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UDC 597.62:619:577.151.042:579.62
BIOCHEMICAL PROFILE IN THE CARDIAC TISSUE OF SEA TROUT (SALMO TRUTTA M. TRUTTA L.) AFFECTED BY FURUNCULOSIS
H. M. Tkachenko, N. N. Kurhaluk
БИОХИМИЧЕСКИЙ ПРОФИЛЬ В СЕРДЕЧНОЙ ТКАНИ ПОРАЖЕННОЙ ФУРУНКУЛЕЗОМ КУМЖИ (SALMO TRUTTA M. TRUTTA L.)
Г. М. Ткаченко, Н. Н. Кургалюк
Motile aeromonads cause diverse pathological conditions that include acute, chronic and covert infections. Numerous studies support the contribution of reactive oxygen species in the pathogenesis of parasite invasion mechanisms, as well as activation of immune system. However, the validation of oxidative stress-related biomarkers in these setting remains to be clarified, and novel association of these biomarkers and metabolic alteration, is just emerging. Oxidative stress has been suggested as a pathogenic factor and therapeutic target in infective mechanisms. The aim of the present study was to examine the responses of biochemical alteration and oxidative stress biomarkers in the cardiac tissue of healthy specimens of sea trout (Salmo trutta m. trutta L.) and naturally furunculosis-affected trout from the Slupia river, the river of the Baltic sea basin where trout are spawning (Northern Poland, Central Pomeranian region). Furunculosis induces production of free radicals in the cardiac tissue of sea trout. High levels of aldehydic and ketonic derivatives of protein oxidation were observed both in furunculosis-affected males and females. High protein oxidation occurred together with an alteration of the biochemical profile (decrease of aspartate aminotransferase and lactate dehydrogenase activities in males, as well as lactate level in females).
sea trout Salmo trutta m. trutta L., furunculosis, oxidative stress, cardiac tissue, aminotransferases, lactate dehydrogenase, lactate, pyruvate
Подвижные аэромонады вызывают различные патологические состояния, которые включают острые, хронические и скрытые инфекции [1]. Многочисленные исследования подтверждают вклад реактивных форм кислорода в патогенез механизмов паразитарной инвазии, а также активацию иммунной системы хозяина [2]. Тем не менее анализ механизмов окислительного стресса, связанных с метаболическими изменениями в организме инфицированных рыб, не выяснен. Окислительный стресс был предложен в качестве патогенного фактора и терапевтической мишени в развитии инфекционных механизмов [2]. Целью настоящего исследования было изучение биохимических изменений и содержания биомаркеров окислительного стресса в ткани сердца здоровых особей морской форели (Salmo trutta m. trutta L.) и зараженных фурункулезом рыб из р. Слупии (река бассейна Балтийского моря), где она нерестится (Поморское воеводство, северная Польша). Фурункулез индуцирует продукцию свободных радикалов в ткани сердца морской форели, что приводит к увеличению альдегидных и
кетоновых производных окислительно-модифицированных белков у зараженных самцов и самок форели. Высокое содержание модифицированных белков изменяет биохимический профиль в сердечной ткани морской форели (снижение активности аспартатаминотрансферазы и лактатдегидрогеназы у самцов, а также снижение уровня лактата у самок).
кумжа Salmo trutta m. trutta L., фурункулез, окислительный стресс, сердечная ткань, аминотрансферазы, лактатдегидрогеназа, лактат, пируват
INTRODUCTION
Furunculosis induced by motile aeromonads is a problem in farming of salmonids (brown and rainbow trout) and various other fish species in Europe over the last few years. Motile aeromonads are often referred to as a complex of disease organisms that are associated with bacterial haemorrhagic septicaemias and other ulcerative conditions in fish [1]. Nevertheless, motile aeromonads are ubiquitous in most freshwater environments and are common in the water column and in the upper layers of sediment [3]. Motile aeromonads cause diverse pathological conditions that include acute, chronic and covert infections. Severity of disease is influenced by a number of interrelated factors, including bacterial virulence, the kind and degree of stress exerted on a population of fish, the physiological condition of the host and the degree of genetic resistance inherent within specific populations. Motile aeromonads differ interspecifically and intraspecifically in their relative pathogenicity or their ability to cause disease [1].
In the acute form of a motile aeromonad disease, a fatal septicaemia may occur so rapidly that fish die before they have time to develop anything but a few gross signs of disease [1]. When clinical signs of infection are present, affected fish may show exophthalmia, reddening of the skin and an accumulation of fluid in the scale pockets [4]. The abdomen may become distended as a result of an oedema and the scales may bristle out from the skin to give a 'washboard' appearance. The gills may haemorrhage and ulcers may develop on the dermis [1]. Ogara et al. (1998) noted severe eye pathology and heavy mortality among yearling and older rainbow trout accompanying a severe outbreak of motile aeromonad septicaemia [5]. Motile aeromonads were isolated from the eyes, liver and kidneys of affected fish. Histopathologically, fish may exhibit epithelial hyperplasia in the foregut, leptomeningeal congestion in the brain, as well as a thrombosis and inflammation in the perisclerotic region and corneal epithelium of the eye. There may also be a severe branchitis, as indicated by leucocytic infiltration and dilation of the central venous sinus [6].
Microbiological infections implicate interaction of host and pathogen [7, 8]. To date, most of the research on Aeromonas hydrophila has still focused on the so-called virulence factors. However, recently, increasing reports have proposed that animal environments that pathogens colonize have likely driven the evolution of new metabolic adaptations to maximize these new nutritional opportunities, and these adaptations may link with bacterial virulence. Research in fish metabolism and parasite invasion mechanisms has advanced the knowledge of the mechanisms whereby parasites evade or cope with fish immune response. Oxidative stress has been suggested as a pathogenic factor and therapeutic target in infective mechanisms [2]. A better understanding of the bacterium-induced oxidative stress in sea trout is required to find a cure. Therefore, the aim of the present study was to examine the responses of oxidative stress biomarkers in the cardiac tissue of healthy specimens of sea trout (Salmo trutta m. trutta L.) and
naturally furunculosis-affected trout from Slupia river, the river of Baltic sea basin where trout are spawning (Northern Poland, Central Pomeranian region). The activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities, lactate and pyruvate levels, as well as oxidative stress biomarkers (2-thiobarbituric acid reactive substances as lipid peroxidation biomarker, aldehydic and ketonic derivatives as protein oxidative modification biomarker), and correlation between those in the cardiac tissue of healthy and furunculosis-affected trout were assayed.
MATERIALS AND METHODS
Fish. Adult sea trout (Salmo trutta m. trutta L.), 3-5 years of age, were collected from site on the Slupia river, Slupsk, Northern Poland. Fish-catching took place in exact co-operation from Landscape Park "The valley of Slupia" as well as the Board of Polish Angling Relationship in Slupsk.
Sampling. The sampling for analysis from healthy males (n=21) and females (n=37) (control group), as well as males (n=34) and females (n=18) of sea trout affected by furunculosis (study group) were collected directly after catch. After catching, microbiological tests were carried out [9]. These tests suggested that Aeromonas spp. complex caused furunculosis.
Tissue homogenate preparation. Specimens in each group were dissected. One fish was used for each preparation. Each cardiac sample was homogenized in cold Tris-HCl buffer (100 mM, pH 7.2) to obtain a 10% (w/v) tissue homogenate. The homogenate was then centrifuged at 5,000g for 15 min. Each supernatant was collected and stored at -20°C until use. The protein content of each sample was determined using Bradford method (1976) and bovine serum albumin as the standard [10].
Biochemical assays. 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.
TBARS assay for lipid peroxidation. Lipid peroxidation level was determined by quantifying the concentration of 2-thiobarbituric acid reactive substances (TBARS), expressed as |imol of malondialdehyde (MDA) per mg of protein, according to Kamyshnikov (2004) [11]. The TBARS level was expressed in nmol MDA per mg protein by using 1.56105 mM-1 cm-1 as molar extinction coefficient.
Carbonyl derivatives of oxidatively modified protein (OMP) assay. The rate of protein oxidative destruction was estimated from the reaction of the resultant carbonyl derivatives of amino acid reaction with DNFH as described by Levine et al. (1990) [12] and as modified by Dubinina et al. (1995) [13]. The carbonyl content was calculated from the absorbance measurement at 370 nm and 430 nm and an absorption coefficient 22,000 M-1xm-1. Carbonyl groups were determined spectrophotometrically from the difference in absorbance at 370 nm (aldehydic derivatives, OMP370) and 430 nm (ketonic derivatives, OMP430) and expressed in nmol per mg of tissue protein.
Alanine aminotransferase (ALT, E.C. 2.6.1.2) and aspartate aminotransferase (ALT, E.C. 2.6.1.1) activities assay. ALT and AST activity were analyzed spectrophotometrically by standard enzymatic method of Reitman and Frankel (1957) [14]. ALT and AST activities were expressed as |imol pyruvate per hour per mg of protein.
Lactate dehydrogenase (LDH, E.C. 1.1.1.27) activity. The colorimetric method of Sevela and Tovarek (1959) was used for the determination of LDH activity [15]. One unit/L of LDH is defined as the formation of 1 |imol of pyruvate per hour at 370C incubation per mg of protein.
Lactate and pyruvate concentration. Lactate and pyruvate concentrations were measured according to the procedure described by Herasimov and Plaksina (2000) [16]. Calibration curves of lactate (0.1-5 mM) and pyruvate (0.1-5 mM) were used, and results were expressed in nmol lactate per mg of protein or nmol pyruvate per mg of protein.
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 contents and enzyme activities (significance level, p<0.05) was examined using Kruskal-Wallis oneway analysis of variance by ranks test. Correlations between parameters at the set significance level were evaluated using Spearman's correlation analysis [17]. All statistical calculation was performed on separate data from each individual with STATISTICA 10.0.
RESULTS
The values of lipid peroxidation in the cardiac tissue of the males and females from control (healthy specimens) and furunculosis-affected trout are summarized in Table 1. No significant differences between healthy and furunculosis-affected males were observed, while TBARS level in cardiac tissue of females was non-significant lower (by 50%, p>0.05) compared to healthy trout (Table 1).
Table. Level of the lipid peroxidation and oxidatively modification of proteins biomarkers in the cardiac tissue of the furunculosis-affected sea trout from the Baltic sea (M±m)
Таблица. Содержание маркеров перекисного окисления липидов и окислительной модификации белков в сердечной ткани самцов и самок здоровой и пораженной
Oxidative stress biomarkers Males Females
Healthy trout Furunculosis-affected trout Healthy trout Furunculosis-affected trout
TBARS, nmol-mg-1 protein 1962.6±317.56 1970.79±266.02 2399.56±254.10** 847.11±63.72**
OMP370, nmol-mg-1 protein 19.14±2.28 55.89±9.85 18.70±1.59** 46.98±5.84**
OMP430, nmol-mg-1 protein 32.45±3.26* 73.67±12.38* 33.28±2.70** 63.61±7.07**
furunculosis-affected males;
**
the significant difference was shown as p<0.05 when compared healthy and furunculosis-affected females;
статистически достоверные изменения (p<0,05) в значениях между группой здоровых и пораженных фурункулезом самцов кумжи;
статистически достоверные изменения (p<0,05) в значениях между группой здоровых и пораженных фурункулезом самок кумжи.
Aldehydic derivatives of oxidatively modified proteins in the cardiac tissue of furunculosis-affected males and females were significantly increased (by 99%, p=0.037 and by 65%, p=0.000, respectively) compared to healthy trout (Fig. 3). Furunculosis caused a significant increase the ketonic derivatives level in the cardiac tissue of males by 63.5% (p=0.043) and females by 48.7% (p=0.000) compared to healthy trout. Higher level of ketonic derivatives of oxidatively modified protein was noted in cardiac tissue both in healthy females (by 26%, p=0.017) and furunculosis-affected females (by 14.5%, p=0.007) compared to respectively males groups (Table 1).
Furunculosis results in decrease of AST and LDH activities in the cardiac tissue of males (by 51.5%, p=0.023 and by 31.7%, p=0.004, respectively) (Fig. 1).
Fig. 1. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) activity in the cardiac tissue of the furunculosis-affected sea trout from the Baltic sea Data are represented as mean ± S.E.M. the significant difference was shown as p<0.05 when compared healthy and
furunculosis-affected males Рис. 1. Активность аланинаминотрансферазы (ALT), аспартатаминотрансферазы (AST) и лактатдегидрогеназы (LDH) в сердечной ткани здоровых и пораженных фурункулезом самцов и самок кумжи с
Балтийского моря
статистически достоверные изменения (p<0,05) в значениях между группой здоровых и пораженных фурункулезом самцов кумжи
Significant decrease of lactate level (by 60%, p=0.001) in the cardiac tissue of females as a consequence of furunculosis-induced oxidative stress were found. Higher lactate level (by 171%, p=0.000) was noted in healthy females compared to healthy males
(Fig. 2).
Fig. 2. Lactate and pyruvale levels in the cardiac tissue of the furunculosis-affected
sea trout from the Baltic sea Data are represented as mean ± S.E.M.
**
the significant difference was shown as p<0.05 when compared healthy and furunculosis-affected females;
# the significant difference was shown as p<0.05 when compared healthy males and
healthy females
Рис. 2. Содержание лактата и пирувата в сердечной ткани здоровых и пораженных фурункулезом самцов и самок кумжи с Балтийского моря статистически достоверные изменения (p<0,05) в значениях между группой здоровых и пораженных фурункулезом самок кумжи;
# статистически достоверные изменения (p<0,05) в значениях между группой
здоровых самок и самцов кумжи.
Several correlations between checked parameters were found (Figs 3 and 4). Cardiac TBARS level in healthy males correlated positively with lactate (r=0.531, p=0.013) and pyruvate (r=0.758, p=0.000) (Fig. 3A). TBARS level correlated inversely with ketonic derivatives of OMP (r=-0.847, p=0.000) and LDH activity (r=-0.664, p=0.000) in furunculosis-affected males (Fig. 3B).
TBARS:Lactate: y = 46.19 + 0.0 46*x; r = 0.531; p = o.of3= 0.282 TBARS:Pyruvate: y = -1.184 + o.fo 99*x; r = 0.758; p = 0.0O0 or.574
1 800
450 400
I 350 !
Д 150
TBARS:OMP430: y = 54.84 - 0.009*x; r = -0.847; p = 0.000; r2 = 0.717 TBARS:LDH: y = 10.279 - 0.0013*x; r = -0.664; P = 0.000; r2 = 0.441
%чОМР430
Ns LDH
E
400 "0
500 1000 1500 2000 2500 3000 3500 4000 450 TBARS,nmobmg protein
1500 2000 2500 3000 3500 4000 4500 5000 TBARS, nmol-mg'1 protein
A B
Fig. 3. Correlations between TBARS, lactate, and pyruvate levels in the cardiac tissue of healthy males (A), as well as between TBARS, ketonic derivatives of oxydatively modified proteins content, and LDH activity in the cardiac tissue of furunculosis-affected males of sea trout (B), which returning to spawn from the Baltic sea Рис. 3. Корреляционные зависимости между маркерами перекисного окисления липидов, содержанием лактата и пирувата (А), между маркерами перекисного окисления липидов и окислительной модификации белков, а также активностью лактатдегидрогеназы (В) в сердечной ткани пораженных фурункулезом самцов кумжи с Балтийского моря
90
14
700
80
12
600
70
10
Î
500 а
60
8
50
6
300
40
4
200
30
20
10
0
0
0
500
0
In both healthy females and furunculosis-affected females, ketonic derivatives of OMP were positively correlated with AST (r=0.907, p=0.000 and r=0.6403, p=0.004, respectively) and LDH activities (r=0.724, p=0.000 and r=0.622, p=0.006, respectively) (Fig. 4).
OMP430:AST: y = 1.34 + 0.131*x; r = 0.907; p = 0.000; r2 = 0.823 OMP^iAST: y = 7.83 + 0.094*x; r = 0.640; p = 0.004; r2 = 0.409
OMP430:LDH: y = 2.646 + 0.044*x; r = 0.724; p = 0.000; r2 = 0.525 OMP^zLDH: y = 1.029 + 0.063*x; r = 0.622; p = 0.006; r2 = 0.386
A B
Fig. 4. Correlations between ketonic derivatives of oxydatively modified proteins content, AST, and LDH activity in the cardiac tissue of healthy females (A), as well as in the cardiac tissue of furunculosis-affected females of sea trout (B), which returning to spawn from the Baltic sea Рис. 4. Корреляционные зависимости между кетоновыми производными окислительно-модифицированных белков, активностью аспартатаминотрансферазы и лактатдегидрогеназы в сердечной ткани здоровых самок (А) и пораженных фурункулезом
самок кумжи с Балтийского моря (В)
DISCUSSION
Our study centers on oxidative stress and biochemical responses in the cardiac tissue of sea trout which returning to spawn from the Baltic sea. The lipid peroxidation measured as TBARS level, the oxidatively modified proteins, such as aldehydic and ketonic derivatives as well as the activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities, lactate and pyruvate levels, and correlations between these indexes are described (Tables 1, Figs 1-4).
Furunculosis induces respiratory burst as response to Aeromonas spp. infection [18]. These authors sequentially challenged rainbow trout representing 15 full-sibling families twice with A. salmonicida causing furunculosis: first as cohabitation and then as injected intraperitoneally. The bleeding procedure prior to challenges caused the outbreak of cold water disease by Flavobacterium psychrophilum. Before and after the outbreak and challenges, 11 immunological parameters were measured from blood samples. The immunological responses predicted the fate of the fish since nearly all the initial responses were lower in individuals which later died from cold water disease than in survivors. Fish died from furunculosis had impaired respiratory burst response to A. salmonicida. The outbreak and challenges resulted in these individuals higher and faster responses compared with initial values. Unlike in mammals, the number of monocytes, but not that of granulocytes, in rainbow trout blood correlated well with the whole blood respiratory burst activity [18]. Moreover, the increase in respiratory burst activities and oxidative stress was non-specific and originated from the increased number of circulating granulocytes and monocytes during vaccination against Aeromonas salmonicida [19]. Results of Nikoskelainen et al. (2007) suggest that the monocytes play a more significant role than the granulocytes in antibody-dependent opsonophagocytosis during vaccination against A. salmonicida [19].
In this study, we have provided evidence that furunculosis induces the production of free radicals in the cardiac tissue of sea trout. Also, the role of ROS in furunculosis-affected trout is proposed. Pathogen invasion leads to oxidative burst and generation of active oxygen species, causing lipid peroxidation, oxidatively modification of proteins and membrane damage, which are believed to be key features of pathogenesis [2]. In the present study, protein oxidation mediated membrane damage was evident (Table 1).
High levels of aldehydic and ketonic derivatives of proteins oxidation were observed both in males and females of sea trout (Table 1). The assays showed that high protein oxidation occurred together with an alteration of the biochemical profile (decrease of AST and LDH activities). Moreover, furunculosis caused a decrease in lactate level in the cardiac tissue of infected sea trout (Fig. 1). This might be due to the inactivation of the aminotransferases and LDH activities by the end products of protein oxidation. The data indicated that these two events occur simultaneously. Therefore, we decided to conduct the correlative analysis between oxidative stress and biochemical biomarkers. In furunculosis-affected males, TBARS level correlated inversely with ketonic derivatives of OMP (r=-0.847, p=0.000) and LDH activity (r=-0.664, p=0.000) (Fig. 3B). In both healthy females and furunculosis-affected females, ketonic derivatives of OMP were positively correlated with AST (r=0.907, p=0.000 and r=0.6403, p=0.004, respectively) and LDH activities (r=0.724, p=0.000 and r=0.622, p=0.006, respectively) (Fig. 4). Therefore, while there is considerable evidence linking ROS accumulation to biochemical alteration, there are a confirm the intrinsic
relationship between ROS accumulation and biochemical activities occurrence and their regulation.
We observed significant decrease of lipid peroxidation in cardiac tissue of furunculosis-affected females; on the other hand, it was possible to note an increase in OMP level at this time. Synergism between AST and LDH activity and aldehydic and ketonic derivales of oxidatively modified proteins was also verified (Fig. 4). These results suggest that oxidative stress in cardiac tissue occurs more significantly in males of furunculosis-affected sea trout and contributes to the oxidative effect (Table 1). Moreover, these results complete the previous our studies reporting an inhibition of antioxidant defense system and activation of oxidative stress biomarkers in the blood, gills, liver, spawn, and muscles of sea trout with furunculosis [20, 21].
In summary, in both males and females, oxidative stress biomarkers (carbonyl derivatives of oxidatively modified proteins) in the cardiac tissue of furunculosis-affected trout showed higher values as compared to the healthy trout. Increase of protein oxidation causes to the inactivation of the aminotransferases and LDH activities and decrease of lactate level. Moreover, the decrease of aminotransferases and LDH modifies lactate-pyruvate ratio and causes to those depletion in cardiac tissue of furunculosis-affected trout. This study encourages efforts to extend the knowledge of oxidative stress biomarkers for the identification of Aeromonas-induced disorders and specific responses of fish typical of the furunculosis. Additionally, these findings are important to a better understanding of the mechanism of furunculosis infection in trout.
this work was supported by grant of the Pomeranian University for Young Scientists
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ИНФОРМАЦИЯ ОБ АВТОРАХ
Ткаченко Галина Михайловна - Институт биологии и охраны среды, Поморская Академия, г. Слупск, Польша, кафедра зоологии и физиологии животных; кандидат биологических наук, докторант; E-mail: tkachenko@apsl.edu.pl
Tkachenko Halyna Mikhailovna - Institute of Biology and Environmental Protection, Pomeranian University, Slupsk, Poland, Department of Zoology and Animal Physiology; Candidate of Biological Sciences, Assistant Professor; E-mail: tkachenko@apsl.edu.pl
Кургалюк Наталия Николаевна - Институт биологии и охраны среды, Поморская Академия, г. Слупск, Польша, кафедра зоологии и физиологии животных; доктор биологических наук, профессор; E-mail: biology.apsl@gmail.com
Kurhaluk Natalia Nikolaevna - Institute of Biology and Environmental Protection, Pomeranian University, Slupsk, Poland, Department of Zoology and Animal Physiology; Doctor of Biology, Professor; E-mail: biology.apsl@gmail.com
