The aim of the present study was to ascertain whether the metallothioneins (MTs) of freshwater mussels can serve as a biomarker of environmental quality in the spontaneously polluted area. Anodonta anatina mussels from an agricultural site (A), the cooling pond of a nuclear power plant (N) and a forestry as a reference site (F) in Western Ukraine were studied during spring, summer and autumn. Concentrations of copper (Cu), zinc (Zn) and cadmium (Cd) in MTs and in the tissue, MTs protein (MT-SH) level, oxidative stress indices and apoptotic activities were determined in the digestive gland. The three sites were clear distinguished by biomarkers. In the A-group, the specimens, despite higher concentrations of Cu and Zn in the tissue, had low level of MT-SH level (in two seasons) making the metal-keeping function of MTs invalid. MTs-bound metals dropped to about 2.3% of Cu, 0.4% of Zn and 9.1% of Cd in the tissue in autumn in this polluted area. Lower level of glutathione redox-index reflected the oxidative stress in this group. The N-group displayed high levels of MT-bound Cu (corresponding to up to 57.7% of the total tissue metal) and caspase-3 activity (up to 7 times higher than in the reference F- group), indicating specific for cooling pond pollution by Cu and thermal effects. In group F, the set of markers showed the characteristic of the reference site. An extremely low redox index of glutathione (RI GSH: 0.45) was observed in the A-group in spring, and high levels of protein carbonyls and TBA-reactive substances in autumn in group N.Hence, multi-marker approach needed to provetoxicityassesments in mussel sexposed byspontaneous and mixed pollution.
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Amiard, J.C., Amiard-Triquet, C., Barka, S., Pellerin, J. and Rainbow, P.S. 2006. Metallothioneins in aquatic invertebrates: Their role in metal detoxification and their use as biomarkers. Aquat. Toxicol., 76:160202. DOİ: 10.1016/j .aquatox.2005.08.015.
Anderson, M.E. 1985Determination of glutathione and glutathione disulphide in biological samples. Met. Enzymol., 113: 548-555. doi: 10.1016/S0076-6879 (85)13073-9.
Atli, G. and Canli, M. 2007. Natural occurrence of metallothionein-like proteins in liver tissues of four fish species from the northeast Mediterranean Sea.Water Environ. Res., 79(9): 958-963. doi: 10.2175/106143007X175780.
Barienė, J. and Rybakovas, A. 2008. Cytogenetic damage in gill and gonad cells of bivalve mollusks. Ekologija, 54: 245250.
Beauchamp, C. and Fridovich, I. 1971. Superoxide dismutase: improved assay and an assay applicable to acrylamide gels. Anal. Biochem., 44(1): 276287. doi: 10.1016/0003-2697(71)90370-8.
Berthet, B., Mouneyrac, C., Amiard, J.C., Amiard-Triquet, C., Berthelot, Y., LeHen, A., Mastain, O., Rainbow, P.S. and Smith, B.D. 2003. Accumulation and solublebinding of cadmium, copper, and zinc in the polychaete Hediste diversicolor from coastal sites with different trace metal bioavailabilities. Arch. Environ. Contam. Toxicol., 45: 468478. doi: 10.1007/s00244-003-0135-0.
Berthet, B., Mouneyrac, C., Pérez, T. and Amiard-Triquet, C. 2005. Metallothionein concentration in sponges (Spongia off icinalis) as a biomarker of metal contamination. Comp. Biochem. Physiol., 141: 306 313. doi: 10.1016/j .cca.2005.07.008.
Bigot, A., Vasseur, P. and Rodius, F. 2010. SOD and CAT cDNA cloning, and expression pattern of detoxification genes in the freshwater bivalve Unio tumidus transplanted into the Moselle river. Ecotoxicology, 19: 369376. doi: 10.1007/s10646- 009-0419-x.
Bonomini, M., Dottori, S., Amoroso, A., Arduini, A. and Sirolli, V. 2004. Increased platelet phosphatidylserine exposure and caspase activation in chronic uremia. J. Thromb. Haemost., 2: 18. doi: 10.1111/j .1538- 7836.2004.00837.x.
Carreira, S., Costa, P.M., Martins, M., Lobo, J., Costa, M.H. and Caeiro, S. 2013.Ecotoxicological heterogeneity in transitional coastal habitats assessed through the integration of biomarkers and sediment-contamination profiles: A case study using a commercial clam. Arch. Environ. Contam. Toxicol., 64(1): 97 109. doi: 10.1007/s00244-012-9812-1.
Cavaletto, M., Ghezzi, A., Burlando, B., Evangelisti, V., Ceratto, N. and Viarengo, A. 2002. Effect of hydrogen peroxide on antioxidant enzymes and metallothionein level in the digestive gland of Mytilus gallop rovincialis. Comp. Biochem. Physiol., 131: 447455. doi: 10.1016/S1532-0456(02)00030-3.
Ciocan, C.M. and Rotchell, J.M. 2004. Cadmium induction of metallothionein isoforms in juvenile and adult mussel (Mytilus edulis). Environ. Sci. Technol., 38: 10731078. doi: 10.1021/es030110g.
Cravo, A., Lopes, B., Serafim, A., Company, R., Barreira, L., Gomes, T. and Bebianno, M.J. 2013. Spatial and seasonal biomarker responses in the clam Ruditapes decussatus. Biomarkers, 18(1): 3043. doi: 10.3109/ 1354750X.2012.730549.
Da Ros, L., Meneghetti, F. and Nasci, C. 2002. Field application of lysosomal destabilisation indices in the mussel Mytilus gallop rovincialis: biomonitoring and transplantation in the Lagoon of Venice (north-east Italy). Mar. Environ. Res., 54(3-5): 817822. doi: 10.1016/S0141-1136(02)00123-X.
Dallinger, R., Berger, B., Gruber, C., Hunziker, P., and Sturzenbaum, S. 2000. Metallothioneins in terrestrial invertebrates: structural aspects, biological significance and implications for their use as biomarkers. Cell Mol. Biol., 46: 331346.
Datkhile, K.D., Mukhopadhyaya, R., Dongre, T.K. and Nathl, B.B. 2009. Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation. Comp. Biochem. Physiol., 149: 500550. doi: 10.1016/j .cbpc.2008.11.003.
Dondero, F., Piacentini, L., Banni, M., Rebelo, M., Burlando, B. and Viarengo, A. 2005.Quantitative PCR analysis of two molluscan metallothionein genes unveils differential expression and regulation. Gene., 345: 259270. doi: 10.1016/j .gene.2004.11.031.
Doyen, P., Bigot, A., Vasseur, P. and Rodius, F. 2008. Molecular cloning and expression study of pi-class glutathione S-transferase (pi-GST) and selenium- dependent glutathione peroxidase (Se-GPx) transcripts in the freshwater bivalve Dreissena polymorpha. Comp. Biochem. Physiol., 147: 6977. doi: 10.1016/j .cbpc.2007.08.002.
Falfushynska, H.I., Gnatyshyna, L.L., Golubev, A.P. and Stoliar, O.B. 2012. Main partitioning criteria for the characterization of the health status in the freshwater mussels Anodonta cygnea from spontaneously polluted area in Western Ukraine. Environ. Toxicol., 27(8): 485494. doi: 10.1002/tox.20663.
Falfushynska, H.I., Delahaut, L., Stolyar, O.B., Geffard, A. and Biagianti-Risbourg, S. 2009. Multi-biomarkers approach in different organs of Anodonta cygnea from the Dnister Basin (Ukraine). Arch. Environ. Contam. Toxicol., 57(1): 8695. doi: 10.1007/s00244-008- 9234-2.
Falfushynska, H.I., Gnatyshyna, L.L., Farkas, A., Vehovszky, A., Gyori, J. and Stoliar, O.B. 2010. Vulnerability of biomarkers in the indigenous mollusc Anodonta cygnea to spontaneous pollution in a transition country. Chemosphere, 81(10): 13421351. doi: 10.1016/j .chemosphere.2010.08.016.
Falfushynska, H.I., Gnatyshyna, L.L, Osadchuk, O.Yu, Farkas, A., Vehovszky, A., Carpenter, D.O., Gyori, J. and Stoliar, O.B. 2014. Diversity of the molecular responses to separate wastewater effluents in freshwater mussels. Comp. Biochem. Physiol., 164: 5158. doi: 10.1016/j .cbpc.2014.04.007.
Fetisov, A.N., Rubanovich, A.V., Slipchenko, T.S. and Shevchenko, V.A. 1992. The structure of Dreissena polymorpha populations from basins adjacent to the Chernobyl atomic power station. Sci. Total Environ., 112: 115 124. doi: 10.1016/0048-9697(92)90242-K.
Geffard, A., Amiard-Triquet, C. and Amiard, J.C. 2005. Do seasonal changes affect metallothionein induction by metals in mussels, Mytilusedulis? Ecotoxicol. Environ. Saf., 61(2): 209-220. doi: 10.1016/j .ecoenv. 2005.01.004.
Giguère, A., Couillard, Y., Campbell, P.G., Perceval, O., Hare, L., Pinel-Alloul, B. and Pellerin, J. 2003. Steady-state distribution of metals among metallothionein and other cytosolic ligands and links to cytotoxicity in bivalves living along a polymetallic gradient. Aquat. Toxicol., 64(2): 185-200. doi: 10.1016/S0166-445X(03)00052-3.
Griffith, O.W. 1980. Determination of Glutathione and Glutathione Disulfide Using Glutathione Reductase and 2-Vinylpyridine. Anal. Biochem., 106: 207212. doi: 10.1016/0003-2697(80)90139-6.
Gruber, C., Sturzenbaum, S., Gehrig, P., Sack, R., Hunziker, P., Berger, B. and Dallinger, R. 2000.Isolation and characterization of a self-sufficient one-domain protein: (Cd)-metallothionein from Eisenia f oetida. Eur. J. Biochem., 267: 573582. doi: 10.1046/j .1432-1327.2000.01035.x.
Guerlet, E., Ledy, K., Meyer, A. and Giambérini, L. 2007. Towards a validation of a cellular biomarker suite in native and transplanted zebra mussels: a 2-year integrative field study of seasonal and pollution- induced variations. Aquat. Toxicol., 81(4): 377388. doi: 10.1016/j .aquatox.2006.12.016.
Hagger, J.A., Jones, M.B., Leonard, D.R.P., Owen, R. and Galloway, T.S. 2006. Biomarkers and integrated environmental risk assessment: Are there more questions than answers? Integr. Environ. Assess. Manag., 2(4): 321329. doi: 10.1897/1551-3793 (2006)2[312:BAIERA]2.0.CO;2.
Hamza-Chaffai, A., Amiard, J.C., Pellerin, J., Joux, L. and Berthet, B. 2000.The potential use of metallothionein in the clam Ruditapes decussatus as a biomarker of in situ metal exposure. Comp. Biochem. Physiol., 127: 185-197. doi: 10.1016/S0742-8413(00)00147-X.
Jenny, M.J., Warr, G.W., Ringwood, A.H., Baltzegar, D.A. and Chapman, R.W. 2006. Regulation of metallothionein genes in the American oyster (Crassostrea virginica): Ontogeny and differential expression in response to different stressor. Gene, 379: 156 165. doi: 10.1016/j .gene.2006.05.004.
Kagi, J.H.R. and Schaffer, A. 1988.Biochemistry of metallothionein. Biochemistry, 27: 85098515. doi: 10.1021/bi00423a001.
Kelly, E., Duncan, R., Ngu, T.T., Chan, J., Salgado, M.T. and Merrifield, M.E. 2006.Peptide Folding, Metal- Binding Mechanisms, and Binding Site Structures in Metallothioneins. Exp. Biol. Med., 231: 1488-1499. doi: 10.1016/j .jinorgbio.2006.09.005.
Lilja, K., Prevodnik, A., Gardeström, J., Elfwing, T., Tedengren, M. and Bollner, T. 2008. Regional differences in mRNA responses in blue mussels within the Baltic proper. Comp. Biochem. Physiol., 148: 101 106. doi: 10.1016/j .cbpc.2008.04.001.
Lowry, O.H., Rosebrough, H.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem., 193: 265275.
Manduzio, H., Monsinjon, T., Galap, C., Leboulenger, F. and Rocher, B. 2004. Seasonal variations in antioxidant defences in blue mussels Mytilus edulis collected from a polluted area: major contributions in gills of an inducible isoform of Cu/Zn-superoxide dismutase and of glutathione S-transferase. Aquat. Toxicol., 70: 8393. doi: 10.1016/j .aquatox.2004.07. 003.
Mead, R., Curnow, R.N. and Hasted, A.M. 2002. Statistical Methods in Agriculture and Experimental Biology, Second Edition. Chapman and Hall, CRC., 488 pp.
Nasci, C., Nesto, N., Monteduro, R.A. and Da Ros, L. 2002. Field application of biochemical markers and a physiological index in the mussel, Mytilus gallop rovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy). Mar Environ. Res., 54(3-5): 811816. doi:10.1016/S0141- 1136(02)00122-8.
Nielson, K.B. and Winge, D.R. 1984. Preferential binding of copper to the beta domain of metallothionein. J Biol. Chem., 259: 49414946.
Ohkawa, H., Onishi, N. and Yagi, K. 1979. Assay for lipid peroxidation in animal tissue by thiobarbituric acid reaction. Anal. Biochem., 95: 351358. doi: 10.1016/0003-2697(79)90738-3.
Piano, A., Valbonesi, P. and Fabbri, E. 2004. Expression of cytoprotective proteins, heat shock protein 70 and metallothioneins, in tissues of Ostrea edulis exposed to heat and heavy metals. Cell. Stress. Chaperones, 9(2): 134142. doi: 10.1379/483.1.
Regoli, F. and Principato, G. 1995. Glutathione, glutathione -dependent and antioxidant enzymes in mussel, Mytilus gallop rovincialis, exposed to metals under field and laboratory conditions: implications for the use of biochemical biomarkers. Aquat. Toxicol., 31(2): 143164. doi:10.1016/0166-445X(94)00064- W.
Reznick, A.Z. and Packer, L. 1994. Oxidative damage to proteins: spectrophotometric method for carbonyl assay. Met. Enzymol., 233: 357363.
Robillard, S., Beauchamp, G. and Laulier, M. 2003. The role of abiotic factors and pesticide levels on enzymatic activity in the freshwater mussel Anodontacygnea at three different exposure sites. Comp. Biochem. Physiol., 135(1): 4959. doi:10.1016/S1532-0456(03) 00049-8.
Roesijadi, G. and Fowler, B. 1991. Purification of invertebrate metallothioneins. Met. Enzymol., 205: 263273.
Romero, A., Este´vez-Calvar, N., Dios, S., Figueras, A. and Novoa, B. 2011. New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus gallop rovincialis. PLoS ONE, 6:1 15. doi: 10.1371/journal.pone.0017003.
Rzymski, P., Niedzielski, P., Klimaszyk, P. and Poniedzialek, B. 2014. Bioaccumulation of selected metals in bivalves (Unionidae) and Phragmites australis inhabiting a municipal water reservoir. Environ. Monit. Assess., 185(5): 31993212. doi: 10.1007/s10661-013-3610-8.
Silayeva, A.A. and Protasov, A.A. 2005. Composition and structure of zoobenthos of Stir River in the zone of Rovenska AES and the evaluation of its effect on the bottom groups. Hydrobiol. J., 41: 2545.
Van Campenhout, K., Infante, H.G., Adams, F. and Blust, R. 2004. Induction and binding of Cd, Cu and Zn to metallothionein in carp (Cyp rinus carp io) using HPLC-ICP-TOFMS. Toxicol. Sci., 80(2): 276287. doi:10.1093/toxsci/kfh149.
Viarengo, A., Lowe, D., Bolognesi, C., Fabbri, E. and Koehler, A. 2007. The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms. Comp. Biochem. Physiol., 146: 281300. doi: 10.1016/j .cbpc.2007.04.011.
Viarengo, A., Ponzano, E., Dondero, F. and Fabbri, R. 1997. A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs. Mar. Environ. Res., 44: 6984. doi:10.1016/S0141- 1136(96)00103-1.
Wang, J., Wei, Y., Li, X., Cao, H., Xu, M. and Dai, J. 2007. The identification of heat shock protein genes in goldfish (Carassius auratus) and their expression in a complex environment in Gaobeidian Lake, Beijing, China. Comp. Biochem. Physiol., 145: 350362. doi:10.1016/j .cbpc.2007.01.018.
Wilhelmsen, T.W., Olsvik, P.A., Hansen, B.H. and