Combined effect of salinity and cadmium in the presence of o ther metals (Zn & Cu) and GSH on lysosomal responses in an arcid blood clam

Exposure of tropical arcid blood clam Anadara granosa to sublethal concentration of Cd, Zn, Cu and GSH (Glutathione) at different salinities (16 32 g kg-') induced various changes in the activities of the lysosomal marker enzymes arylsulphatase and acid phosphatase. Changes in the lysosomal enzyme activity on exposure either to Cd or GSH was inversely related to ambient salinity. Cu inhibited enzyme activity at both salinities. Zn, however, caused no significant changes. GSH together with Cd reduced the enzyme activity at both salinities whereas the presence of Zn or Cu significantly (P<0.001) increased it. So also Cd in combination with Zn and Cu stimulated (total and free) the arylsulphatase activity significantly (P<0.001) at lower salinity, however, at higher salinity it was not affected appreciably. GSH together with Cd, Zn and Cu ameliorated the enzyme activity within the range of control. The lysosomal bound latent activity in all the cases of Cd exposure except the one with GSH at lower salinity reduced irrespective of salinity changes. Thus at lower salinity the enzyme activity was not depended upon the levels of metals in the ambient salinity. Further the salinity and speciation of these metals played a significant role in the toxicity of Cd. Moreover, GSH in the presence of Cd, Zn and Cu subsided the toxic effect of Cd in marine water Introduction Heavy metals are highly reactive with SH groups of functionally important cellular molecules including aminoacids peptides and proteins (De Bruin, 1976; George and Young, 1986). Bioaccumulation of these metals can induce S37nthesis of metallothioneins (Greorge et al., 1979; Frankenne et al., 1980; Ray and White, 1981; Kohler and Riisgard, 1982) low molecular weight cytoplasmic proteins containing 25-30% cysteine residues that normally bind 7-10 g atoms per molecule of Cd (George et al., 1979; Kohler and Riisgard, 1982). Glutathione (GSH), a tripeptide is also involved in the uptake, transport and excretion of heavy metals, like cadmium and mercury (Cherian and Vostal, 1977; Congorjiu, et al., 1978; Takafumi et al., 1988). Intracellular concretions of metals in many shellfish species have been designated as a store house and possible detoxif3dng device having function similar to those of metallothioneins and related proteins (Engel, 1983). K. Anthony and B. Patel 312 Lysosomal system is also reported to play an important role in sequestration and detoxification of metals (George et al., 1979). Thus lysosomal latency and other biochemical ad haematological parameters have been successfully monitored in evaluating toxicity interaction of trace metals and other pollutants at sub cellular levels (Eisler, 1979; Moore, 1980; Harrison, 1979). This has led to recognition of the importance of biochemical and cytochemical studies in evaluating the effect of variety of pollutants (Moore, 1980; Patel and Eapen, 1989). In view of the salinity changes and also due to increased input of domestic and industrial wastes into the coastal waters of Mumbai, it was felt desirable to evaluate the combined effect of salinity and cadmium in the presence of other metals (Zn and Cu) and a tripeptide, glutathione (GSH) on a commercially harvested benthic clam, Anadara granosa. Moreover, this information was desired since the chemicals and their toxic effect on tropical benthos is poorly understood. Work presented in this paper was therefore, designed to give a better understanding of the possible impact of cadmium and its detoxification potential on benthic ecosystem. Material and methods About 200 Anadara gronosa of 3 6 cm in shell length and 2 3 yr old were collected from intertidal mud flats along the Trombay coast (Mumbai). Extraneous matter was removed by gentle scrubbing followed by acclimation to the laboratory conditions for a period of ca. 48 h at a salinity ca 33%c and temperature 23 ± 2''C in aerated, filtered sea water. A static bioassay technique was followed. About eight to ten clams were exposed in duplicate sets in glass aquaria containing Cd (as CdCl2, 1.2 pg ml-i), Zn (as ZnSO^, 1.8 pg ml"'), Cu (as CuSO^, 0.2 pg ml-i) and GSH (10.0 pg ml"') individually and in combination at 16 and 32 g kg-^ salinity for 96 h. Concentration of chemicals for exposure were chosen on the basis of a 96 h LCgg determined separately. Control group of clams was also maintained, in duplicate, without any addition of chemicals. Each set of experiments was repeated at least five times. No freeding was made during the experimental period. The physiological behaviour such as movements of foot and shell, secretion of mucus and bleeding of test and control group was monitored visually at 0800 h intervals. Bleeding and moribund clams, if any, were removed. Digestive glands from both control and exposed clams were separated from the visceral mass by keeping in a crushed ice bath. Enzyme-arylsul-phatase (E.C.3.1.6.1) and acid phosphatase (E.C.3.1.3.2.) assays were conducted following techniques described by Patel and Patel (1985). The pooled digestive gland was homogenised in 0.25 M sucrose (pH 7.2) with 10 strokes using a teflon pestel revolving at 500 rpm. Enzymes in the homogenate were assayed after appropriate dilution with sucrose solution. Enzyme activity is expressed in terms of international units (lU), defined as the amount of enzyme required to liberate 1 p mol of product per min per g tissue. Percentage of latent (lysosomal/particle bound) arylsulphatase activity was calculated from the differences between levels Combined effect of salinity & cadmium on lysosomal responses in Anadara granosa 313 observed with (total) and without (free activity) addition of the non-ionic detergent Triton x 100. Protein levels in tissue homogenates were estimated by the standard Biuret method. Levels of Cd in dry tissue {ca. 100°C) were measured following Energy Dispersive XRF Technique (Patel and Patel, 1985). About 160 g dry tissue homogenate was irradiated with amrecium-241. X-ray spectra of Cd in the tissue samples were obtained using a Germanium low energy resolution of 500 eV at 122 Ke V. Final Cd concentration was calculated by comparison with the standard (Patel and Patel, 1985). Results Arylsulphatase Combined effect of salinity and Cd, Zn, Cu and GSH administered individually and in combination on arylsulphatase activity is given in Table 1. Enzyme activity increased on exposure to CD or GSH at lower saHnity (16 g kg'i), whereas Cu inhibited it. At higher salinity (32 g kg'^) arylsulphatase acti-vity was inhibited by Cd, Cu and GSH. Exposure to GSH alongwith Cd at lower salinity inhibited enzyme activity by 27%, whereas in the presence of Zn or Cu, the enzyme activity increased significantly (P<0.001, Table 1). Similarly GSH in the presence of TABLE 1. Anadara granosa. Effect of salinity on arylsulphatase activity in the digestive glands after 96 h exposure to CdCl^ , ZnSO^, CuSO^ and OSH. Figures in parenthesis denote % increase (+) or decrease (-) over control. Mean ± S.D. n = 6 Levels in water (Hg ml')