Effect of Triiodothyronine on the Activity and Sensitivity of Glycosidases to Heavy Metals (Cu, Zn, and Pb) in Juvenile Blue Bream Ballerus Ballerus (L.)

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Effect of triiodothyronine on the activity and sensitivity of glycosidases to heavy metals (Cu, Zn, and Pb) in juvenile blue bream Ballerus ballerus (L.)

Article in Inland Water Biology · July 2017 DOI: 10.1134/S1995082917030063

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Andrey Filippov Aleksey Bolotovskiy Russian Academy of Sciences Russian Academy of Sciences

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The user has requested enhancement of the downloaded file. ISSN 1995-0829, Inland Water Biology, 2017, Vol. 10, No. 3, pp. 305–307. © Pleiades Publishing, Ltd., 2017. Original Russian Text © A. Filippov, A.A. Bolotovskiy, B.A. Levin, I.L. Golovanova, 2017, published in Biologiya Vnutrennykh Vod, 2017, No. 3, pp. 62–65. ECOLOGICAL PHYSIOLOGY AND BIOCHEMISTRY OF HYDROBIONTS

Effect of Triiodothyronine on the Activity and Sensitivity of Glycosidases to Heavy Metals (Cu, Zn, and Pb) in Juvenile Blue Bream Ballerus ballerus (L.) A. Filippov, A. A. Bolotovskiy, B. A. Levin, and I. L. Golovanova* Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, 152742 Russia *е-mail: [email protected] Received October 14, 2016

Abstract⎯The influence of exogenous triiodothyronine (0.25 ng/mL) on the activity glycosidases (maltase, amylolytic activity) and their sensitivity to Cu, Zn and Pb ions (25 mg/L) has been studied in the whole organism of juvenile blue bream Ballerus ballerus (L.). We have found that the treatment of fish with triiodothyronine resulted in an increase in amylolytic activity. Maltase activity is not affected. In addition, the exposure to exogenous triiodothyronine results in a decrease in the sensitivity of glycosidase to the in vitro action of the ions of biogenic metals Cu and Zn and in an increase in the sensitivity of the maltase to the action of Cu, Zn, and Pb ions.

Keywords: Cyprinidae, blue bream, digestive glycosidases, triiodothyronine, heavy metals, Cu, Zn, Pb DOI: 10.1134/S1995082917030063

INTRODUCTION It was revealed that Т3 may affect the activity of glycosidases in the intestine of roach Rutilus rutilus (L.) Thyroid hormones (THs) are one of the most (fam. Cyprinidae) [3]. Another cyprinid species, blue important groups of hormones that start to function in bream Ballerus ballerus (L.), exhibits the lowest level fish since early ontogenesis. These hormones play an of Т3 in the blood plasma among all studied fish spe- important role in the metabolic and morphogenetic cies; i.e., this species is TH-deficient [9]. In this processes (including metamorphosis), in regulating respect, the study on the influence of Т3 on the activity the reproductive system, and in the regeneration of of glycosidases in the TH-deficient species is espe- certain organs and tissues [8, 11, 13]. It is believed that cially interesting. thyroxin is a prohormone, while 3,5,3'- triiodothy- The goal of this paper is to study the effect of ronine (Т3) is a more active form of thyroid hormones formed by the deiodination of thyroxin [8]. triiodothyronine on the activities of glycosidases and their sensitivity to the effects of Cu, Zn, and Pb ions in Aquatic organisms are subject to a range of anthro- juvenile blue bream Ballerus ballerus (L.). pogenic factors, such as salts of heavy metals strongly affecting natural ecosystems. Copper, zinc, and lead are the priority pollutants of ambient waters [5]. MATERIALS AND METHODS Unlike lead, copper and zinc are essential micronutri- Juvenile blue breams were exposed to T3. The gam- ents participating in many biochemical reactions. etes were obtained from one female and three males However, in large quantities the latter two elements are caught by seine net in the Rybinsk Reservoir near toxic to aquatic life. In the areas affected by techno- Borok (Yaroslavl oblast, Russia) in early May 2015. genic pollution, concentrations of these metals reach Fish eggs were fertilized by the dry method, placed on several milligrams per 1 L of water [4]. Entering organ- pieces of glass, and incubated for 8 days in a 3-L plastic ism with water and food, copper, zinc, and lead may container at a water temperature of 11.2–15.8°С. Fol- affect fish digestive enzymes both directly and indi- lowing hatching, the larvae were transferred into a 60-L rectly [1, 7]. The lysosomal hydrolases of tissues of aquarium. The larvae and fries were maintained at food organisms (prey) may considerably contribute to natural regimes of temperature fluctuations from 16.5 the processes of digestion in fish [10]. This is why it is to 24.1°С and illumination (12 h light : 12 h dark). necessary to study the effects of heavy metals not only After switching to exogenous feeding, the fries were fed upon the digestive hydrolases of a consumer, but also ad libitum with Sera Micron dry food for aquarium upon the enzymes in the prey organisms. fishes, live Artemia sp. nauplii, and frozen Chironomus sp.

305 306 FILIPPOV et al.

Table 1. Activity of glycosidases in the whole organism of solutions of substrates (18 g/L starch and 50 mM/L juvenile blue bream of control and test (Т3) groups upon maltose) were prepared using the same Ringer’s solu- exposure to Cu, Zn and Pb ions in vitro tion. The solutions of enzymatically active prepara- Metal ions Activity of glycosidases, μM/(g min) tions and substrates were incubated for 20–30 min at 20°С, рН 7.4 at constant mixing. To study the effects (25 mg/L) amylolytic maltase of heavy metals, the homogenates were preliminarily incubated with salts of copper (CuSO4 ⋅ 5H2O), zinc 1.34± 0.01а 2.16± 0.05а Absence (ZnSO4 ⋅ 7H2O), and lead (Pb(NO3)2 for 1 h. The con- 1.53± 0.07а 2.14± 0.02а centrations of Cu, Zn, and Pb ions, as calculated by ± c ± а the content of metal in salt, was 25.0 mg/L. The amy- Cu 0.92 0.02 1.93 0.15 c b lolytic activity reflecting the sum activities of the 1.09± 0.02 1.98± 0.05 enzymes hydrolyzing starch, α-amylase EC 3.2.1.1, ± b ± a glucoamylase EC 3.2.1.3, and maltase EC 3.2.1.20, Zn 1.13 0.03 2.23 0.03 a b was assessed by the gain of hexoses following modified 1.39± 0.04 1.95± 0.03 Nelson’s technique [6]. To determine the maltase ± b ± a Pb 1.17 0.03 2.31 0.04 activity by the glucose oxidase method, the Photoglu- 1.25± 0.04b 1.89± 0.02c cosa (OOO Impact, Russia) test for clinical biochemistry was used. The optical density was read on a Means and errors of means are given; different letters superscript indicate statistically significant differences between the parame- Lambda 25 (PerkinElmer, United States) spectropho- ters in a row (ANOVA, LSD-test), p < 0.05. Control is on top; test tometer at wavelengths of 670 nm (amylolytic activity) is on bottom. and 505 nm (maltase activity). The activities of enzymes were expressed in micromolar of the reaction product formed for 1 min incubation per 1 g wet tissue once a day. At the age of 22 days post fertilization, the weight (μM/(g ⋅ min)). fish were split into two groups, with 35 individuals in each group. The fish of the first group were maintained The results are given as means and errors of means in an aquarium with clean water (control); those of the (M ± m). The normality of distribution of studied second group were in an aquarium with triiodothy- parameters was assessed using Shapiro–Wilk test. Sta- ronine (Т ) at a concentration of 0.25 ng/mL (test vari- tistical significance of differences was assessed using 3 single-factor analysis (ANOVA followed by LSD-test) ant). The conditions (temperature, illumination, and at ≤ 0.05. feeding) were the same for both groups. Every day, p 1/3–1/2 of the water volume in the aquarium was changed, with the Т3 concentration kept constant. RESULTS AND DISCUSSION The duration of the experiment was 85 days (fish age of 107 days after fertilization). No differences in either length or weight in juvenile fish of two experimental groups were revealed by the After the termination of exposure, Т3 was extracted end of experiment. Body length and weight in control from the whole fish (12 individuals from control and test fish were 29.8 ± 0.7 mm and 0.30 ± 0.02 g, respec- groups) tissue homogenates using lysing buffer (0.1 M tively; in the test group it was 31.8 ± 1.4 mm and 0.41 ± phosphate buffered saline pH 7.4, 0.1% Triton X-100, 0.05, respectively. By the end of experiment, the con- 1 mM propylthiouracil – Sigma Aldrich). The sam- centration of Т3 in the tissues of test-group fish was ple-to-buffer ratio was 1 : 1 by weight. After homoge- higher by a factor of three (0.75 ± 0.05 ng/mL) than in nization and centrifuging (for 30 min at 10000 g) of the control (0.25 ± 0.05 ng/mL). the samples, the homogenates were analyzed follow- Amylolytic activity in the test-group fish was 26% ing the standard technique of enzyme-linked immu- higher than in the control; maltase activity did not dif- nosorbent assay (ELISA) for commercial kits of total fer in compared groups (see table 1). In the presence of T3 (tT3, Monobind Inc., CA, USA). According to the Cu, ion amylolytic activity in the control fish data provided by the kit producer, the sensitivity of the decreased by 32% when compared to the fish not technique is 0.04 ng/mL. The results were read on a exposed to the metal; in the test fish the activity Stat Fax 303 Plus (Awareness Technology, United decreased by 25%. After exposure to Zn ions, the States) microstrip photometer using two optic filters activity decreased by 13% only in the control fish; after for 450-nm and 630-nm wavelengths. exposure to Pb ions, activity decreased by 13% in both To determine the glycosidase activities, enzymati- groups. The studied ions did not change maltase activ- cally active preparations were prepared. Sum whole- ity in the control blue bream; in the fish of the test organism samples from 12 fish individuals of either group, enzymatic activity statistically significantly control or test groups were homogenized in a glass declined by 5% (exposure to Cu), 6% (Zn), and 9% homogenizer with the addition of Ringer’s solution for (Pb). poikilotherms (110 mM NaCl, 1.9 mM KCl, 1.3 mM It was revealed earlier that the exposure of juvenile CaCl2, рН 7.4) chilled to 2–4°С at 1 : 9 ratio. The roach (age from 1.5 to 9 month) to T3 leads to a rise in

INLAND WATER BIOLOGY Vol. 10 No. 3 2017 EFFECT OF TRIIODOTHYRONINE ON THE ACTIVITY AND SENSITIVITY 307 amylolytic activity; a similar rise was revealed in the 2. Golovanova, I.L., Filippov, A.A., Bolotovskiy, A.A., experiments with sterlet Acipenser ruthenus L. [3]. It and Levin, B.A., Characterization of the intestinal was suggested upon studies on closely related species digestive glycosidases in plankton- and benthos-feed- blue bream and white-eye bream Ballerus sapa (Pallas) ing species of the fish genus Ballerus (Cyprinidae), Zh. that the higher activity of glycosidases (amylolytic Evol. Biokhim. Fiziol., 2015, vol. 51, no. 1, pp. 17–20. activity, maltase, and sucrose) in the latter species may 3. Kuz’mina, V.V., Levin, B.A., and Lyu, Vei,Rusa- relate to the higher level of Т in this species [2]. In the novaP.V., Effect of thyroid hormones on activity 3 dynamics of enzymes of intestinal mucosa of juvenile blue bream exposed to Т3, the tissue content of this roach Rutilus rutilus, J. Ichthyol., 2010, vol. 50, no. 5, hormone was also higher, along with higher amylolytic pp. 396–401. (but not maltase) activity. These differences may be 4. Perevoznikov, M.A. and Bogdanova, E.A., Tyazhelye related to different effects of triiodothyronine on amy- metally v presnovodnykh ekosistemakh (Heavy Metals in lolytic activity (reflecting the sum activities of α-amy- Freshwater Ecosystems), St. Petersburg: Gos. NII lase, glucoamylase, and maltase) and the activity of Ozer. Rech. Ryb. Khoz., 1999. maltase. Exposure to triiodothyronine changing amy- 5. Perechen’ rybokhozyaistvennykh normativov: predel’no lolytic activity may change both the rate of carbohy- dopustimykh kontsentratsii (PDK) i orientirovochno bezo- drate metabolism in juvenile fish and the potential pasnykh urovnei vozdeistviya (OBUV) vrednykh vesh- contribution of prey enzymes to the digestion of con- chestv dlya vody vodnykh ob”ektov, imeyushchikh rybok- sumers. hozyaistvennoe znachenie (List of Fishery Standards: Maximum Permissible Concentrations (MPCs) and The data of the present study, in combination with the Estimated Safe Exposure Levels (SELs) of Pollut- the results of other research [2, 3], indicate that Т3 ants to Water of Aquatic Objects of Fishery Impor- somehow regulates carbohydrate metabolism in fish. tance), Moscow: Vseros. NII Ryb. Khoz. Okeanogr., This may be important from the point of view of 1999. phenotypic plasticity [12, 14], when an elevated level 6. Ugolev, A.M., Iezuitova, N.N., Masevich, Ts.G., et al., of hormone allows a species not only to adapt mor- Issledovanie pishchevaritel’nogo apparata u cheloveka. Obzor sovremennykh metodov (Investigation of the phologically and behaviorally to changing conditions, Digestive Tract in Humans: Overview of Modern but also to undergo physiological transformation by Methods), Leningrad: Nauka, 1969. changing or widening the feeding spectrum. Out 7. Filippov, A.A. and Golovanova, I.L., Separate and results indirectly confirm the hypothesis [9] according joint effects of copper and zinc on the intestine carbo- to which Т3 may serve as a trigger for changes at the hydrase activity in vitro in freshwater teleosts, Inland initial stages of speciation as for trophic resource par- Water Biol., 2010, vol. 3, no. 1, pp. 96–104. titioning. 8. Blanton, M.L. and Specker, J.L., The hypothalamic– pituitary–thyroid (HPT) axis in fish and its role in fish development and reproduction, Crit. Rev. Toxicol., CONCLUSIONS 2007, vol. 37, pp. 97–115. Exogenous triiodothyronine (0.25 ng/mL) 9. Bolotovskiy, A.A. and Levin, B.A., Thyroid hormone increases amylolytic activity, but has no effect on mal- divergence between two closely related but ecologically tase activity in juvenile blue bream. The effect of diverse cyprinid fish species (Cyprinidae), Biochem. triiodothyronine decreases the sensitivity of starch- Syst. Ecol., 2015, vol. 59, pp. 305–310. hydrolyzing glycosidases to the in vitro impacts of bio- 10. Kuzmina, V.V. and Golovanova, I.L., Contribution of prey proteinases and carbohydrases in fish digestion, genic metal ions of Cu and Zn and, to a certain extent, Aquaculture, 2004, vol. 234, nos. 1–4, pp. 347–360. increases the sensitivity of maltase to impacts of Cu, 11. Leatherland, J.F., Environmental physiology of the Zn, and Pb ions. teleostean thyroid gland: a review, Environ. Biol. Fish, 1982, vol. 7, pp. 83–110. ACNOWLEDGMENTS 12. Scheiner, S.M., Genetics and evolution of phenotypic plasticity, Annu. Rev. Ecol. Evol. Syst., 1993, vol. 24, This study was supported by Russian Foundation pp. 35–68. for Basic Research nos. 15-34-20416 and 15-04- 13. Sekimizu, K., Tagawa, M., and Takeda, H., Defective 03586a. fin regeneration in medaka fish (Oryzias latipes) with hypothyroidism, Zool. Sci., 2007, vol. 24, no. 7, pp. 693–699. REFERENCES 14. West-Eberhard, M.J., Phenotypic plasticity and the 1. Golovanova, I.L. and Urvantseva, G.A., Effect of lead origins of diversity, Annu. Rev. Ecol. Evol. Syst., 1989, on the activity of glycosidases of intestinal mucous vol. 20, pp. 249–278. membrane of fish, Tr. Karel. Nauch. Tsentra RAN, 2014, no. 5, pp. 195–199. Translated by D. Pavlov

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