Matkovics, Béla

Nour, Abd El Aziz Mousa

Rady, Amal A.

Saber, Nadia

English

Saber Nadia

Rady Amal A.

Matkovics Béla

Nour Abd El Aziz Mousa

University of Szeged

A C T A BIOL SZEGED. 3 8 . p p . 1 1 - 1 8 ( 1 9 9 2 ) METALS EFFECT ON FISH TISSUES n . T H E EFFECT OF CHRONIC ZINC A N D CADMIUM TREATMENT ON Y O U N G TILAPIA TISSUE ENZYMES A N D LIPID PEROXIDATION NADIA SABER1 . AMAL A . R A D Y 2 , B . MATKOVICS3 a n d A . M . NOUR 4 'National Institute of Oceanography and Fisheries, Anfoushy, Alexandria, Egypt 2Department of Biochemistry, Faculty of Veterinary Medicine, Alexandria University, Alexandria, Egypt 'Biological isotope Laboratory, József Attila University, H-6701 Szeged. P. O. B. 539. Hungary (Correspondence and reprint request) 'Department of Animal production. Faculty of Agriculture, Alexandria University. Alexandria. Egypt (Received: May 12, 1992) Abstract Metals effect on liver and muscle tissues of young Tilapia (Oreochromis niloticus) were studied under separate and simultaneous effects of ZnCI, and CdCI, during 120 days, with special regards to the respective ion containing active site of the enzymes. Besides the enzymes, changes in lipid peroxidation value in both tissues were also studied. Key words: Young Tilapia. chronic treatment, zinc chloride and cadmium chloride, tissue enzyme activity, lipid peroxidation. Introduction Due to industrial development and the extensive use of chemicals in agricul-ture, heavy metals are widely distributed in aquatic systems and can affect fish populations, reducing their growth, reproduction and causing significant mortality ( V I N I K O U R et al. , 1 9 8 0 ) . Similarities in chemical reactivity of cadmium (Cd) and zinc (Zn) lead to similar metabolic pathways in biological systems. Whereas . Zn is an important essential element, acting as co-factor for many enzymes, neces-sary for D N A synthesis and others. Cd is best known for its toxicity and metabolic antagonism of Zn and other essential elements. Anemia , bone de-mineralizaton and kidney damage are principal adverse effects of Cd ingested in moderate amount , its higher levels can lead to death (VALLEE et al. , 1 9 7 2 ) . In general Cd can inhibit several key enzymes metabolic processes and it can reduce their ability in osmoregulation. It has been suggested that the environmental contamination of Cd found even in low concentration in the nature , interferes 12 N . SABER. A . A . R A D Y . B . MATXOVICS. a n d A . M . N O U R with the basal metabolism of birds and mammals (SUZUKI et al. 1990a. b. c; M E N E N D E R - B O T E T et al. 1991). The aim of the present work was to study the effects of different concentrat ions of cadmium and zinc (both used in plant protection) on the activity of metabolic enzymes, protein values and level of the lipid peroxidation of tilapia (Oreochromis niloticus). Materials and Methods Fish: Two separate experiments were carried on Tilapia IOreochromis nilolicus) fingerlings weighing about 4—5 g/fish in average, obtained from the Marjout Fish Farming Company, Alexan-dria Governorate . Egypt. They were kept in glass jars (105 litre capacity) at temperature of 28 °C and fed for seven days on basic diet. At the start of the experiment healthy fish were weighed and distributed in the experimental jars. Fourteen jars of 105 litres capacity each were used in the experiment and filled with tap water stored for two days before use. Water was changed every three days (about third part changed every day). Water temperature was thermostatically controlled at 28 °C± 1 °C. The formulated basic diet consisted of fish meal, soybean meal, yellow corn and bone meal, fish oil, vitamins and minerals, and was used as control diet RADY et al. , (1992) without the addition of stock sol. of A or B. Experimental diets: Stock solutions of trace metals: cadmium chloride (CdCl_. and zinc chloride (Zn Cl2) of 100 ppm concentrations were prepared. They were prepared by dissolving 0.203 g of CdCl, hydrate with 5 ml of conc. H N O j in 1 litre of distilled waater (stock solution A) and one gram of ZnCl, and 20 ml conc. HC1 in one litre of distilled water (stock solution B) suggested by CHAPMAN et al (1961) . The experimental diets were prepared by the addition of 5. 10 and 15 ml from stock solution A per kg of basic diet to give diet 5, 10 and 15 ppm of cadmium, respectively, and 10. 20 and 30 ml of stock solution B added 1 kg of basic diet to give diets of 10. 20 and 30 ppm of zinc concentration. Mixture of CdCl, (solution A) and ZnCI, (solution B) were prepared by adding 5,10 and 15 ml of solution A and 30, 20 and 10 ml of solution B respectively. Feeding Strategy: Fish in every jar were fed on experimental diet (two jars) for each diet at a rate of 8 per cent on the basis of live body weight, first week, then feeding level was reduced to 6 per cent from the third week on . and to 4 per cent from the 7th week. (Daily feed was divided into 100 equal portions and offered for each jar and adjusted every 7-day intervals to the fresh body weight.) The glass jars were cleaned daily t o prevent accumulation of faeces. Specimens of different treatments were collected and taken alive to the laboratory in aerated plastic bags. The apparent examination showed that all fishes were somatically healthy and parasite-free. Fish sampling: At the end of the experiments five fish from each jar were taken sacrificed by vertebral rupture , then liver and parts f rom the muscle tissues were rapidly removed. A part of each tissue were weighted and homogenized with 0.64 per cent sodium chloride for biochemical analysis. Biochemical assays: Total protein was determined by the method of LOWRY et al. (1951), for preparation of calibration curve bovine serum albumin was used. Lipid peroxidation (LP): Malondialdehyde (MDA) was used as an indicator for lipid peroxides. It was determined by the method described by PLACER et al (1966). Calibration curve was prepared by using malondialdehyde diethyl acetate (Merck. Germany). Alkaline and acid phosphatase (AlPh-as; E C 3.1.3.1) (AcPh-ase: E C 3.1.3.1) activities were measured activities by the method of BERGMEYER (1974). Phenol released through enzyme hydrolysis Metals effect on fish tissues II. 13 from phenyl phosphate was determined under defined conditions, colorimetrically at 400 nm. Time and p H were also measured. Lactate dehydrogenase (LDH: E C 1.1.1.27) activity was measured also colorimetrically by the method of ANON (1971). The method is based on reduction of pyruvate by incubation with the enzyme in the presence of N A D H . The reaction was arrested by adding dinitrophenyl hydrazine to the solution which reacts wiht the remaining pyruvate, the colour produced was measured at 510 nm. Glutathione S-transferase (GSH-S-Tr-ase; E C 2.51.18) enzyme activity expressed as (imol of 4-chloro-l ,3 dini trobenzene (CDNB) conjugated /minute/ mg protein according to the method of VESSEY et a l . ( 1 9 8 4 ) . Results In Fig. 1 it can be seen that AlPh-ase activity of liver tissues is increasing steadily af ter the effect of ZnCl2 , while it is gradually decreasing affected by CdCl2 , both in the function of concentrat ion. Combination of the two metals, however, do not show any characteristic effect . AcPh-ase activities are elevated by increased ZnCl2 concentrations, while activating effect of CdCl2 is decreasing along with its concentrat ions increases. The same characteristic is demonstra ted by the simultaneous effects of the two metals, etc. of CdCl2 is demonstrated in Figure 2, which is prevailing, though to a lesser extent upon the treatment with the two metals. Effect of ZnCl2 and CdCl2 , respectively and in combination on GSH-S-Tr-ase activity can also be seen in Fig. 2. Here , Zn has an inhibitory effect , as well as that of the higher concentrations of CdCl2 . Liver parenchyma LP is decreased by ZnCl2 , while increased by CdCl2 in the function of the concentration of the latter. Changes in the activities of the same enzymes in muscle tissues are shown in Figs 3 and 4. Here , striking are the low enzyme activities of muscle-tissues and that all enzyme activities were increased at low- while inhibited at higher concentration of CdCl2. No noticeable is the LP increasing effect of the highest concentrat ion of CdCl2 in muscle tissues. The results in Table 1 showed that most of Cd2+ an Zn more accumulated mainly, in the liver. Increasing the level of Cd2* and Zn in the diet significantly increased its accumulation in liver and muscle. Zn supplementat ion greatly reduced Cd2 4 in the tissues of tilapia. Discussion Cadmium occurs widely in the nature in close association with Zn . Cadmium as a human pollutant (Steel industry, waste incineration, volcanic action, Zn production etc. . . ) continuously added to soil, water and air. The main feature of Cd2 + toxicity are (i) accumulation in soft tissues (liver and kidney) (ii) inter 16 N . SABER. A . A . R A D Y . B . MATXOVICS. a n d A . M . NoUR Alkal -Ph-ase pmol/mg prot. 4 0 -3.0 2 . 0 -1 . 0 -¿1 i l l A rfl r C 1 2 3 C 1 2 3 C 1 2 3 Acid -Ph- ase ĵmol I mg prot. 6 . 0 V0-2 0 - •fl m i l r 4i C 1 2 3 C 1 2 3 C 1 2 3 1. Effects of metal treatments on liver tissue enzyme activities, first row of columns demonstrate the effect of 10. 20 and 30 ppm ZnCl, on AlPh-ase and AcPh-ase activities in tilapia liver compared with the control. The second row of columns shows the influence of CdCl ; of 5. 10 and 15 ppm. respectively, on the enzymes. The third row of columns illustrate the simultaneous effect of ZnCI, and CdCl, (1 = 3 0 ppm ZnCl ,+5 ppm CdCl,: 2 = 20 ppm ZnCl ,+ 10 ppm CdCl, : 3 = 1 0 ppm ZnCI,+ 15 ppm CdCl,: 3 = 1 0 ppm ZnCI :+15 ppm CdCl,) treatment on enzyme activities. •and** = p 0.01 and P 0.001 significances, respectively. Metals effect on fish tissues II. 15 LDH pmol Img prot 0 20 016 012 006 004 QSH-S-tronstergse pmol/min/mg prot 020 I 016 012 008 004 IP nmpl MQA mg prot 100 80 6 0 40 20-] T r f l r f j c d " C 1 2 3 C 1 2 3 C 1 2 3 C 1 2 3 C 1 2 3 C 1 2 3 C 1 2 3 C 1 2 3 C 1 2 3 Fig. 2. Sums up the effects of t reatments listed in Fig. 1 on L D H and GSH-S-Tr-ase activities and on LP in liver homogenates. action with other divalent cations (mainly with Zn) (iii) Long half-live time (iv) Very slow elimination. Its metabolism and transport is bound to metallothionein. As to follow up our eaarlier investigatins with carp (RADY et al. 1988) in the present work it was studied how ZnCL and CdCL, respectively, at three different concentrations, and in combination at various defined concentrations, affect liver and muscle biochemical parameters of young Tilapia. It could be observed that upon the t reatment with the two metals, ei ther Table 1. The distribution of cadmium and zinc in liver and muscle of tilapia IOrochromis nilolicus) Hg/gm w. t. Treatment Cadmium Zinc Muscle Liver Muscle Liver Control - - 0.260 2.43 CdCl2 mg/kg feed 5 0.245 13.28 0.245 2.320 10 0.265 14.18 0.135 1.820 15 0.318 14.61 0.125 1.500 ZnCI; mg/kg feed 10 — — 0.365 6.070 20 — — 0.790 7.120 30 - - 1.180 9.082 C d C k Z n C L mg/kg feed 5:30 0.225 10.17 1.250 6.370 10:20 0.240 12.70 0.813 6.330 15:10 0.312 13.57 0.409 5.250 16 N. SABER. A. A . RADY. B. MATKOVICS. and A. M. NOUR Alka t -Ph-ase pmol / mg prot. 0 . 6 -0 V 0 2 -C 1 2 3 Acid-Ph-ase ¿jmol/mg prot. 20-1.5-1.0-0.5 r f * » » 4-T-H C 1 2 3 •ft Ml C 1 2 3 fell C 1 2 3 C 1 2 3 C 1 2 3 Fig. 3. Effects of t reatments detailed in Figure 1 on muscle tissue phosphatases activities. separately or together, the effect of CdCl2 dominated , which is in line with earlier papers reporting that Cd ion is able to ousting Zn f rom several important loci of i ts e f f e c t (SUZUKI e t a l . , 1990a; HELLAWELL, 1986 a n d HEATH, 1987). In additin to lead to significantly enzyme inhibition of white muscle, due to its accumulation increase oxidation of molecular oxygen to superoxide radical, this reaction would act as source of H 2 0 2 , which inicitiates polymerisation of specific membrane protein. The present series of investigations might be important f rom the fact that the enzymes studied contain Zn atom in their active centres. Metals effect on fish tissues II. 17 I D H >jmol I rriQ prot 016 0 1 2 -0 0 8 0.0V C 1 2 3 r 1 G S H - S - t r o m f t r m t pmol/min / mg prot 016 012 008 00V C 1 2 3 C 1 2 3 C 1 2 3 C 1 2 3 LP nmol HQA rag prot U0 30 2 0 10 C 1 2 3 M , M + L C 1 2 3 f M * 1 C 1 2 3 C 1 2 3 Fig. 4. Changes of L D H and GSH-S-Tr-ase activities and LP values in muscle tissue homogenates upon separate and simultaneous t rea tments with Zn and Cd. References ANON, L. (1971): Photometr ic determinat ion of lactic dehydrogenase in blood serum. — Z . klin. Chem. klin. Biochem. a8, 658 - 6 6 2 . BERGMEYER. U. (1974): Methoden der enzymatischen Analyse. Verlag Chemie. Weinheim. CHAPMAN, H. D . and PRATT, P. F. (1961): Methods of analysis for soils, plants and water. Agricultural Publisher, Berkeley C A . U S A . HEATH. A. G . (1987). Water pollution and fish physiology. C R C Press. Boca Ra tom. USA. HELLAWELL, J. M. (1986): Biological indicators of freshwater pollution and environmental manage-ment . 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(1984): Differential activation and inhibition of different forms of rat liver glutathione S-transferase by the herbicides 2.4-dichlorophenoxyacetat (2.4-D) and 2.4.5-trichloro-phenoxyacetat (2.4.5-T). - Toxicol. Appl. Pharmacol. 73. 492 - 499. VINIKOUR. W . S . . GOLDSTEIM. R . M . a n d ANDERSON. R . V . ( 1 9 8 0 ) . B i o c o n c e n n t e r a t i o n p a t t e r n s o f zinc, copper, cadmium and lead in selected fish species from the Fox river, illinois. Bull. Environm. Contam. Toxicol. 24. 727—734. 

Metals effect on fish tissues II. The effect of chronic zinc and cadmium treatment on young Tilapia tissue enzymes and lipid peroxidation

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Metals effect on fish tissues II. The effect of chronic zinc and cadmium treatment on young Tilapia tissue enzymes and lipid peroxidation

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