THE IN VITRO EFFECTS OF HUMIC COMPOUNDS ON FISH CATALASE
VARANKA, ZS., SZEGLETES, T., SZEGLETES, ZS., KOTORMÁN, M., NEMCSÓK, J. and ÁBRAHÁM, M.
Department of Biochemistry, József Attila University, Szeged, Hungary, Institute of Biophysics, Biological Research Center, Szeged, Hungary
Earlier in vivo studies showed that humic compounds may induce oxidative stress in freshwater fish: the rate of lipid peroxidation, the activity of catalase, etc. increased in the tissues of common carp after intraperitoneal treatment (10 mg/kg). It's well known that phenolics derived from the plant cell wall can function both in plants and animals as antioxidants, e.g. free radical scavengers or enzyme inducers, but they may act as enzyme inhibitors too. The main aim of this study was to compare the in vitro effects of a series of phenolics with similar structures on fish catalase, a major antioxidant enzyme, and to establish the possible toxicity of phenolcarboxy acids.
These phenolic compounds dose-dependently inhibited carp catalase in vitro and the IC50 values were compared to determine the structure-inhibitory properties. It was concluded that the number of substituents in the phenolic compound molecule is not the primary factor in the inhibitory effect of phenolics, as was considered earlier. The assay revealed that the configuration of the substituents plays the key role in the formation of the inhibitory effect, and the number of substituents has only a secondary role. Tannic acid was found to be a very toxic substance relative to the other examined phenolics.
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A FAHÉJSAV ÉS A TANNINSAV IN VIVO HATÁSÁNAK ÖSSZEHASONLITÁSA PONTY ANTIOXIDÁNS VÉDEKEZÖ-RENDSZERÉRE
VARANKA Zsolt, KOTORMÁN Márta, NEMCSÓK János, ÁBRAHÁM Magdolna
József Attila Tudományegyetem Biokémiai Tanszék, Szeged
A növényeket felépitö polihidroxilált aromás vegyületek közül (fenol-karbonsavak, flavonoidok) ma már több ezer szerkezete és biológiai hatása ismert. A laboratóriumi kisérleteket elsösorban emlösökön végzik annak érdekében, hogy az eredményeket minél elöbb fel lehessen használni a humán medicinában. Az újabb kutatások ugyanis sorra alátámasztják a több száz éve használt hagyományos gyógynövények kedvezö élettani hatását, ami sok esetben antioxidáns komponenseiknek köszönhetö. Ezen vegyületek in vitro hatása (gyökfogó aktivitás, komplexképzés, enzimgátlás) többek között a hidroxi-, metoxi-, oxo-, karboxil- stb. ligandumok sajátos térbeli elrendezödésének függvénye, az in vivo hatások oka (enzimindukció, csökkentik a szervezet zsirfelvételét stb.) még nem kellöen tisztázott.
A növényi szövetek elbomlása során végsö soron humusz-vegyületek, ill. fenol-karboxil savak keletkeznek, melyek a vizi ökoszisztémára is hatással vannak. Érdeklödésünk középpontjában a halak állnak a vizi szervezetek közül. Jelen tanulmányunk az i.p. adagolt fahéjsav ill. tanninsav pontyra kifejtett in vivo hatásáról ad tájékoztatást.
A májban a fahéjsav a lipid peroxidáció (LP) szintjét a kezelést követöen 24 óra múltán szignifikánsan (*) megemelte. A kataláz és az össz-SOD aktivitás 24 és 48 óránál megemelkedett, a Mn-SOD aktivitás 24 óránál szintén megnött (*) és a továbbiakban is magasabb maradt a kontrollhoz képest. A szöveti GSH koncentrációja 24 óránál maximális (*), a glutation-peroxidáz (GPx) aktivitás növekedés (*) ezt 48 óránál követi. A tanninsav ugyanakkor az LP értékét 24 és 48 óránál is csökkenti (*), a katalázra gátlóan hat, az össz-SOD aktivitás nem változott, 24 óránál a Mn-SOD-ot gátolja, a GSH szint viszont 48 óránál megnövekszik, amit a GPx aktivitás növekedése (*) követ.
Az eredmények tehát azt mutatják, hogy a fahéjsav in vivo pro-, mig a tanninsav antioxidáns sajátságú, mely enzimgátló hatással párosul.
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DERIVATIVES OF THE PLANT CELL WALL. PRO- OR ANTIOXIDANTS FOR FISH?
Varanka Zs, Kotormán M, Nemcsók J, Ábrahám M
Department of Biochemistry, József Attila University, Szeged, Hungary
Phenolcarboxylic acids, flavonoids and tannins are widely distributed in nature, both in the soil and in water. They are enzyme inhibitors and inducers, but they are currently at the centre of interest because of their antioxidant features. Studies have been published concerning their effects on algae, bacteria and mammals, but the effects on fish, at the top of the food chain of the water ecosystem, have been less well examined. This paper reports data relating to this question.
In in vivo experiments on fish, the levels of MDA and tissue GSH and the activities of antioxidant enzymes were elevated by cinnamic acid treatment. Tannic acid had a dual effect: it is an effective antioxidant and decreased the level of lipid peroxidation, but it increased the GSH concentration; nevertheless it behaved as a potent inhibitor of all the antioxidant enzymes examined, except glutathione-peroxidase.
Accordingly, cinnamic acid proved to be a prooxidant phenolic compound for fish, while tannic acid was found to be an effective antioxidant with considerable enzyme inhibitory properties.
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A COMPARATIVE IN VITRO STUDY OF THE PRO/ANTIOXIDANT PROPERTIES OF THE COPPER(II)-ELLAGIC ACID AND COPPER(II)-TANNIC ACID COMPLEXES. CONCENTRATION DEPENDENCE
Zsolt Varanka, Aranka Deér, János Nemcsók and Magdolna Ábrahám (László Vereczkey)
Department of Biochemistry, József Attila University, Szeged, Középfasor 52, H-6726 Hungary
(Chemical Research Center Hungarian Academy of Sciences, Budapest, P.O.Box. 17. H-1525, Hungary)
Plant cell wall degradation products such as phenolcarboxylic acids and tannins are widely distributed in nature, both in the soil and in water (humic substances). A number of them are enzyme inhibitors or inducers. Nevertheless, they are currently at the centre of interest because of their antioxidant features, which are considered partly due to their metal chelating ability (Kono et al., Biosci. Biotechnol. Biochem. 62(1): 22-27, 1998). Some publications report not only on the antioxidant properties, but also on the prooxidant capacities of metal-phenolic complexes. The objective of this study was to examine the in vitro pro/antioxidant properties of the copper(II)-ellagic acid and copper(II)-tannic acid complexes relative to those of ellagic acid, tannic acid or CuSO4 solutions toward glutathione (GSH) in in vitro "biological" and "chemical" systems. Ellagic acid, tannic acid, a CuSO4 solution, Cu(II)-ellagic acid mixture, and a Cu(II)-tannic acid mixture (final concentrations: 20, 2 and 0.2 mikrog/ml) were incubated for 30 min at 20 °C with a carp liver homogenate supernatant ("biological" system) or with glutathione dissolved in distilled water ("chemical" system, final concentration: 50 mikrog/ml). Each reaction had its own control, with the same ingredients but without incubation. After the incubation period, the GSH concentration was determined according to Sedlak and Lindsay (Anal. Biochem. 16: 359-364, 1968). There was no change in glutathione concentration in the cases of ellagic acid and tannic acid, but a significant concentration dependence was observed for copper(II), the Cu(II)-ellagic acid complex and the Cu(II)-tannic acid complex: a GSH loss of about 40-50% was detected at 20 mikrog/ml, and of 8-15% at 2 mikrog/ml, in both "biological" and "chemical" systems. At 0.2 mikrog/ml, the change was minimal. In conclusion, the results of in vitro experiments indicate, that the natural antioxidants can become prooxidant complexes on chelating with copper(II). It is possible that these complexes may act as prooxidants in the animal organism. Preliminary in vitro studies may therefore contribute to an explanation of the in vivo effects of phenolics and their metal complexes on animal antioxidant defence systems.
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THE EFFECTS OF CRUDE OIL ON THE CYTOCHROME P450-DEPENDENT MONOXYGENASE ACTIVITY AND ANTIOXIDANT DEFENCE SYSTEM OF CARP (CYPRINUS CARPIO L.) LIVER
Aranka K. Deér, Zsolt Varanka, Lajos Banka, Márta Kotormán, János Nemcsók and Magdolna Ábrahám (László Vereczkey)
Department of Biochemistry, József Attila University, Szeged, Középfasor 52, H-6726 Hungary (Chemical Research Center Hungarian Academy of Sciences, Budapest, P.O.Box. 17. H-1525, Hungary)
Large amounts of crude oil often enter the seas or rivers in consequence of disasters involving tankers. Although tankers do not operate on Hungarian fresh waters, other motor-driven vessels cause local oil pollution, especially near ports on lakes. Oil well drilling can pollute the soil and precipitation can lead to oil-contaminated water entering rivers or lakes and damaging the water ecosystem. The present aim was to investigate the effects of crude oil on the common carp (Cyprinus Carpio L.) xenobiotic metabolizing enzyme system and antioxidant defence system. Fish were injected intraperitoneally with crude oil (2 or 4 ml/kg-1) for 3 or 8 days, the control fish receiving same the amount of corn oil. Ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD), antioxidant enzymes and antioxidant stress biomarkers (lipid peroxidation (LP), catalase, superoxyde dismutase (SOD), glutathione peroxydase (GPx), and glutathione level (GSH) were then assayed in the liver. The crude oil resulted in a significant induction of the activities both CYT P450 isoenzymes treatment for 3 days. After 8 days, the activity levels following 2 ml/kg-1 treatment were the same as in the control, whereas 4 ml/kg-1 treatment resulted in the same induced activity level as was measured after 3 days. In these in vivo experiments, the LP decreased. This might be due to an interaction between malondialdehide and the reactive compounds of the oil. During the shorter exposure time the lower amount of crude oil elevated the tissue glutathione level and elicited enzyme induction, excluding catalase. During the longer exposure time, the signs of enzyme inhibition appeared as in the case of application of the higher amount of the crude oil. In conclusion, the crude oil resulted in the induction of cytochrome P450-dependent enzyme activities and the fish antioxidant defence system. For purposes of comparison, we analysed carp liver tissue samples from a port on Lake Balaton and samples taken 100 m from the shore. Both EROD and ECOD activities were much higher in the liver of fish collected from the port area of the lake. The same results were found for the antioxidant defence system, while a higher oxidative stress was detected in fish collected from the port area.
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Can ellagic acid detoxify copper(II) in fish?
Zs. Varanka, M. Kotormán, J. Nemcsók and M. Ábrahám
Department of Biochemistry, József Attila University, Szeged, Hungary
Background: Plant derived phenolics are present not only in food and the soil, but even in living waters (about 1-70 mg/l). Their complexing ability with metal ions and their pro/antioxidant features in in vitro systems have been well examined. The goal of this study was to evaluate the possible pro/antioxidant capacity of a metal-phenolic complex under in vivo conditions.
Methods: Carp (Cyprinus carpio L.) were injected intraperitoneally (i.p.) with 10 mg/kg ellagic acid and/or 10 mg/kg Cu(II) in the form of CuSO4 (pH~7.0). Control fish received tridistilled water (i.p.). After exposure for 24 or 48 h the livers were removed and studied. The activities of catalase (CAT), superoxide dismutase (SOD) and glutathione-peroxidase (GPx) and the concentrations of thiobarbituric acid reactive substances (TBARS) and tissue glutathione (GSH) were determined.
Results: The amount of TBARS was decreased by ellagic acid, but not changed significantly by CuSO4, but the Cu(II)-ellagic acid complex elevated the lipid peroxidation level. While the CAT activity was increased 24 and 48 h after treatment with ellagic acid, it was inhibited by Cu(II) and the Cu(II)-ellagic acid complex. Total SOD and Mn-SOD activation was observed 24 and 48 h following ellagic acid treatment. The SOD activity was elevated by Cu(II) and the Cu(II)-ellagic acid chelate after one day, but decreased after two days. The effects on Mn-SOD were similar, but its inhibition by Cu(II) or the Cu(II)-ellagic acid complex was more marked. Ellagic acid resulted in a higher GSH level, whereas Cu(II) and the Cu(II)-ellagic acid complex had the opposite effect. All three compounds inhibited the GPx activity relative to the controls.
Conclusions: Ellagic acid act as an antioxidant in fish. Cu(II), a strong prooxidant and enzyme inhibitor, causes deleterious effects with characteristic physiological symptoms in fish. Ellagic acid-Cu(II) chelate exerts noxious effects like Cu(II) but it is more prooxidant for lipids and a stronger enzyme inhibitor than Cu(II) itself.
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The humic acid content of living water influences the antioxidant parameters of fish
Zs. Varanka, I. Varanka, L. Tölg, A. K. Deér, J. Nemcsók and M. Ábrahám
Department of Biochemistry, József Attila University, Szeged, Hungary
Balaton Limnological Research Institute, Tihany, Hungary
Background: The level of humic compounds dissolved in living waters or freshwaters may be 1-70 mg/l, but in "blackwaters" it may even attain 100 mg/l. Several studies have reported the enzyme inhibitory or inducibility features and pro- or antioxidant capacity of humics. The aim of this study was to evaluate the antioxidant parameters of fish living in water with different humus contents.
Methods: Bream (Abramis brama L.) were collected in two regions of Lake Balaton: about 150 m from the shore near Fonyód and from the western catchment basin (Kis-Balaton I. reservoir). Both of the two regions are free of significant pollution, but the humic content of the water at Kis-Balaton is significantly higher than that at Fonyód. The lipid peroxidation level (LP), the glutathione (GSH) concentration and the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione-peroxidase (GPx) were determined from the liver of the fish.
Results: The LP level was lower in the fish from Kis-Balaton, but the CAT activity was higher in that region. Interestingly, the SOD activity was lower in the bream from Kis-Balaton. A higher GSH concentration and a higher GPx activity were detected in the fish from Kis-Balaton.
Conclusions: The results agreed well with our previous experiments: the decreased malondialdehyde concentration in fish living in humus-rich water was considered due to the antioxidant capacity of humics toward lipids. The higher CAT activity was due to the hydrogen peroxide decomposing activity of the humics or small amounts of humic-transition metal complexes. Under these conditions the decreased SOD activity was due to inhibition and the antioxidant capacity of the humics, present in the water. Humics can trigger a higher GSH level, which often resulted in a higher GPx activity.
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articles:
Biochemical and Morphological Changes in Carp (Cyprinus carpio L.) liver following exposure to Copper Sulfate and Tannic Acid
Zsolt Varanka, Imre Rojik, István Varanka, János Nemcsók and Magdolna Ábrahám
Department of Biochemistry and Department of Comparative Physiology, University of Szeged, P.O. Box 533, H-6701, Szeged, Hungary
Balaton Limnological Research Institute, P.O. Box 35, H-8237, Tihany, Hungary
As a consequence of human activity various toxicants reach the aquatic ecosystems; humics may interact with them and may change their toxicity. Many fish are exposed to a considerable concentration of humics and pollutants. Because of paucity of data on the biochemical action of tannins in the presence of the fungicide CuSO4 a comparative study was undertaken. The alterations of redox-parameters in carp liver were monitored and tissue necrosis was followed by measuring the plasma transaminase activities and by electron microscopy. Tannic acid, a representative phenolic/humic compound exerted prooxidant effects in carp, which may be partially due to formation of prooxidant intermediates/end-products via its biotransformation. Alternatively, tannic acid may partially inhibit the antioxidant enzymes of fish. The response to CuSO4 was more severe. Although, tannic acid alone acted as a prooxidant in fish, electron micrographs demonstrated that it reduced the necrotizing effect of copper, which may be due to the complexing activity of tannic acid with the biomolecules of the hepatocytes and to the H2O2-degrading activity of tannin-CuSO4 combination. Our results indicate that the heavy metal-detoxifying capacity of tannin may be significant; however, tannin-exposure alone or combined with metals may be toxic for fish due to enzyme inhibition and oxidative stress induction.
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Influence of the polyphenolic tannic acid on the toxicity of the insecticide deltamethrin to fish. A comparative study examining both biochemical and cytopathological parameters
Zs. Varanka, K. Aranka Deér, I. Rojik, I. Varanka, Kinga László, T. Bartók, J. Nemcsók and Magdolna Ábrahám
Department of Biochemistry, University of Szeged, P.O. Box 533, H-6701, Szeged, Hungary
Department of Comparative Physiology, University of Szeged, P.O. Box 533, H-6701, Szeged, Hungary
Balaton Limnological Research Institute, P.O. Box 35, H-8237, Tihany, Hungary
Cereal Research Institute, P.O. Box 391, H-6701, Szeged, Hungary
Both humics and pesticides are present in aquatic environment and the toxicological consequences of their chemical interaction is well studied. However, there are only few data concerning the mechanism of the biochemical action of humic-pesticide combinations, especially in vertebrates. Thus we choose to study the in vivo effects of the plant polyphenolic tannic acid and the pyrethroid insecticide deltamethrin [Decis] alone or in combination on hepatic xenobiotic-metabolizing enzyme activities and the associated redox-parameters in carp, as the complex assessment of these systems are regarded to serve as a relevant biomarker of environmental pollution. Stress effects and tissue damage were followed by determination of the plasma glucose level, the activities of plasma transaminases, and by electron microscopy. Tannic acid alone exerted weak prooxidant effect due to its marked antioxidant enzyme inhibitory activity. Deltamethrin, applied in a very low dose, induced oxyradical production in fish via activation of cytochrome P450 isozymes. This effect was promoted by the antioxidant enzyme inhibitory action of tannic acid, when the two chemicals were combined; however, the ultrastructural damage of the hepatocytes was reduced by the common cytoprotective capacity of the phenolic. Numerous humics are known to alter the toxicity of pesticides and their influence depends on their type and concentration. Therefore, our work taken together with other comparative studies may contribute to the assessment of the impact of humics in nature, especially in case of environmental pollution.