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UDC 57.044:577.3:639.3:612.062
IMPACT OF CHLORAMINE-T TREATMENT ON BIOCHEMICAL ENZYMES' ACTIVITY IN THE MUSCLE TISSUE OF RAINBOW TROUT, ONCORHYNCHUS MYKISS (WALBAUM)
HALYNA TKACHENKO1, JOANNA GRUDNIEWSKA2
1Department of Zoology and Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University in Slupsk, Poland
2Department of Salmonid Research, Stanislaw Sakowicz Inland Fisheries Institute, 83-330 Zukowo, Poland
(Поступила в редакцию 29.01.2016)
Резюме. В статье изучено влияния дезинфекции хлорамином Т на содержание биомаркеров окислительного стресса (уровень продуктов реагирующих с 2-тиобарбитуровой кислотой, ТБК-активные продукты) и активность биохимических ферментов [аланин- и аспартатаминотрансферазы (АЛТ и АСТ), лактатдегидрогеназа (ЛДГ)] в мышечной ткани радужной форели (Oncorhynchusmykiss Walbaum). Полученные данные позволяют мониторировать последствия профилактического дезинфицирующего купания с хлорамином Т (9 мг/л, погружение в течение 20 минут, три раза каждые 3 дня) для этого вида рыб. Наши результаты показали, что дезинфекция хло-рамином-Т незначительно снижает перекисное окисление липидов на фоне снижения активности трансаминаз и ЛДГ. Уровень перекисного окисления липидов коррелирует с активностью АЛТ и АСТ в мышечной ткани контрольной группы. Активность ЛДГ положительно коррелирует с АЛТ и АСТ в мышечной ткани форели обработанной дезинфектантом хлорамином-Т. Хлорамин-Т заметно влияет на метаболизм лактата и пирувата, что приводит к снижению активности ЛДГ. Эти параметры могут быть эффективно использованны в качестве потенциальных биомаркеров токсичности хло-рамина-Т в аквакультуре. Наши исследования показали, что хлорамин-T в дозе 9 мг на литр может по крайней мере частично ослабить окислительный стресс и может быть использован для профилактической дезинфекции радужной форели. Тем не менее, нужны более подробные исследования по использованию этих конкретных биомаркеров для мониторинга дезинфицирующих мероприятий в аквакультуре.
Ключевые слова: Хлорамин-Т, дезинфекция, радужная форель (Oncorhynchusmykiss-Walbaum), мышечная ткань, перекисное окисление липидов, аминотрансферазы, лак-татдегидрогеназа.
Summary. The aim of the present study was to examine the effects of exposure to chlora-mine-T on the muscle tissue of rainbow trout (Oncorhynchus mykiss Walbaum) using oxidative stress biomarkers (level of 2-thiobarbituric acid reactive substances, TBARS) and biochemical enzymes activity [alanine- and aspartate aminotransferases (ALT and AST), lactate dehydro-genase (LDH)] to observe the its toxic effects. The endpoints obtained from this study will be useful to monitor the effects of disinfectant bathing with chloramine-T for this species of fish.In the disinfectant exposure, rainbow trout (n=11) were exposed to chloramine-T in final concentration 9 mg per L. Control group of trout (n=11) were handled in the same way as chlora-mine-T exposed groups. Fish were bathed for 20 min and repeated three times every 3 days. Two days after the last bathing fish were sampled. Our results showed that chloramine-T caused the decrease of the lipid peroxidation with non-significant decrease of ALT and AST activity as well as significantly decreased LDH activity. Moreover, lipid peroxidation level is
correlated with ALT and AST activity in the muscle tissue of unhandled control group. In the muscle tissue of trout disinfected by chloramine-T, LDH activity is correlated positively with ALT and AST.Chloramine-T markedly affects on lactate and pyruvate metabolism and resulted to decrease of LDH activity. These parameters could be effectively used as potential bi-omarkers of chloramine-T toxicity to the fish in the warning signal for pharmaceutical exposure to aquatic organisms. Our studies indicated that chloramine-T in dose 9 mg per L could at least partly attenuate oxidative stress and can be used for prophylactic disinfecting treatment of rainbow trout. However, more detailed studies on using of these specific biomarkers to monitor the disinfectant treatment in aquaculture are needed.
Key words: Chloramine-T disinfection, rainbow trout (Oncorhynchus mykiss Walbaum), muscle tissue, lipid peroxidation, aminotransferases, lactate dehydrogenase.
Introduction. The use of pharmaceutical substances is rather limited in fish compared to mammalian therapeutics. It is basically restricted to anaesthetic agents and anti-infective agents for parasitic and microbial diseases. The anti-infective agents are used for controlling diseases and the choice of drug depends on efficacy, ease of application, human safety, target animal safety including stress to the fish, environmental impact, regulatory approval, costs, and implications for marketing the fish (Burka et al., 2007). In fish aquaculture, disinfectants are used against bacterial and protozoal infections. These compounds cause oxidative stress that may stimulate the generation of reactive oxygen species, and subsequently the alteration in anti-oxidant systems of exposed organisms (Stara et al., 2014).
Chloramine-T, as an anti-microbial agent, has had widespread use in a broad range of practices, including medical, dental, veterinary, food processing, and agricultural. As a disinfectant, it is used to disinfect surfaces and instruments. Chloramine-T has a low degree of cytotoxicity and has been used in direct contact with tissues. It is easy to use and effective against many bacteria (both Gram-negative and -positive), viruses (enveloped and naked), fungi, algae, yeast, and parasites (Toxicological Summary for Chloramine-T[127-65-1] andp-Toluenesulfonamide [70-55-3]).
Chloramine-T, a widely used chemotherapeutic and chemoprophylactic treatment for gill diseases in the freshwater aquaculture industry (Thorburn and Moccia, 1993) was found to increase freshwater bathing efficacy and reduced amoeba survival (Powell and Clark, 2003). Other studies also suggest that Chloramine-T in seawater is as effective in seawater as in fresh water (Harris et al., 2004, 2005). Chloramine-T has been widely used in the treatment of gill diseases in the freshwater aquaculture industry (Thorburn and Moccia, 1993).
Sanchez et al. (1998) concluded that although chloramine-T and formalin may continue to be useful in the aquaculture industry they cause potentially harmful alterations to fish skin. Juvenile rainbow trout were exposed to therapeutic concentrations of formalin or chloramine-T to assess the effects of these chemicals on the morphology of the piscine epidermis and its mucous
coat. Repeated treatment, once weekly for 4 weeks, with either chemical did not affect the mucous coat of the epithelium or the degree of folding of the basal lamina. However, treated fish had increased numbers of highly dense vesicles within the apical portions of epithelial cells. The epidermal mucous cells of chloramine-T-treated fish were significantly smaller than in controls. This effect was not noted in formalin-treated fish. Treatment with either chemical resulted in a significantly thinned epidermis (Sanchez et al., 1998).
Therefore, the aim of the present study was to examine the effects of exposure to chloramine-T on the muscle tissue of rainbow trout (Oncorhynchus mykiss Walbaum) using oxidative stress biomarkers (level of 2-thiobarbituric acid reactive substances) and biochemical enzymes activity (alanine- and as-partate aminotransferases, lactate dehydrogenase) to observe the its toxic effects. The endpoints obtained from this study will be useful to monitor the effects of disinfectant bathing with chloramine-T for this species of fish.
Materials and methods. Fish. Twenty two clinically healthy rainbow trout were used in the experiments. The study was carried out in a Department of Salmonid Research, Inland Fisheries Institute near the village of Zukowo, Poland. Experiments were performed at a water temperature of 16±2°C and the pH was 7.5. The dissolved oxygen level was about 12 ppm with additional oxygen supply. All biochemical assays were carried out at Department of Zoology and Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University (Slupsk, Poland).
The fish were divided into two groups and held in 250-L square tanks (70 fish per tank) supplied with the same water as during the acclimation period (2 days). On alternate days, the water supply to each tank was stopped. In the disinfectant exposure, rainbow trout (n=11) were exposed to chloramine-T in final concentration 9 mg per L. Control group of trout (n=11) were handled in the same way as chloramine-T exposed groups. Fish were bathed for 20 min and repeated three times every 3 days. Two days after the last bathing fish were sampled. Fish were not anesthetized before tissue sampling.
Muscle tissue isolation. Muscle tissue were excised, weighted and washed in ice-cold buffer. The minced tissue was rinsed clear of blood with cold isolation buffer and homogenized in a glass Potter-Elvehjem homogenising vessel with a motor-driven Teflon pestle on ice. The isolation buffer contained 100 mM tris-HCl; pH of 7.2 was adjusted with HCl.
Analytical methods. All enzymatic assays were carried out at 25±0.5°C using a Specol 11 spectrophotometer (Carl Zeiss Jena, Germany). The enzymatic reactions were started by adding the homogenate suspension. The specific assay conditions are presented subsequently. Each sample was analyzed in triplicate. The protein concentration in each sample was determined according to Bradford (1976) using bovine serum albumin as a standard.
TBARS assay for lipid peroxidation.LPO levelwas determined by quantifying the concentration of 2-thiobarbituric acid reactive substances (TBARS) according to Kamyshnikov (2004). The TBARS level was expressed in nmol MDA per mg protein by using 1.56 105 mM"1 cm-1 as molar extinction coefficient.
Biochemical enzymes activity.Alanine aminotransferase (ALT, E.C. 2.6.1.2) and Aspartate aminotransferase (AST, E.C. 2.6.1.1) activities were analyzed spectrophotometrically by standard enzymatic method (Reitman and Frankel, 1957). The colorimetric method of Sevela and Tovarek (1959) was used for the determination of lactate dehydrogenase (LDH, E.C. 1.1.1.27) activity.
Statistical analysis. The mean ± S.E.M. values was calculated for each group to determine the significance of inter group difference. All variables were tested for normal distribution using the Kolmogorov-Smirnov and Lilliefors test (p>0.05). Significance of differences between the oxidative stress biomarkers level (significance level, p<0.05) was examined using Kruskal-Wallis one-way analysis of variance by ranks test. Correlations between parameters at the set significance level were evaluated using Spearman's correlation analysis (Zar, 1999). All statistical calculation was performed on separate data from each individual with STATISTICA 10.0.
Results. Influence of chloramine-T on lipid peroxidation biomarker, measured as 2-thiobarbituric acid reactive substances in the muscle tissue of rainbow trout are presented in Fig. 1A. Non-significantly lower TBARS level (by 6%, p>0.05) in fish disinfected by chloramine-T compared to control group was observed (Fig. 1A).
TBARS
545
'3 540
0 &
7 535 «
S
2 530
525
I 'nhandled control group Chloramine-T exposed group
X
I
B
Ql'iiliandlcd i'onIrciI group OChloramme-T exposed group
Fig. 1. Influence of chloramine-T on lipid peroxidation biomarker, measured as 2-thiobarbituric acid reactive substances (TBARS, A), as well as alanine and aspartate aminotransferases, lactate dehydrogenase (B) in the muscle tissue rainbow trout (Oncorhynchus
mykiss Walbaum)
* the significant difference was shown as p<0.05 when compared control and chloramine-T exposed groups
LDH activity in the muscle tissue of chloramine-T-exposed trout were significantly lower by 339 % (p=0,017) compared to controls (Fig. 1B).
Several correlations between checked parameters were found (Fig. 2). Muscle TBARS level is correlated positively with ALT (r=0,858, p=0.001) and AST (r=0,896, p=0,000) in unhandled control group (Fig. 2A). LDH activity is correlated positively with ALT (r=0,689, p=0,019) and AST (r=0.852, p=0.000) in the muscle tissue of rainbow trout exposed by Chlo-ramine-T (Fig. 2B). A
TBARS:ALT: y = 0.3137 + 0.0 273*x; r = 0.8577; p = 0.0007; r2 = 0.7356 TBARS:AST: y = 3.156 + 0.041*x; r = 0.896; p = 0.0002; r2 = 0.8036
TBARS, nmol-mg"1 protein
B
LDH:ALT: y = 1.0629 + 0.4585*x; r = 0.6890; p = 0.0190; r 2 = 0.4747 LDH:AST: y = 9.2014 + 0.4762*x; r = 0.8521; p = 0.0009; r2 = 0.7260
28 26
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g 20 1 18
I 16
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32 30
28 .5
1
26 & bp
24 | 22 1 20 s;
18 is
16 i
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14 16 18 20 22 24 26 28 30 32 34 36 38 40 LDH, pmol pyruvate-h -mg protein
Fig. 2. Correlations between TBARS, ALT and AST activity in the muscle tissue of unhandled control group (A), and LDH, ALT and AST activity in the muscletissue of trout disinfected by chloramine-T
Discussion. Our results showed that chloramine-T bathing caused the decrease of the lipid peroxidation with non-significant decrease of ALT and AST activity as well as significantly decreased LDH activity (Figs 1A and IB). Moreover, lipid peroxidation level is linked with ALT (r=0,858, p=0,001) and AST activity (r=0,896, p=0,000) in the muscle tissue of unhandled control group (Fig. 2A). In the muscle tissue of trout disinfected by chloramine-T, LDH activity is correlated positively with ALT (r=0,689, p=0,019) and AST (r=0.852, p=0,000) (Fig. 2B).
Skeletal muscle of fishes plays an important role in the post-exercise processing of lactate through the gluconeogenic and glycogenic pathways, a situation very different from that in mammals, where the enzymatic machinery for regenerating glucose is primarily found in the liver (Suarez et al., 1986; Moon, 1988; Gleeson, 1996). The muscle of fish is thus more multi-functional than that of mammals, possessing a wider array of enzymes and greater inherent metabolic flexibility than mammalian muscle, including a greater potential for biosynthesis (Gleeson, 1996). In fact, fish skeletal muscle has been shown to sequester lactate post-exercise (Gleeson, 1996), facilitating the regeneration of glucose within the muscle and enhancing post-exercise recovery (Torres et al., 2012).
Skeletal muscle acts as a clearing house for lactate produced in brain, heart and liver during anaerobiosis. Lactate is taken up by muscle and con-
12
10
12
10
12
verted to pyruvate by LDH. Pyruvate reaching the muscle via the bloodstream and that produced during glycolytic activity in the muscle is processed by the pyruvate dehydrogenase (PDH) pathway within the mitochondrion to produce acetaldehyde and CO2; acetaldehyde is converted to ethanol within the cytosol, and ethanol can then diffuse out of the cell to be excreted at the gills (Shoubridge and Hochachka, 1980).
In our study, correlation between pyruvate recovery through aminotrans-ferases (ALT and AST activity) and lactate conversion to pyruvate through LDH was shown (Fig. 2B). In our previous study (Tkachenko et al., 2012), chloramine-T bathing markedly decrease aldehydic and ketonic derivatives of oxidative protein, and aminotransferases activity only in rainbow trout liver, and their elevation is a compensatory mechanism to impaired metabo-lism.No significant changes were found in oxidative stress biomarkers between control and chloramine-treated brown trout. For grayling, chlora-mine-T exposure caused significantly elevation in the levels of severe oxi-dative stress biomarkers.Increased aldehydic and ketonic derivatives of oxi-dative protein could modify lactate and pyruvate levels, aminotransferases and lactate dehydrogenase activities, principally causing increased enzymes activity due to oxidative stress in the liver of chloramine-exposed fish (Tkachenko et al., 2012).
Boran and Altinok (2014) assessed the effects of therapeutic, and higher concentrations of Chloramine-T on the antioxidant enzyme system and genetic structure in juvenile rainbow trout Oncorhynchus mykiss (Walbaum). Red blood cells acetylcholinesterase, A-aminolevulinic acid dehydratase, paraoxonase and liver glutathione S-transferase activity were increased at 10 and 20 mg per L Chloramine-T-exposed fish, while they were decreased at 30 mg per L Chloramine-T-exposed fish. On the other hand, liver catalase activity and liver protein levels increased at 10 mg per L and decreased at 20 and 30 mg per L concentrations of Chloramine-T. Liver superoxide dis-mutase activity decreased at 10 mg per L and 20 mg per L Chloramine-T and increased at 30 mg per L of Chloramine-T. Compared to control, comet assay indicated that Chloramine-T did not cause significant DNA damage to red blood cells of the fish. Results indicate that 10 or 20 mg per L Chlora-mine-T can be safely used to prevent or treat external parasitic and bacterial infection of rainbow trout (Boran and Altinok, 2014).
Studies of Powell and Clark (2003) have shown that Chloramine-T is effective at killing Neoparamoeba spp. and its addition to seawater can produce efficacy levels similar to that found in freshwater treatment (Harris et al., 2004). In study of Leef et al. (2007), treatment with Chloramine-T at 10 mg per L appeared to briefly mitigate the rise in standard metabolic rates (RS), as there was an approximately 30 % drop (not statistically significant) in RS following treatment.
In rainbow trout Oncorhynchus mykiss, acute Chloramine-T exposure at 9 mg per L in fresh water induces both respiratory and acid-base disturbances that may be directly related to hyperventilation (Powell and Perry, 1997) and an increase in branchial mucus production due to the irritant effect on the gills (Powell and Perry, 1996). Oxygen consumption rates also increases following Chloramine-T exposure at 9 mg per L (Powell and Perry, 1999).
Accumulating evidence has shown that chloramine-T causes oxidative stress by inducing the generation of reactive oxygen species (ROS) (Tatsu-mi and Fliss, 1994; Sakuma et al., 2009; Stanley et al., 2010). The data suggest that HOCl and monochloramine can increase endothelial permeability by causing very rapid cytoskeletal shortening and cell retraction, possibly as a result of the oxidation of intracellular sulfhydryls (Tatsumi and Fliss, 1994). Sakuma et al. (2009) assessed the influence of monochloramine on the conversion of xanthine dehydrogenase into xanthine oxidase in rat liver in vitro. When incubated with the partially purified cytosolic fraction from rat liver, monochloramine (2,5-20 microM) dose-dependently enhanced xanthine oxidase activity concomitant with a decrease in xanthine dehydro-genase activity, implying that monochloramine can convert xanthine dehy-drogenase into the ROS producing form xanthine oxidase. It was found that monochloramine could increase ROS generation in the cytoplasm of rat primary hepatocyte cultures, and that this increase might be reversed by an xanthine oxidase inhibitor, allopurinol. These results suggest that mono-chloramine has the potential to convert xanthine dehydrogenase into xan-thine oxidase in the liver, which in turn may induce the ROS generation in this region (Sakuma et al., 2009).
Conclusion.Chloramine-T in dose 9 mg per L has no influence on the level of lipid peroxidation in the muscle tissue of rainbow trout. Chlora-mine-T markedly affects on lactate and pyruvate metabolism and resulted to decrease of LDH activity. These parameters could be effectively used as potential biomarkers of chloramine-T toxicity to the fish in the warning signal for pharmaceutical exposure to aquatic organisms. Our studies indicated that chloramine-T in dose 9 mg per L could at least partly attenuate oxidative stress and can be used for prophylactic disinfecting treatment of rainbow trout. However, more detailed studies on using of these specific biomarkers to monitor the disinfectant treatment in aquaculture are needed.
This work was supported by grant of the Pomeranian University for Young Scientists.
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