochim. Biophys. Acta 1666, 142-157. https://doi.org/10.1016/j.bbamem.2004.08.002 ; Thyrring et al., 2015Thyrring J, Juhl BK, Holmstrup M, Blicher ME, Sejr M. 2015. Does acute lead (Pb) contamination influence membrane fatty acid composition and freeze tolerance in intertidal blue mussels in arctic Greenland? Ecotel. 24, 2036-2042. https://doi.org/10.1007/s10646-015-1539-0 ). Indeed, FAs, as the main constituent of the cell membrane, play crucial roles in several physiological functions as well as in the maintenance of membrane structures (Neves et al., 2015Neves M, Castro BB, Vidal T, Vieira R, Marques JC, Coutinho JAP, Gonçalves F. 2015. Biochemical and populational responses of an aquatic bioindicator species, Daphnia longispina, to a commercial formulation of an herbicide (primextraç gold TZ) and its active ingredient (S-metolachlor). Ecol. Indic. 53, 220-230. https://doi.org/10.1016/j.ecolind.2015.01.031 ). It therefore appears that lipids and their constitutional components could be closely involved in cellular responses to pollutants such as Hg in aquatic organisms (Ferrain et al., 2018Ferain A, Bonnineau C, Neefs I, Das K, Larondelle Y, Rees JF, Debier C, Lemaire B. 2018. Transcriptional effects of phospholipid fatty acid profile on rainbow trout liver cells exposed to methylmercury. Aquat. Toxicol. 199, 174-187.). In this context, it has been proven that the susceptibility of individual FA to peroxidation increases exponentially with an increasing number of double bonds on the carbon chain (Holman, 1954Holman TR. 1954. Autoxidation of fats and related substances. In Progress in the Chemistry of Fats and Other Lipids. Academic Press, New York. 2, 491-514.). Thus, polyunsaturated fatty acids (PUFAs) like eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) are not only targets that are damaged by ROS, but also play a key role in enhancing an organism’s adaptation to environmental stress (Munro et al., 2016Munro D, Banh S, Sotiri, Tamanna N, Treberg J. 2016. The thioredoxin and glutathione‐dependent H2O2 consumption pathways in muscle mitochondria: Involvement in H2O2 metabolism and consequence to H2O2 efflux assays. Free Radic. Biol. Med. 96, 334-346. https://doi.org/10.1016/j.freeradbiomed.2016.04.014 ). Given this, FAs were recently argued to be promising bio-indicators to assess stress exposure and ecosystem health in a marine environment (Silva et al., 2017Silva CO, Simões T, Novais SC, Pimparel I, Granada L, Soares AM, Barata C, Lemos MF. 2017. Fatty acid profile of the sea snail Gibbula umbilicalis as a biomarker for coastal metal pollution. Sci. Total Environ. 15 (586), 542-550. https://doi.org/10.1016/j.scitotenv.2017.02.015 ).
Nowadays, most of the available data regarding the use of FA as biomarkers for marine pollution monitoring refers to bivalves, micro and macroalgae and fish (Filimonova et al., 2016Filimonova V, Gonçalves F, Marques JC, De Troch M, Gonçalves ANN. 2016. Fatty acid profiling as bioindicator of chemical stress in marine organisms: A review. Ecol. Indic. 67, 657-672. https://doi.org/10.1016/j.ecolind.2016.03.044 ; Gonçalves et al., 2016Gonçalves A, Mesquita A, Verdelhos T, Coutinho J, Marques J, Gonçalves F. 2016. Fatty acids profiles as indicator of stress induced by of a common herbicide on two marine bivalves species: Cerastoderma edule (Linnaeus, 1758) and Scrobicularia plana (da Costa, 1778). Ecol. Indic. 63, 209-218. https://doi.org/10.1016/j.ecolind.2015.12.006 ); however, little is known about other marine organisms such as holothurians. Our previous study (Telahigue et al., 2019Telahigue K, Rabeh I, Hajji T, Trabelsi W, Bejaoui S, Chouba L, El Cafsi M, Soudani N. 2019. Effects of acute mercury exposure on fatty acid composition and oxidative stress biomarkers in Holothuria forskali body wall. Ecotox. Environ. Safe. 169, 516-522. https://doi.org/10.1016/j.ecoenv.2018.11.051 ) investigated for the first time, the impact of acute mercury exposure on several biochemical parameters including fatty acid profile in the Holothuria forskali body wall. Holothurians, commonly known as sea cucumbers, represent an important marine resource due to their numerous nutraceutical and pharmaceutical proprieties (Bordbar et al., 2011Bordbar S, Anwar F, Saari N. 2011. High-value components and bioactives from sea cucumbers for functional foods-A Review. Mar. Drugs 9 (10), 1761-1805. https://doi.org/10.3390/md9101761 ). The sea cucumber fishery and aquaculture trade have been an active industry in Asia for a long time. In recent years, the sea cucumber trade has also flourished in some Mediterranean sea and NE Atlantic Ocean countries (Sicuro and Levine, 2011Sicuro B, Levine J. 2011. Sea cucumber in the Mediterranean: a potential species for aquaculture in the Mediterranean. Rev. Fish. Sci. 19, 299-304. https://doi.org/10.1080/10641262.2011.598249 ). The black sea cucumber or cotton-spinner Holothuria forskali, which is one of the most common sea cucumber species in the Mediterranean sea, is listed as a new target resource for future farming industries. (Sicuro and Levine, 2011Sicuro B, Levine J. 2011. Sea cucumber in the Mediterranean: a potential species for aquaculture in the Mediterranean. Rev. Fish. Sci. 19, 299-304. https://doi.org/10.1080/10641262.2011.598249 ). Like most of other sea cucumbers species, H. forskali is a deposit feeder and may ingest a large amount of sediment (Navarro et al., 2014Navarro PG, García-Sanz S, Tuya F. 2014. Contrasting displacement of the sea cucumber Holothuria arguinensis between adjacent nearshore habitats. J. Exp. Mar. Biol. Ecol. 453, 123-130. https://doi.org/10.1016/j.jembe.2014.01.008 ) which make it particularly prone to heavy metal pollution. Several authors have reported that sea cucumbers could be considered as a potential bioindicator for heavy metal pollution (Turk Culha et al., 2016Turk Culha S, Dereli H, Karaduman FR, Culha M. 2016. Assessment of trace metal contamination in the sea cucumber (Holothuria tubulosa) and sediments from the Dardanelles Strait (Turkey). Environ. Sci. Pollut. R. 23 (12), 11584-11597.). Indeed, it is well established that mercury tends to accumulates in tissue in a specific manner depending on the organ’s physiological role and its regulatory ability. Generally, some tissues/organs such as body wall and respiratory tree have received particular attention to evaluate the impact of noxious stimuli in holothurians. Nonetheless, little information is available on the longitudinal muscles. In fact, muscle constitutes an interesting organ to analyze owing to the energy supply function and to its major role in locomotion and contraction movements in response to environmental stimuli and to its being a target tissue for bioaccumulation of merury (as confirmed by unpublished data obtained in our laboratory). The current study was mainly designed to evaluate the impact of Hg contamination on the fatty acid composition of H. forskali muscle. The results may provide valuable data concerning the underlying toxicity mechanism of Hg in lipid metabolism and to verify the validity of the FA profile as a biomarker of mercury intoxication in sea cucumber. Other parameters such as levels of H2O2 and LOOH were also assessed in order to verify the induction of an oxidative stress state in H. forskali muscle tissue following acute Hg exposure.
2. MATERIALS AND METHODS
⌅2.1. Chemicals
⌅Mercury chloride (HgCl2 > 99.5% purity) was obtained from Sigma-Aldrich (St. Louis, MO, USA). It was dissolved in pure water for stock concentrations, and the desired test solutions used in the present study were prepared by diluting the stock solution with double-distilled water. Reduced glutathione (GSH), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), and thiobarbituric acid (TBA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were purchased from standard commercial suppliers.
2.2. Experimental design
⌅Young H. forskali, with an average body length of 20.25 ± 2.75 cm, were collected by hand during scuba diving off the Bizerte coast (North east of Tunisia), a relatively clean sea shore. The sea cucumbers were transported to the laboratory in cooler boxes with water from the collection site. Once in the laboratory, the specimens were kept in tanks filled with aerated seawater (18 °C ± 1; photoperiod of 12:12 h) for three days prior to experimentation. The animals were acclimated and then randomly divided into 4 groups of 12 specimens each (triplicate design) in order to ensure the reproducibility of the results. As we proceeded in our previously published research (Rabeh et al., 2018Rabeh, Telahigue K, Bejaoui S, Hajji T, Nechi S, Chelbi E, EL Cafsi M, Soudani N. 2019. Effects of mercury graded doses on redox status, metallothione in levels and genotoxicity in the intestine of sea cucumber Holothuria forskali. Chem. Ecol. 35 (3), 204-218.https://doi.org/10.1080/02757540.2018.1546292 ), the animals were exposed to a range of mercury chloride (HgCl2) concentrations as follows: (controls (0), D1 (40 μg·L-1), D2 (80 μg·L-1) and D3 (160 μg·L-1). Tested doses were chosen based on studies that investigated the effects of the acute exposure of marine invertebrates to mercury, using similar concentrations (Bhamre et al., 2010Bhamre PR, Thorat SP, Desai AE. 2010. Evaluation of acute toxicity of mercury, cadmium and zinc to a freshwater mussel Lamellidens consobrinus. Our Nat. 8, 180-184.; Oliveira et al., 2015Oliveira P, Lopes-Lima M, Machado J, Guilhermino L. 2015. Comparative sensitivity of European native (Anodonta anatina) and exotic (Corbicula fluminea) bivalves to mercury. Estuar. Coast. Shelf Sci. 167, 191-198. https://doi.org/10.1016/j.ecss.2015.06.014 ). The lowest concentration (40 μg·L-1) was in the range of concentrations that can be found in some heavily contaminated water bodies near industrialized areas (Alinnor, 2005; Guilherme et al., 2008); while the highest tested concentrations (60 and 80 μg·L-1), were selected to guarantee the observable effects of Hg and to elucidate the responsiveness of sea cucumber to the worst-case-scenario of mercury contamination. To ensure water quality, 50% of the water was replaced every 24 h and the concentrations of mercuric chloride were re-established. The changed volume of water was replaced by an equal amount of water containing the initial concentration of HgCl2. During the exposure period, sea cucumbers were not fed to avoid prandial effects and to prevent the deposition of feces. No mortalities were observed throughout the experimental period in any group. At the end of experiments, nine animals from each treatment were removed from the tanks by dip-net and weighed. Muscle was carefully dissected and immediately frozen in liquid nitrogen and then stored at −80 °C until the biochemical assays were carried out.
2.3. Hydrogen peroxide generation assay
⌅The amount of hydrogen peroxide (H2O2) generation in tissues was monitored by the ferrous ion oxidation xylenol orange method of Ou and Wolff (1996)Ou P, Wolff SP. 1996. A discontinuous method for catalase determination at near physiological concentrations of H2O2 and its application to the study of H2O2 fluxes within cells. J. Biochem. Biophys. Meth. 31 (1-2), 59-67. The amount of H2O2 produced was determined using the extinction coefficient of 2.67×105 cm−1M−1 and results were expressed as nmol per gram of tissue.
2.4. Lipid hydroperoxide assay
⌅Lipid hydroperoxide levels were estimated using the ferrous oxidation in xylenol orange assay (FOX assay) described by Jiang et al., (1992)Jiang ZY, Hunt JV, Wolf SP. 1992. Detection of lipid hydroperoxides using Fox method. Anal. Biochem. 202, 384-389.. The amount of hydroperoxides produced was calculated using the molar extinction coefficient of 4.6 × 9 × 104 M−1 cm−1, and the results were expressed as nanomoles per milligram of protein.
2.5. Lipid extraction
⌅Total lipids in each sample were extracted according to the Folch, Lees, and Sloane-Stanley (1957)Folch J, Lees M, Sloane-Stanley GA. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226 (1) 497-509. method with the solvent mixture chloroform-methanol (2:1, v/v). Following solvent evaporation under nitrogen, lipids were transferred to pre-weighed 2ml vials. The solvent mixture was again evaporated under nitrogen, and the extracts were further dried overnight at ambient temperature (18 °C) in a vacuum desiccator. Once weighed, the lipids were re-dissolved in chloroform-methanol (2:1, v/v) with 0.01% butylated hydroxy toluene (BHT, Sigma-Aldrich) added as an antioxidant to minimize the risk of lipid oxidation.
2.6. Fatty acid analysis
⌅Fatty acids from total lipid extracts were transesterified according to the Cecchi et al., (1985)Cecchi G, Basini S, Castano C. 1985. Méthanolyse rapide des huiles en solvant. Rev. Franc. Corps Gras 4, 63-164. method. Methyl nonadecanoate C19:0 (Sigma) was added as internal standard. The resulting fatty acid methyl esters (FAME) were extracted using sodium methylate (NaOCH3) in the presence of hexane and sulfuric acid (H2SO4). Separation of FAMEs was carried out on a HP 6890 gas chromatograph (Agilent Technologies, Sacramento,CA, USA) with a split/splitless injector equipped with a flame ionization detector at 275 °C, and a 30m HP. Innowax capillary column with an internal diameter of 250 μm and a 0.25 μm film thickness. The injector temperature was held at 250 °C. The oven was programmed to rise from 50 to 180 °C at a rate of 4 ºC·min-1, from 180 to 220 °C at 1.33 ºC·min-1 and to stabilize at 220 °C for 7 min. The carrier gas was nitrogen. Identification of FAMEs was based on the comparison of their retention times with those of a mixture of methyl esters (Supelco 47085U PUFA No: 3 and Supelco 37 component FAME mix 47885-U). Fatty acid peaks were integrated and analyzed using Hewlett-Packard ChemStation software. All fatty acid data are reported as percentage of total fatty acids.
2.7. Calculation of indices and statistical analysis
⌅The indices of desaturase activities were estimated as the product/precursor ratios of individual fatty acids according to the following formulas:
D9D: Δ9-desaturase = stearoyl-CoA-desaturase = (C18:1n-9)/C18:0); D5D: Δ5-desaturase = C20:5n3/C20:4n3, D6D: Δ6-desaturase =22:6n-3/ C20:5n-3 (Da costa et al., 2015Da Costa F, Robert R, Quéré C, Wikfors GH, Soudant P. 2015. Essential fatty acid assimilation and synthesis in larvae of the bivalve Crassostrea gigas. Lipids 50 (5), 503-511. https://doi.org/10.1007/s11745-015-4006-z ; Rabei et al., 2018Rabei A, Hichami A, Beldi H, Bellenger S, Khan NA, Soltani N. 2018. Fatty acid composition, enzyme activities and metallothioneins in Donax trunculus (Mollusca, Bivalvia) from polluted and reference sites in the Gulf of Annaba (Algeria): pattern of recovery during transplantation. Environ. Pollut. 237, 900-907. https://doi.org/10.1016/j.envpol.2018.01.041 ).
The indices of the elongase (Elovl 6) activity were calculated using the C18:0/C16:0 ratio (Kotronen et al., 2010Kotronen A, Seppanen-Laakso T, Westerbacka J, Arola J, Ruskeepaa AL, Yki-Jarvinen H, Oresic M. 2011. Comparison of lipid and fatty acid composition of the liver, subcutaneous and intra-abdominal adipose tissue, and serum. Obesity 18, 937-944).
Mercuric chloride effects on fatty acid composition were analyzed by one way ANOVA of variance (ANOVA) followed by the Tukey’s test in order to compare results between doses. Results were expressed as mean ± SD and differences were considered significant at p < 0.05. First the data for each variable was checked for normality and homogeneity of variance by Kolmogorov-Smirnov and Levene’s tests, respectively. Raw values were arcsine-transformed or log10-transformed (if necessary) to meet the requirements for normal distribution and homogeneity of the variances. Student unpaired t-test was also used when comparison between two groups was required. All statistical analyses were run by the statistical software program R version 3.0.2 (R Core Team2017). Principal Component Analysis (PCA) was applied to evaluate the relationship between mercury treatments and fatty acid composition in the muscle of the control and the exposed groups of H. forskali.
3. RESULTS
⌅3.1. Lipid hydroperoxide (LOOHs), and hydrogen peroxide (H2O2) levels
⌅Significant increases (p < 0.05) in H2O2 and LOOH levels were recorded in the treated H. forskali, respectively, compared to the controls (Figure 1).
3.2. Fatty acids composition
⌅As shown in Table 1, a significant decrease in lipid content was recorded in D1 however, in D2 and D3 a substantial increase was noticed compared to the control. The Fatty acid profiles of the muscle in the control and HgCl2-treated H. forskali are presented in Table 1. The untreated sea cucumber group showed a significantly higher proportion of polyunsaturated fatty acids (PUFA) (52.36% of total FA) followed by monounsaturated fatty acids (MUFA) and saturated fatty acids (SFA) with 25.79 and 21.84%, respectively. Over all, we noticed that exposure to the nominal dose of Hg (D1, 40 µg·L-1) substantially changed the main fatty acid groups. In fact, significant increases in SFA levels with a decrease in unsaturated fatty acid levels (MUFA and PUFA) were observed compared to the controls. A closer examination of data showed that the recorded increase in the SFA group, which almost doubled under D1 treatment, was mainly due to the increased levels of C15:0 and C16:0. As for unsaturated fatty acids, we recorded that C18:1n-7 and C20:1n-9 were the most affected FAs by the Hg treatments and to a lesser extent in D2 and D3. In the PUFA group, the primary source of variation was due to the decrease in the n-6 group level in D1. In fact, the levels of C18:2n-6 and C20:4n-6 dropped by 70 and 90%, respectively, compared to the control group. Regarding the n-3 group, significant decreases of 70 and 40% were recorded for the EPA (C20: 5n-3) levels in D1 and D2 groups, respectively. The EPA level remained statistically unchanged in the D3 group in comparison to the control. Significant changes were also observed for DHA (C22:6 n3) in all treatments. This fatty acid tended to rise in D1 and diminish at higher doses.
| Fatty acids | Control | Hg (D1) | Hg (D2) | Hg (D3) |
|---|---|---|---|---|
| C14:0 | 5.46 ± 0.4 a | 3.61 ± 0.05 b | 4.93 ± 0.86 d | 4.61±0.51c |
| C15:0 | 1.15 ± 0.15 a | 4.77 ± 0.22 b | 3.93 ± 0.01 c | 2.08±0.20b |
| C14:1 | 0.22 ± 0.23 a | 0.52 ± 0.08 b | 1.00 ± 0.50 c | 1.31±0.35 c |
| C15:1 | 1.40 ± 0.14 a | 5.75 ± 0.12 b | 1.87 ± 0.08 a | 0.88±0.54c |
| C16:0 | 4.86 ± 1.4 a | 9.74 ± 0.34 b | 5.35 ± 0.75 c | 5.02±0.13 c |
| C16:1n-9 | 4.37 ± 0.17 a | 3.67 ± 0.17 b | 3.74 ± 1.63 a | 7.29 ± 1.32 b |
| C16:1n-7 | 8.72 ±1.42 a | 3.97 ± 0.29 b | 8.60 ±0.32 a | 4.83±1.16 c |
| C16:2n-4 | 2.42 ± 0.13 a | 1.50 ± 0.05 b | 2.89 ± 0.28c | 1.82±0.31 a |
| C17:0 | 0.4 ± 0.15a | 1.56± 0.27 b | 1.20±0.08 b | 2.70±0.44 c |
| C16:4 | 2.81 ± 0.30 a | 1.78 ± 0.36 b | 1.51 ± 0.05 b | 2.46±0.15 a |
| C16:3n-4 | 3.35 ± 0.5 a | 6.37 ± 0.80 b | 2.25 ± 0.30 b | 2.24±0.26 b |
| C18:0 | 5.62 ± 0.49 a | 6.83 ± 0.13b | 5.12±1.06 c | 4.55±0.52 c |
| C18:1n-9 | 2.38 ± 0.01a | 1,15 ± 0.10 b | 1.06 ± 0.03 b | 2.04±0.25 c |
| C18:1n-7 | 1.92 ± 0.2 a | 0.62 ± 0.18 b | 1.36 ± 0.09c | 2.46±0.57d |
| C18:2n-6 | 11.6± 1.23a | 4.42 ± 0.65b | 11.48 ±0.40 a | 13.79 ± 1.90d |
| C18:4n-3 | 2.05 ± 0.29 a | 6.93 ± 0.51b | 4.25 ± 0.08 c | 1.35 ± 0.10 a |
| C18:3n-3 | 2.08 ± 0.05 a | 4.85 ± 0.42 b | 2.46 ± 0.42 a | 1.92 ± 0.15 c |
| C20:0 | 3.73 ± 0.45 a | 3.19 ± 0.40 b | 4.19 ± 0.06 a | 2.60 ± 0.28 b |
| C20:1n-9 | 6.69 ± 0.8 a | 3.04 ± 0.04 b | 7.87 ± 0.34 a | 7.74 ± 0.35 c |
| C20:3n-6 | 4.94 ± 0.09 a | 1.97 ± 0.06 b | 6.16 ±0.12 a | 7.70 ± 0.53 c |
| C20:2n-6 | 5.91 ± 0.24 a | 9.07 ± 0.05 b | 5. 87 ± 0.13 c | 4.25 ± 0.84 d |
| C20:4n-6 | 4.68 ± 0.03 a | 0.57 ± 0.06 b | 2.35 ± 0.12 c | 6.06 ± 0.93 d |
| C20:3n-3 | 3.31 ± 0.01 a | 2.25 ± 0.20 b | 3.89 ± 0.28 a | 2.41 ± 0.12 c |
| C20:5n-3 | 3.13 ± 0.43 a | 1.30 ± 0.06 b | 1.27 ± 0.11 b | 3.17 ± 0.41 a |
| C22:0 | 0.38 ± 0.08 a | 1.01 ± 0.07 b | 0.62 ± 0.05 b | 0.69 ± 0.08 b |
| C22:1 | 0.36 ± 0.02 a | 0.11 ± 0.01 b | 0.16 ± 0.04 b | 0.09 ± 0.01 b |
| C22:5n-6 | 2.46 ± 0.08 a | 1.14 ± 0,20 b | 0.99 ± 0.07 b | 2.02 ± 0.47 a |
| C22:6n-3 | 3.62 ± 0.02 a | 7.05 ± 1.18 b | 1.72 ± 0.17 c | 2.79 ± 0.38 a |
| ∑SFA | 21.85±0.89 a | 31.72±0.94b | 24.23±0.77 a | 22.09±5.55 c |
| ∑MUFA | 25.79±0.72 a | 19.11±0.42b | 27.70±3.08 a | 27.02±5.90 a |
| ∑PUFA | 52.36±3.26 a | 49.17±1.38b | 48.07±3.03b | 51.15±11.45 a |
| ∑PUFA (n-3) | 14.20±1.21 a | 22.38±1.94b | 13.59±2.29 a | 11.63±0.09 c |
| ∑PUFA (n-6) | 23.67±1.20 a | 13.57±0.3b | 26.85±0.24 c | 34.15±13.08d |
| n-3/n-6 | 0.59±0.05 a | 1.67±0.01 b | 0.5±0.08 a | 0.34±0.03 c |
| Total lipid (mg/g ww) | 5.64±0.16 a | 0.99±0.03 b | 7.5±0.41 c | 10.19±1.02 d |
Data
are expressed as percentage of major fatty acids (mean ± standard
deviation; n=9); Means followed by different letters in the same line
are significantly different (p < 0.05) by Tukey’s test.
SFAs:
saturated fatty acids, MUFAs: monounsaturated fatty acids, PUFAs:
polyunsaturated fatty acids
3.3. Estimated activity of fatty acid desaturases and elongases
⌅Desaturase and elongase activities were determined in the muscle of H. forskali exposed to gradual doses of mercury (Table 2). The obtained results showed that D9D (C18:1n9/C18:0), D5D (C20:4n6/C20:3n-6) and Elongase 6 (C18:0/C16:0) activities decreased significantly at the lower doses (40 and 80 µg·L-1) but remained unaffected at the highest dose (Table 2). The activity of the D6D:Δ6-desaturase (C22:6n-3/C20:5n-3) was significantly influenced by Hg exposure, expressing much higher values at D1 when compared to the control group.
| Treatments and parameters | Control | D1 | D2 | D3 |
|---|---|---|---|---|
| D9D(18:1n9/18:0) | 0.4±0.07 | 0.18±0.14** | 0.30 ±0.03* | 0.45±0.02 |
| D5D(20:4n6/20:3n6) | 0.79±0.05 | 0.27±0.049** | 0.38±0.05** | 1.183±0.2** |
| D6D(22:6n3/20:5n3) | 1.15±0.01 | 5.4±1.04*** | 1.39±0.06 | 0.87±0.08* |
| Elovl6(18:0/C16:0) | 1.23.16±0.04 | 0.7±0.08** | 1.14±0.03 | 0.62±0.08* |
Results
are given in means ± standard deviation of nine replicates in each
group.
D1, D2, and D3 vs control: *p < 0.05; **p < 0.01; ***p <
0.001 (using Tukey’s test)
D9D: Δ9-desaturase (stearoyl-CoA-desaturase=18:1n9/18:0);
D5D: Δ5-desaturase (20:4n6/20:3n-6);
D6D: Δ6-desaturase (22:6n-3/20:5n-3);
Elovl 6: Elongase 2 (18:0/C16:0).
3.4. Principal component analysis
⌅The PCA carried out on the fatty acid composition data matrix produced a two-dimensional pattern which explained 77.7% of total variance (Figure 2). Those two components explained 56.7 (PC1) and 21.0% (PC2) of the total variance. The PCA plot showed a considerable distinction between control and treated groups along the PC2 axis. In addition, we noticed that the group exposed to the lowest dose (D1) was separated from all other groups along the PC1 axis. The most highly-sensitive fatty acids were C18:4n.3 and C14:1 for the PC1 axis and C17:0 and C20:0 for the PC2 axis.
4. DISCUSSION
⌅Heavy metals such as mercury are potential stressors that exert toxic effects through redox cycling which results in the uncontrolled production of ROS and a failure in antioxidant defense systems (Lushchak, 2011Lushchak VI. 2011. Environmentally induced oxidative stress in aquatic animals. Aquatic Toxicol. 719 (101), 13-30.). In the current study, an over production of ROS was confirmed by the increase in the H2O2 level in all Hg-treated groups. Our results are consistent with several studies which reported a close correlation between the generation of intracellular ROS and the toxicity exerted by the inorganic Hg in several marine organisms (Verlecar et al., 2008Verlecar XN, Jena KB, Chainy GBN. 2008. Modulation of antioxidant defences in digestive gland of Pernaviridis (L.), on mercury exposures. Chemosphere 71, 1977-1985. https://doi.org/10.1016/j.Chemosphere.2007.12.014 ; Wang et al., 2016Wang P, Wang R, Wang C, Qian J, Hou J. 2016. Exposure-dose-response relationships of the freshwater bivalve Corbicula fluminea to inorganic mercury in sediments. J. Comput. Theor. Nanosci. 13, 5714-5723. https://doi.org/10.1166/jctn.2016.5476 ). In this context, Patrick (2002)Patrick L. 2002. Mercury toxicity mercury toxicity and antioxidants: Part 1: role of glutathione and alpha-lipoic acid in the treatment of mercury toxicity. Altern. Med. Rev. 7 (6), 456-471. reported that exposure to Hg promotes the synthesis of H2O2, which could be converted into a reactive hydroxyl radical and lipid peroxidation (LPO) products in mitochondrial membranes. Furthermore, Hg ions are able to increase the action potential in the inner membrane of the mitochondria which regulates HO-, O2 - and H2O2, and negatively adjusts the defense enzymes SOD, CAT, GPx, as well as GSH (Flores et al., 2019Flores JS, Torres-Jasso JH, Rojas-Bravo D, Reyna-Villela ZM, Erandis D, Torres-Sánchez ED. 2019. Effects of Mercury, Lead, Arsenic and Zinc to Human Renal Oxidative Stress and Functions: A Review. J. Heavy Met. Toxicity Dis. 4, 1-2. https://doi.org/10.21767/2473-6457.10027 ).
Besides biomarker analysis, the determination of the fatty acid composition has proven to be a good indicator of stress and ecosystem health, due to their high sensitivity to stress factors and environmental changes (Gonçalves et al., 2016Gonçalves A, Mesquita A, Verdelhos T, Coutinho J, Marques J, Gonçalves F. 2016. Fatty acids profiles as indicator of stress induced by of a common herbicide on two marine bivalves species: Cerastoderma edule (Linnaeus, 1758) and Scrobicularia plana (da Costa, 1778). Ecol. Indic. 63, 209-218. https://doi.org/10.1016/j.ecolind.2015.12.006 ). Indeed, changes in lipid metabolism are known to be a common biochemical response to pollutant exposure and accumulation in marine organisms (Dailianis et al., 2011Dailianis S. 2011. Environnemental impact of anthropogenic activities: the use of mussels as a reliable tool for monitoring marine pollution. In: McGevin, L.E. (Ed.), Mussels: Anatomy. Habitat and Environmental Impact. Nova Science Publishers. Inc. pp. 1-30.). Our results showed significant changes in the FA composition of treated sea cucumbers indicating an evident harmful impact of Hg on lipid metabolism. Thereby, after exposure to Hg, the FA profile of sea cucumber showed directional tendency anchored by an increase in SFAs, and the decrease in MUFAs and PUFAs levels. The observed changes are thought to be a compensatory process to defend the integrity of biological membranes and reduce cytotoxicity (Signa et al., 2015Signa G, Di Leonardo R, Vaccaro A, Tramati CD, Mazzola A, Vizzini S. 2015. Lipid and fatty acid biomarkers as proxies for environmental contamination in caged mussels Mytilus galloprovincialis. Ecol. Indic. 57, 384-394.). It should also be noted that the most prominent changes in the FA composition of the Hg-treated H. forskali were mostly recorded at the lowest tested dose as confirmed further by the PCA analysis. Such findings can be attributed to a hormeosis response and suggest that low Hg dose response may be considered as a manifestation of the plasticity of biological systems. In fact, hormeosis, which is a dose-response phenomenon characterized by a low-dose stimulation and a high-dose inhibition, was observed for Algae exposed to silver ions and nanoparticles (Tayemeh et al., 2020Tayemeh MB, Esmailbeigi M, Shirdel I, Joo HS, Johari SA, Banan A, Nourani H, Mashhadi H, Jami MJ, Tabarrok M. 2020. Perturbation of fatty acid composition, pigments, and growth indices of Chlorella vulgaris in response to silver ions and nanoparticles: A new holistic understanding of hidden ecotoxicological aspect of pollutants. Chemosphere 238, 124576 https://doi.org/10.1016/j.chemosphere.2019.124576 ). The increased proportions of SFA mostly resulted from the increment in palmitic acid (C16:0) and may reflect an inflammation response to Hg intoxication. Indeed, the last cited FA is particularly known as a potent agent involved in the activation of proinflammatory signaling pathways (Ma et al., 2018Ma XZ, Pang ZD, Wang JH, Song Z, Zhao LM, Du XJ, Deng XL. 2018. The role and mechanism of KCa3.1 channels in human monocyte migration induced by palmitic acid. Exp. Cell Res. 369, 208-217. https://doi.org/10.1016/j.yexcr.2018.05.020 ). Another interesting finding recorded in the current study is the decrease in the MUFA group mainly due to the diminution of the oleic acid (C18:1n-9, OA) level. The latter is a nonessential FA which derives from the desaturation of stearic acid (C18:0) by the stearoyl-CoA desaturase (Scd). The recorded decline in the OA level is probably due to the inhibition of the Δ9-desaturase activity by Hg ions as reflected by the product-to-precursor ratio used in the current study. Furthermore, OA was probably used for energy production through β-oxidation and/or in modulation of membrane fluidity (Funari et al., 2003Funari SS, Barceló F, Escribá PV. 2003. Effects of oleic acid and its congeners, elaidic and stearic acids, on the structural properties of phosphatidylethanolamine membranes. J. Lipid R. 44, 567-575. https://doi.org/10.1194/jlr.M200356-JLR200 ) and/or in detoxification related mechanisms as suggested by Ruiz-Gutiérrez et al., (1999)Ruiz-Gutierrez V, Muriana FJ, Guerrero A, Cert AM, Villar J. 1996. Plasma lipids, erythrocyte membrane lipids and blood pressure of hypertensive women after ingestion of dietary oleic acid from two different sources. J. Hypertens 14, 1483- 1490.. Also worthy of note, a significant depletion of the n-6 group and particularly ARA (C20:4n-6) and its precursor LA (C18:2n-6), which are presumably required for activation of eicosanoid synthesis through the arachidonic cascade. In fact, ARA is largely reported in the literature to be an important mediator involved in inflammation, immune response, and adaptation to stress through eicosanoid pathways (Calder, 2015Calder PC. 2015. Marine omega-3 fatty acids and inflammatory processes: effects, mechanisms and clinical relevance. Biochim. Biophys. Acta Mol. Cell Biol Lipids 1851, 469-484. https://doi.org/10.1016/j.bbalip.2014.08.010 ). Likewise, we note the expenditure of EPA, another precursor to eicosanoids, which may reflect a compensatory mechanism to maintain membrane fluidity and to promote the adaptive response of the organism to stress (Delaporte et al., 2006Delaporte M, Soudant P, Moal J, Kraffe E, Marty Y, Samain J.F .2005. Incorporation and modification of dietary fatty acids in gill polar lipids by two bivalve species Crassostrea gigas and Ruditapes philippinarum. Comp. Biochem. Phys. A 140, 460-470.). Another PUFA, docosahexaenoic acid (DHA), which is a main component of membrane phospholipids, was found to increase in Hg-treated animals. Such findings might reflect the ability of H. forskali to cope with Hg injuries through the induction of Δ6-desaturase activity as reflected by the enhanced 22:6n-3/22:5n-3 ratio. Indeed, it has been proven that the DHA may act as an antioxidant to reduce lipid peroxidation (Mayurasakorn et al., 2016Mayurasakorn K, Niatsetskaya ZV, Sosunov SA, Williams JJ, Zirpoli H, Vlasakov I, Ten VS. 2016. DHA but not EPA emulsions preserve neurological and mitochondrial function after brain hypoxia-ischemia in neonatal mice. PLOS One 11. https://doi.org/10.1371/journal.pone.0160870 ). Furthermore, Vanse et al. (2002) reported that DHA could be involved in modulating the inflammatory response and regulating membrane homeoviscous to protect the cell from oxidative disruption caused by Hg intrusion. Similar pictures were observed for other marine organisms following exposure to metals and organic pollutants (Telahigue et al., 2019Telahigue K, Rabeh I, Hajji T, Trabelsi W, Bejaoui S, Chouba L, El Cafsi M, Soudani N. 2019. Effects of acute mercury exposure on fatty acid composition and oxidative stress biomarkers in Holothuria forskali body wall. Ecotox. Environ. Safe. 169, 516-522. https://doi.org/10.1016/j.ecoenv.2018.11.051 ; Silva et al., 2017Silva CO, Simões T, Novais SC, Pimparel I, Granada L, Soares AM, Barata C, Lemos MF. 2017. Fatty acid profile of the sea snail Gibbula umbilicalis as a biomarker for coastal metal pollution. Sci. Total Environ. 15 (586), 542-550. https://doi.org/10.1016/j.scitotenv.2017.02.015 ).
To further explore the impact of Hg on lipid metabolism, LOOH levels, primary breakdown products of long-chain fatty acid peroxidation, were determined. The increased LOOH levels recorded in all Hg-treated groups clearly reflected the dysfunction of the mitochondrial respiratory chain. Indeed, being rich in polyunsaturated fatty acids (PUFA) as other marine organisms (Liu et al., 2017Liu X, Wang L, Feng Z, Song X, Zhu X. 2017b. Molecular cloning and functional characterization of the fatty acid delta 6 desaturase (FAD6) gene in the sea cucumber Apostichopus japonicus. Aquac. Res. 48, 4991-5003.), sea cucumber seemed to be vulnerable to lipid peroxidation. The continued oxidation of fatty acid, and the fragmentation of peroxides to produce aldehydes may eventually lead to losses in membrane integrity by alteration of its fluidity.
5. CONCLUSIONS
⌅In the current study, the acute exposure to Hg promoted a state of oxidative stress as denoted by increased hydrogen peroxide and lipid hydroperoxide levels. The changes in the FA profiles of treated sea cucumber clearly indicated an evident toxic effect of Hg on the longitudinal muscle. The palette of acids, including SFA, MUFA and PUFA, determined in the muscle tissue, can be used in ecotoxicological research as an efficient test of the effect of mercury exposure. Furthermore, the prominent effects observed at the lowest dose, which is probably due to a hormeosis response, must be interpreted with caution since no information is available concerning the mechanistic difference between the toxicity and inhibitory effects of mercury to fatty acid pathways in holothurians. Lastly, our findings extend the understanding of the adaptive mechanisms of sea cucumbers and the usefulness of the FA analysis as a biomarker for Hg contamination.