Molluscan Research 32(1): 16–20 http://www.mapress.com/mr/
ISSN 1323-5818 Magnolia Press
Heavy metals accumulation and antioxidant defence system in Helix pomatia (Pulmonata: Helicidae) ANNA NOWAKOWSKA*1, TOMASZ ŁACIAK2 & MICHAŁ CAPUTA1 1
Department of Animal Physiology, Institute of General and Molecular Biology, N. Copernicus University, Toruń, Poland 2 Department of Zoology of Vertebrates and Human Biology, Institute of Biology, Pedagogical University of Krakow, Poland *Corresponding author: Email:
[email protected]
Abstract We present a study on the association between accumulation of heavy metals and activities of antioxidant enzymes, concentrations of glutathione (GSH) and thiobarbituric acid-reactive substances (TBARS) as products of lipid peroxidation in selected tissues of Helix pomatia living in a natural habitat. Hepatopancreatic accumulation of Zn, Cd and Mg was many times higher than that in the kidney and foot, and was connected to an extremely high concentration of TBARS and activity of glutathione transferase. Moreover, the organ concentration of GSH was inversely proportional to TBARS. Activity of glutathione reductase was unaffected by organ type. In contrast to the metals mentioned above, concentrations of Pb, Cu and Ni were extremely high in the kidney and shell and low in the hepatopancreas. The results confirm the crucial role of the hepatopancreas and kidney in the detoxification process and point to the shell as a ‘sink’ for some heavy metals. Key words: lipid peroxidation, oxidative stress, digestive gland, foot, kidney, shell
Introduction Land snails are known as accumulators of heavy metals because of their limited ability to excrete the metals (Dallinger 1993). Toxic effects of the metals disturb various cellular processes, especially the functioning of lipid membranes (Pinto et al. 2003; Valko et al. 2005). Heavy metals, accumulated in the cells, induce overproduction of reactive oxygen species (ROS) in biological systems, causing not only disturbances in reproduction and development, but also enhancing susceptibility to diseases as a result of immunosupression or inhibition of immune system development (Carey and Bryant 1995; Carey et al. 1999; Alford and Richards 1999; Loumbourdis et al. 1999). Reactive oxygen species are continuously generated in cellular processes but the state when their generation overrides their decomposition is called oxidative stress. In land snails the main cause of the stress is arousal from both winter and summer torpor (Nowakowska et al. 2009a; Nowakowska et al. 2009b). Defence against oxidative stress consists of enzymatic and non-enzymatic components. In stylommatophoran land snails, activities of the antioxidant enzymes change in response to natural environmental stressors and/or physiological processes (Ramos-Vasconcelos and HermesLima 2003; Ramos-Vasconcelos et al. 2005; Nowakowska et al. 2009a; Nowakowska et al. 2009b; Nowakowska et al. 2010). The snails are normally able to maintain a balance between production and neutralization of ROS. However, exposure to heavy metals can disturb the antioxidant defence system, resulting in oxidative stress. Anthropogenic sources of the metals are industrial and agricultural activities, traffic, smelting, and combustion of fossil fuels (Pinto et al. 2003). The regulation of the activity of antioxidant enzymes and the concentration of non-enzymatic antioxidants seems to be the main biochemical mechanism supporting normal functioning 16
of the organism during the accumulation of heavy metals (Radwan et al. 2010a; Radwan et al. 2010b). Helix pomatia Linnaeus, 1758 is an important snail species, not only from the scientific point of view (Dallinger and Wieser 1984; Manzl et al. 2004; Nowakowska et al. 2009a; Nowakowska et al. 2009b; Nowakowska et al. 2010), but it also has economic value in Europe. This study evaluates profiles of the accumulation of heavy metals and the response of the antioxidant defence systems in Helix pomatia taken from natural habitat. Another reason to use this species in the present investigation was our many years’ practice in testing the defence system of the species. The specific aim of the present study was to examine the modulation of activities of antioxidant enzymes in response to organ-specific accumulation of the heavy metals. Concentrations of zinc, nickel, copper, cadmium, iron, magnesium and lead were measured in the hepatopancreas, kidney, foot and shell. Activities of antioxidant enzymes such as catalase (CAT), total glutathione preoxidase (GPX), selenium-dependent peroxidase (se-GPX), glutathione reductase (GR) and glutathione s-transferase (GST) and concentration of reduced glutathione (GSH), as a nonenzymatic antioxidant, in the hepatopancreas, kidney and foot were also evaluated. Moreover, to estimate oxidative damage to the snails’ organs, concentration of thiobarbituric acid-reactive substances (TBARS), as products of lipid peroxidation, was recorded.
Materials and methods A total of 12 adult (all with well-defined lip on the shell) specimens of the Helix pomatia, each weighing approximately 24 g, were used in this study. Active snails were collected in summer from a natural habitat near Toruń (central Poland 53°02’N, 18°35’E) (permission of the Polish
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HEAVY METALS AND ANTIOXIDANT DEFENCE SYSTEM IN HELIX
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Ministry of Environmental Protection No. WsiR.II.KLD6631-209/05). After collection they were divided into two groups. Members of one group (n=6) were tested for antioxidant defence and lipid peroxidation and those of the second group (n=6) were used to examine concentrations of heavy metals. The hepatopancreas (i.e., the digestive gland), kidney and part of the foot were used post mortem for determination of both concentrations of heavy metals and activities of antioxidant enzymes and concentration of GSH and TBARS. For determination of the concentrations of the metals, a part of the shell of each specimen (within the second group) was also taken. The samples of the first group were used for assessment of protein content, catalase activity, reduced glutathione concentration, activities of the glutathionerelated enzymes and TBARS concentration. The organ samples were homogenized in potassium phosphate buffer at pH 7.4 using a Potter homogenizer with a teflon piston at 200 rotations per minute. After being centrifuged for 10 min at 12 000 g the supernatants were collected in Eppendorf tubes and were stored until they were used for the biochemical analysis. Samples of the second group were dried for measurement of the metals concentration (see below). The methods used to determine the activities of antioxidant enzymes, concentration of reduced glutathione and concentration of TBARS were described in detail in our previous papers (Nowakowska et al. 2009a, Nowakowska et al. 2009b, Nowakowska et al. 2010). Briefly, catalase activity was determined by decomposition of 54 mM H2O2 at 240 nm (Bartosz 2004). Activity of the enzyme was expressed as U/g protein. The unit of catalase is defined as the reduction of 1μmol/l-1 of the peroxide per minute. Glutathione peroxidase activity was determined as previously described by Chiu et al. (1976), using cumene hydroperoxide for oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm. Selenium-dependent glutathione peroxidase activity was determined according to the method described by Pagila and Valentine (1967), using H2O2 as a substrate. Glutathione reductase activity was determined by the method of Szczeklik (1974) by following the consumption of NADPH per minute at 340 nm by oxidised glutathione (GSSG). Glutathione transferase activity was determined by following the conjugation of GSH with 1chloro-2,4 dinitrobenzene (CDNB) in phosphate buffer (pH 6.5) at 340 nm (Habig et al. 1974). Reduced glutathione concentration was assayed using the method described by Ellman (1959), based upon the reaction of 5,5-dithio-bis (2nitrobenzoic) acid (DTNB) with sulfhydryl groups of GSH at 412 nm. GSH concentration was expressed in μmols per gram of wet mass. Thiobarbituric acid-reactive substances were assayed using the methods of Rice-Evans et al. (1991). TBARS concentration was expressed in mmols/g of wet mass. All assays were performed using a Shimadzu spectrophotometer (UV-1601). To obtain the concentrations of heavy metals, the samples, dried at 105°C, were digested in HNO3, and after
complete dissolution were diluted with water and neutralised pyrrolidine with saturated NaHCO3. Ammonium ditiocarbaminate was added to the samples and they were shaken for 15 min. Then 2 ml samples were collected for measurement of heavy metal concentrations. The heavy metal content was estimated by AAS a BUCK 200A spectrophotometer with air-acetylene flame and appropriate hollow cathode lamps. Analytical efficiency of the measurements was checked using certified reference material (Bovine Liver BCR 185R) and analysed in the same way as the samples. The recovery of the metals in this method ranges from 95 to 105%. Each measurement was repeated three times and the results were averaged. Moreover, standard curves were determined using standarized samples (Atomic Absorption Standard; BUCK Scientific, USA). Each enzyme activity was calculated considering the total protein content of the organ extracts. The protein concentration was estimated by Folin-Phenol methods described by Lowry et al. (1951) using bovine serum albumin (Sigma Chemical, Steinheim, Germany) as a standard. Chemicals used included cumene hydroperoxide, reduced glutathione (GSH), oxidised glutathione (GSSG), 5,5-dithio-bis (2-nitrobenzoic) acid (DTNB), 1-chloro-2,4dinitrobenzene (CDNB), etylenediaminetetraacetic acid (EDTA). They were purchased from Sigma Chemical (Steinheim, Germany). Trichloroacetic acid (TCA), tris(hydroxymrthyl)aminomethane (TRIS), disodium versenate dihydrate were purchased from Polskie Odczynniki Chemiczne (Gliwice, Poland). All other reagents were of analytic grade. CDNB was dissolved in ethanol and DTNB was diluted in methanol. The remaining solutions were dissolved in deionized water. Data are presented as mean value ± standard error (SE). Analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to analyse activity of the glutathionerelated enzymes, activity of catalase and concentrations of reduced glutathione and TBARS as well. ANOVA was also applied to analyse the concentration of heavy metals in the organs and in the shell. The threshold of statistical significance was P
