231

Słupskie Prace Biologiczne

Nr 13 ss. 231-252 2016

ISSN 1734-0926 Przyjęto: 7.11.2016

© Instytut Biologii i Ochrony Środowiska Akademii Pomorskiej w Słupsku Zaakceptowano: 16.01.2017

OXIDATIVE STRESS BIOMARKERS IN THE MUSCLE

TISSUE OF GRAYLING (THYMALLUS THYMALLUS LINCK)

AFTER CHLORAMINE-T DISINFECTION

Halyna Tkachenko1

Joanna Grudniewska2

1 Pomeranian University in Słupsk, Poland Institute of Biology and Environmental Protection Department of Zoology and Animal Physiology Arciszewski St. 22B, 76-200 Słupsk e-mail:tkachenko@apsl.edu.pl 2 Department of Salmonid Research

Stanislaw Sakowicz Inland Fisheries Institute 83-330 Żukowo, Poland

ABSTRACT

Chloramine-T is a widely used disinfectant for the treatment of gill diseases of fish

in freshwater, and more recently attention has turned to its use in seawater. However,

despite the wide use of chloramine-T, only few studies have examined its toxicity to

fish (Powell and Harris 2004). Therefore, the aim of the current study was to examine

the effects of disinfection by Chloramine-T on the muscle tissue of grayling (Thy-

mallus thymallus Linck) using oxidative stress biomarkers [levels of 2-thiobarbituric

acid reactive substances (TBARS) and oxidative modified protein (OMP) derivatives]

and antioxidant defense (superoxide dismutase, catalase, glutathione reductase, gluta-

thione peroxidase, total antioxidant capacity) to observe the its toxic effects. The end-

points obtained from this study will be useful to monitor the effects of disinfectant

bathing with Chloramine-T for this species of fish. Our results showed that Chlora-

mine-T bathing markedly decrease lipid peroxidation with non-significant decrease of

aldehydic and ketonic derivatives of oxidatively modified proteins. However, reduced

lipid peroxidation results in decrease of total antioxidant capacity. Moreover, de-

creased lipid peroxidation level causes decrease of aldehydic (r = 0.854, p = 0.002)

and ketonic derivatives of oxidatively modified proteins (r = 0.852, p = 0.002). The

present study has revealed that fish developed tissue-specific enzyme responses, such

as decrease in superoxide dismutase and catalase activity as well as total antioxidant

capacity in muscle tissue with decrease of lipid peroxidation as response to the Chlo-

ramine-T disinfection. This data are compatible with lower antioxidant defense. Cor-

232

relative analysis has revealed positive correlations between oxidative stress biomarkers

(aldehydic and ketonic derivatives of oxidatively modified proteins, TBARS as marker

of lipid peroxidation) and antioxidant defenses. 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 treatment of grayling. However, more detailed studies on using of these

specific biomarkers to monitor the disinfectant treatment in aquaculture are needed.

Key words: Chloramine-T, disinfection, grayling Thymallus thymallus, muscle tis-

sue, lipid peroxidation, oxidatively modified proteins, antioxidant defense, total an-

tioxidant capacity

INTRODUCTION

With the continued expansion of cultured fish and shellfish species, aquaculture

has become a key component of the animal health industry (Shao 2001). The infec-

tious diseases in aquaculture are of major concern to the industry and are typically

controlled by eradication of the pathogen, treatment with antibiotic or chemothera-

peutics, and/or by preventative measures such as the use of probiotics or vaccines.

To limit the use of chemicals and antibiotics, good farm management is highly rec-

ommended. In terms of treatment, chemicals and antibiotics should be evaluated to

establish recommended doses and withdrawal periods, otherwise alternative treat-

ments should be developed (Chinabut and Puttinaowarat 2005). The ever-increasing

demand for quality pharmaceutical and biological products, to combat bacterial and

viral infections, calls for the development of modern formulations and novel drug

delivery systems (Shao 2001). The anti-infective agents are used for controlling dis-

eases 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. 1997).

Chloramine-T, as an anti-microbial agent, has had widespread use in a broad

range of practices, including medical, dental, veterinary, food processing, and agri-

cultural, as well as a disinfectant for disinfection surfaces and instruments. In agri-

cultural practices, Chloramine-T has been approved as a broadspectrum biocide for

foot-and-mouth disease, swine vesicular disease, diseases of poultry, and tuberculo-

sis, while in medicine in the treatment of burns, in whirlpools for the treatment of

wounds, and as an oral mouthwash (Chloramine-T…).

Chloramine-T (n-chloro-para-toluene sulfonamide sodium salt) has traditionally

been used to treat external bacterial infections in salmonid aquaculture (Bullock et

al. 1991). It has also been reported to be effective for the treatment of monogenean

trematodes (skin and gill flukes). Chloramine-T functions by slowly breaking down

to hypochlorous acid, with the release of oxygen and chlorine. The active ingredient

is p-toluene sulfonamide (Booth and MkDonald 1988). Toxicity, as well as effective

dose, is dependent upon water hardness, pH and temperature (Rach et al.1988).

Bacterial gill disease is caused by a variety of Gram-negative bacteria (myxobac-

teria, aeromonads, and pseudomonads) (Chloramine-T…). The disease is highly

contagious among cultured salmonids and can lead to substantial fish losses. An ap-

233

proved therapeutant to control bacterial gill disease is needed to enable the produc-

tion of salmonids for restoration of fish stocks and for sport and commercial fisher-

ies (Chloramine-T…). As a therapeutic agent, it is used as an effective treatment of

bacterial gill disease in freshwater or marine aquaria, garden ponds, or other aquatic

systems at concentrations ranging from 6.5 to 10.0 mg/l [23.1 to 35.5 µM] (Bullock

et al. 1991, Powell and Perry 1996) and as a preventative, prophylactic, and disin-

fectant treatment in many fresh water hatcheries (Chloramine-T…, Powell and Perry

1996, Thorburn and Moccia 1993).

Organic chlorine compounds (N-chloro compounds), which contain the = N – Cl

group, show microbicidal activity. Examples of such compounds, are chloramine-T,

dichloramine-T, halazone, halane, dichloroisocyanuric acid, sodium and potassium

dichloroisocyanurates and trichloroisocyanuric acid. All appear to hydrolyze in wa-

ter to produce an imino (= NH) group (Powell and Harris 2004, Principles…).

Their action is claimed to be slower than that of the hypochlorites, although this

can be increased under acidic conditions (LeChevallier et al. 1988). Experiments

where equal weights of disinfectants were used suggested that the greater penetrat-

ing power of monochloramine compensated for its limited disinfection activity.

Studies of LeChevallier and co-workers (1988) showed that monochloramine was as

effective as free chlorine for inactivation of biofilm bacteria. In fish studies, chlora-

mine was poorly absorbed from water, and that which was absorbed was rapidly me-

tabolized to the residue marker, p-Toluenesulfonamide. A second, as of yet unidenti-

fied, metabolite may also exist (Chloramine-T…).

However, despite the wide use of chloramine-T, only few studies have examined

its toxicity to fish (Powell and Harris 2004). Therefore, the aim of the current study

was to examine the effects of disinfection by Chloramine-T on the muscle tissue of

grayling (Thymallus thymallus Linck) using oxidative stress biomarkers (levels of 2-

thiobarbituric acid reactive substances and oxidative modified protein derivatives)

and antioxidant defense (superoxide dismutase, catalase, glutathione reductase, glu-

tathione peroxidase, total antioxidant capacity) to observe the its toxic effects. The

endpoints obtained from this study will be useful to monitor the effects of disinfect-

ant bathing with Chloramine-T for this species of fish.

234

MATERIALS AND METHODS

Fish. Twenty clinically healthy grayling (Thymallus thymallus) were used in the

experiments. The study was carried out in a Department of Salmonid Research, In-

land Fisheries Institute (Rutki, 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 Environ-

mental Protection, Pomeranian University in Słupsk (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 al-

ternate days, the water supply to each tank was stopped. In the disinfectant exposure,

grayling (n = 10) were exposed to Chloramine-T in final concentration 9 mg per l.

Control group of grayling (n = 10) 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 samples were removed from grayling af-

ter decapitation. One grayling was used for each homogenate preparation. Briefly,

muscle tissue were excised, weighted and washed in ice-cold buffer. The minced tis-

sue was rinsed clear of blood with cold isolation buffer and homogenized in a ho-

mogenizer H500 with a motor-driven 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 con-

centration in each sample was determined according to Bradford (1976) using bo-

vine serum albumin as a standard (Bradford 1976).

TBARS assay for lipid peroxidation. The level of lipid peroxidation was deter-

mined by quantifying the concentration of TBARS with the Kamyshnikov method

(2004) for determining the malondialdehyde (MDA) concentration. This method is

based on the reaction of the degradation of lipid peroxidation product, MDA, with

TBA under high temperature and acidity to generate a colored adduct that is meas-

ured spectrophotometrically. Briefly, 0.1 ml of sample was added to 2 ml of distilled

water, 1 ml of 20% TCA and 1 ml of 0.8% TBA. The mixture was heated in a boil-

ing water bath for 10 minutes. After cooling, the mixture was centrifuged at 3,000 g

for 10 minutes. The nmol of MDA per 1 mg of tissue protein was calculated by us-

ing 1.56 · 105 mM-1 · cm-1 as extinction coefficient.

The carbonyl derivatives content of protein oxidative modification (OMP) as-

say. 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 and co-workers (1990) and as modified by Dubinina and co-workers (1995).

DNPH was used for determining carbonyl content in soluble and insoluble proteins.

Briefly, 1 ml of 0.1M DNPH (dissolved in 2M HCl) was added to 0.1 ml of the sam-

235

ple after denaturation of proteins by 20% TCA. After addition of the DNPH solution

(or 2M HCl to the blanks), the tubes were incubated for a period of 1 h at 37oC. The

tubes were spun in a centrifuge for 20 min at 3,000 g. After centrifugation, the su-

pernatant was decanted and 1 ml of ethanol-ethylacetate solution was added to each

tube. Following mechanical disruption of the pellet the tubes were allowed to stand

for 10 min and then spun again (20 min at 3,000 g). The supernatant was decanted

and the pellet washed thrice with ethanol-ethylacetate. After the final wash, the pro-

tein was solubilized in 2.5 ml of 8 M urea solution. To speed up the solubilization

process, the samples were incubated in at 90oC water bath for 10-15 min. The final

solution was centrifuged to remove any insoluble material. The carbonyl content

was calculated from the absorbance measurement at 370 nm and 430 nm, and an ab-

sorption coefficient 22,000 M-1 · cm-1. Carbonyl groups were determined spectro-

fotometrically 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.

Superoxide dismutase activity assay. Superoxide dismutase (SOD, E.C. 1.15.1.1)

activity was assessed by its ability to dismutate superoxide produced during querce-

tin auto-oxidation in an alkaline medium (pH 10.0) by Kostiuk and co-workers

(1990) method. Briefly, 1.0 ml of C reagent was mixed with 0.1 ml of sample. C re-

agent was made ex tempore (mixture of equal volumes of 0.1M K,Na-phosphate

buffer, pH 7.8 and 0.08 M EDTA solution); pH of C reagent was adjusted to 10.0 by

adding TEMED. Distilled water (0.1 ml) was added to blank vials instead of blood

sample. The total volume of all samples was brought up to 2.4 mL using distilled

water. The reaction was initiated by adding 0.1 ml of quercetin (1.4 μM dissolved in

dimethyl sulphoxide). Absorbance at 406 nm was measured immediately and after

20 min addition of quercetin solution. Activity is expressed in units of SOD per mg

of tissue protein.

Catalase activity assay. Catalase (CAT, E.C. 1.11.1.6) activity was determined by

measuring the decrease of H2O2 in the reaction mixture using a spectrophotometer at

the wavelength of 410 nm by the method of Koroliuk and co-workers (1988). The

reaction was initialized by adding 0.1 ml of sample into the incubation medium

(2 ml of 0.03% H2O2 solution) and to 1.0 ml of 4% ammonium molybdate dissolved

in 12.5 mM H2SO4 solution (blank sample). The duration of reaction was 10 min at

room temperature. The reaction was terminated by rapid adding 1.0 ml of 4% am-

monium molybdate dissolved in 12.5 mM H2SO4 solution to incubation medium and

1 ml of 125 mM H2SO4 to all samples. All samples were centrifuged at 3,000 g for

5 min. The absorbance of the obtained solution was measured at 410 nm and com-

pared with that of the blank. One unit of catalase activity is defined as the amount of

enzyme required for decomposition of 1 μmol H2O2 per min per mg of tissue pro-

tein.

Glutathione reductase activity assay. Glutathione reductase (GR, EC 1.6.4.2) ac-

tivity in the tissue was measured according to the method described by Glatzle and

co-workers (1974). The enzymatic activity was assayed spectrophotometrically by

measuring NADPH2 consumption. In the presence of GSSG and NADPH, GR re-

duces GSSG and oxidizes NADPH2, resulting in a decrease in the absorbance at 340

nm. The enzyme assay mixture contained 2.4 ml of 67 mM sodium phosphate buffer

236

(pH 6.6), 0.2 ml of 7.5 mM oxidized glutathione, and 0.1 ml of sample. The rate of

NADPH oxidation was followed spectrophotometrically at 340 nm. Quantification

was performed based on a molar extinction coefficient of 6.22 mM-1·cm-1 of NADPH.

The GR activity was expressed as nmol NADPH per min per mg of tissue protein.

Glutathione peroxidase activity assay. Glutathione peroxidase (GPx, EC

1.11.1.9) activity was determined by detecting the nonenzymatic utilization of GSH

(the reacting substrate) at an absorbance of 412 nm after incubation with 5,5-

dithiobis-2-nitrobenzoic acid (DTNB) according by the method of Moin (1986). The

assay mixture contained 0.8 ml of 0.1 M Tris-HCl buffer with 6 mM EDTA and

12 mM sodium azide (pH 8.9), 0.1 ml of 4.8 mM GSH, 0.2 ml of sample, 1 ml of

20 mM t-butylhydroperoxide, and 0.1 ml of 0.01 M 5,5-dithiobis-2-nitrobenzoic

acid. The rate of GSH reduction was followed spectrophotometrically at 412 nm.

GPx activity is expressed as μmol GSH per min per mg of tissue protein.

Total antioxidant capacity assay. The TAC level in the sample was estimated by

measuring the TBARS level following Tween 80 oxidation. This level was deter-

mined spectrophotometrically at 532 nm by Galaktionova and co-workers (1998).

Plasma inhibits the Fe2+/ascorbate-induced oxidation of Tween 80, resulting in a de-

crease in the TBARS level. Briefly, 0.1 ml of sample were added to 2 ml of 1%

Tween 80 reagent, 0.2 ml of 1 mM FeSO4, and 0.2 mM of 10 mM ascorbic acid. In

the blank assay, 0.1 ml of distilled water were used instead of the sample. The mix-

ture was heated in a boiling water bath for 48 h at 37oC. After cooling, 1 ml of 20%

TCA was added. The mixture was centrifuged at 3,000 · g for 10 min. After centrif-

ugation, 2 ml of supernatant and 2 ml of 0.25% of TBA reagent were mixed. The

mixture was heated in a boiling water bath at 95oC for 15 min. The absorbance of

the obtained solution was measured at 532 nm. The absorbance of the blank was de-

fined as 100%. The level of TAC in the sample (%) was calculated with respect to

the absorbance of the blank.

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 (signifi-

cance level, p < 0.05) was examined using Mann-Whitney U test. Correlations be-

tween parameters at the set significance level were evaluated using Spearman’s cor-

relation analysis (Zar 1999). All statistical calculation was performed on separate

data from each individual with STATISTICA 8.0.

RESULTS

Influence of chloramine-T on lipid peroxidation biomarker, measured as

2-thiobarbituric acid reactive substances in the muscle tissue of grayling are present-

ed in Fig. 1А. Significantly lower TBARS level (by 39%, p = 0.004) in grayling dis-

infected by chloramine-T compared to control group was observed (Fig. 1А).

237

A

B

Fig. 1. Influence of Chloramine-T on lipid peroxidation biomarker, measured as 2-thio-

barbituric acid reactive substances (А), as well as aldehydic and ketonic derivatives of oxi-

datively modified proteins (B) in the muscle tissue of grayling (Thymallus thymallus)

Data are represented as mean ± S.E.M. * the significant change was shown as p < 0.05 when compared values of unhandled (n = 10)

and disinfected groups (n = 10)

Source: own research

Aldehydic and ketonic derivatives of oxidatively modified proteins in the muscle

tissue of grayling disinfected by chloramine-T were non-significantly lower com-

pared to controls (Fig. 1В).

The main enzymatic antioxidants include superoxide dismutase (SOD), glutathione

peroxidase (GPx), and catalase (CAT). Each of these enzymes is responsible for the

nm

ol

∙ m

g-1

pro

tein

nm

ol

∙ m

g-1

pro

tein

238

reduction of different reactive oxygen species (ROS), and they are located in different

cellular compartments (Nunes-Silva and Freitas-Lima 2015). Antioxidant defense in

the muscle tissue of grayling disinfected by Chloramine-T are shown in Fig. 2.

A B

C D

Fig. 2. Antioxidant enzymes’ activities in the muscle tissue of grayling disinfected by Chlo-

ramine-T

Data are represented as mean ± S.E.M. * the significant change was shown as p < 0.05 when compared values of unhandled (n = 10)

and disinfected groups (n = 10)

Source: own research

There were no statistically significant alterations in the activities of antioxidant de-

fenses instead CAT and SOD activity in the muscle tissue of grayling disinfected by

Chloramine-T (Fig. 2A, 2B). The CAT activity was lower by 72% (p = 0.001) after

Chloramine-T influence. The SOD activity in the muscle tissue of grayling disinfected

by Chloramine-T was also decreased by 15% (p = 0.001) compared to the controls. SOD

acts on superoxide radicals to form oxygen and the lesser reactive non-radical species,

hydrogen peroxide, while GR can regenerate oxidized glutathione (GSSG, glutathione

disulfide) to reduced glutathione (GSH). The CAT is located mainly in peroxisomes and

mitochondria and also removes H2O2. CAT requires iron as a cofactor, and similar to

GPx and SOD, its activity is highest in highly oxidative muscle fibers (Gomes et al.

2012).

Significant decrease of TAC level (by 13%, p = 0.026) in the muscle tissue of

grayling as a consequence of bathing with Chloramine-T was found (Fig. 3).

239

Fig. 3. Influence of Chloramine-T on total antioxidant capacity in the muscle tissue of gray-

ling (Thymallus thymallus)

Data are represented as mean ± S.E.M. * the significant change was shown as p < 0.05 when compared values of unhandled (n = 10)

and disinfected groups (n = 10)

Source: own research

Several correlations between checked parameters were found (Fig. 4). Muscle alde-

hydic derivatives of OMP correlated positively with TBARS (r = 0.854, p = 0.002) and

catalase activity (r = 0.734, p = 0.016), ketonic derivatives of OMP correlated positively

with TBARS (r = 0.852, p = 0.002) and aldehydic derivatives of OMP (r = 0.965,

p = 0.000), catalase activity correlated positively with TBARS (r = 0.735, p = 0.015) and

GR activity (r = 0.714, p = 0.020), as well as GR activity correlated positively with

TBARS (r = 0.827, p = 0.003) and GPx activity (r = 0.725, p = 0.018) (Fig. 4).

OMP370:TBARS: y = 40.55 + 3.74*x; r = 0.854; p = 0.002; r2 = 0.729

OMP370:Catalase: y = 0.145 + 0.149*x; r = 0.734; p = 0.016; r2 = 0.539

5 10 15 20 25 30 35 40

Aldehydic derivatives of OMP, nmol·mg-1 protein

40

60

80

100

120

140

160

180

TB

AR

S,

nm

ol·

mg

-1 p

ro

tein

-1

0

1

2

3

4

5

6

Ca

tala

se

, µ

mo

l H

2O

2·m

in-1

·mg

-1 p

ro

tein

TBARS

Catalase

240

OMP430:TBARS: y = 32.35 + 7.54*x; r = 0.852; p = 0.002; r2 = 0.725

OMP430:OMP370: y = -1.736 + 1.947*x; r = 0.965; p = 0.000; r2 = 0.931

2 4 6 8 10 12 14 16 18

Ketonic derivatives of OMP, nmol·mg-1 protein

40

60

80

100

120

140

160

180T

BA

RS

, n

mo

l·m

g-1

pr

ote

in

5

10

15

20

25

30

35

40

Ald

eh

yd

ic d

er

iva

tiv

es

of

OM

P,

nm

ol·

mg

-1 p

ro

tein

TBARS

OMP370

Catalase:TBARS: y = 54.249 + 15.79*x; r = 0.735; p = 0.015; r2 = 0.540

Catalase:GR: y = 4.22 + 1.868*x; r = 0.714; p = 0.020; r2 = 0.510

-1 0 1 2 3 4 5 6

Catalase, µmol H2O2·min-1·mg-1 protein

40

60

80

100

120

140

160

180

TB

AR

S,

nm

ol·

mg

-1 p

ro

tein

0

2

4

6

8

10

12

14

16

Glu

tath

ion

e r

ed

uc

tas

e,

nm

ol

NA

DP

H2·m

in-1

·mg

-1 p

ro

tein

TBARS

GR

241

GR:TBARS: y = 31.41 + 6.79*x; r = 0.827; p = 0.003; r2 = 0.684

GR:GPx: y = 146.86 + 11.07*x; r = 0.725; p = 0.018; r2 = 0.526

0 2 4 6 8 10 12 14 16

Glutathione reductase, nmol NADPH2·min-1·mg-1 protein

40

60

80

100

120

140

160

180T

BA

RS

, n

mo

l·m

g-1

pr

ote

in

140

160

180

200

220

240

260

280

300

320

340

360

Glu

tath

ion

e p

er

ox

ida

se

, µ

mo

l G

SH

·min

-1·m

g-1

pr

ote

in

TBARS

GPx

Fig. 4. Correlations between TBARS, aldehydic and ketonic derivatives of oxidatively modi-

fied proteins content and antioxidant enzymes activity in the muscle tissue of grayling disin-

fected by chloramine-T (n = 10)

Source: own research

DISCUSSION

Our results showed that chloramine-T bathing markedly decrease lipid peroxida-

tion with non-significant decrease of aldehydic and ketonic derivatives of oxidative-

ly modified proteins (Figs 1А and 1В). However, reduced lipid peroxidation results

in decrease of total antioxidant capacity (Fig. 3). Moreover, decreased lipid peroxi-

dation level causes to decrease of aldehydic (r = 0.854, p = 0.002) and ketonic deriv-

atives of oxidatively modified proteins (r = 0.852, p = 0.002) (Fig. 4).

In our previous study (Tkachenko et al. 2012, Tkachenko and Grudniewska

2015, Tkachenko and Grudniewska 2016a-e), we assessed the influence of chlora-

mine-T on oxidative stress biomarkers and metabolic alterations in various tissues of

grayling and rainbow trout. Chloramine-T bathing markedly decrease aldehydic and

ketonic derivatives of oxidative protein, and aminotransferases activity only in rain-

bow trout liver, and their elevation is a compensatory mechanism to impaired me-

tabolism. No significant changes were found in oxidative stress biomarkers between

control and chloramine-treated brown trout. For grayling, Chloramine-T exposure

caused significantly elevation in the levels of severe oxidative stress biomarkers in

the liver. Increased aldehydic and ketonic derivatives of oxidative protein could

242

modify lactate and pyruvate levels, aminotransferases and lactate dehydrogenase ac-

tivities, principally causing increased enzymes activity due to oxidative stress in the

liver of chloramine-exposed fish (Tkachenko et al. 2012). Our results also showed

that chloramine-T bathing markedly increase aldehydic and ketonic derivatives of

oxidative protein in hepatic tissue, while significantly decrease of carbonyl deriva-

tives in cardiac tissue of grayling was observed (Tkachenko and Grudniewska 2015,

2016c). In the muscle tissue of grayling, chloramine-T bathing markedly decrease

lipid peroxidation with non-significant decrease of aldehydic and ketonic derivatives

of oxidative proteins. However, reduced lipid peroxidation results in decrease of to-

tal antioxidant capacity. Moreover, decreased lipid peroxidation level causes de-

crease of aldehydic and ketonic derivatives of oxidatively modified proteins (Tka-

chenko and Grudniewska 2016d). Our results also showed that Chloramine-T non-

significantly decrease lipid peroxidation as well as aldehydic and ketonic derivatives

of oxidative proteins in the gills of grayling. No statistically significant alterations in

the activities of antioxidant defenses instead catalase and superoxide dismutase ac-

tivity in the gill tissue of grayling disinfected by Chloramine-T were noted (Tka-

chenko and Grudniewska 2016b).

The effects of disinfection by Chloramine-T using oxidative stress biomarkers

(levels of 2-thiobarbituric acid reactive substances and derivatives of oxidatively

modified proteins) and biochemical enzymes’ activity [alanine- and aspartate ami-

notransferases (ALT and AST), lactate dehydrogenase (LDH)] were assessed in the

muscle tissue of rainbow trout (Oncorhynchus mykiss Walbaum) (Tkachenko and

Grudniewska 2016a). Our results showed that Chloramine-T bathing caused the de-

crease of the lipid peroxidation as well as ALT and AST activity and significant de-

crease of LDH activity (by 339%, p = 0.017) compared to controls. Chloramine-T

markedly affects on lactate and pyruvate metabolism and resulted to decrease of

LDH activity. Correlative analysis revealed that the lipid peroxidation level is corre-

lated 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 activity. Thus, the skeletal muscles of fish play an im-

portant role in the processing of lactate through the gluconeogenic and glycogenic

pathways including a greater potential for biosynthesis (Tkachenko and Grudniew-

ska 2016a, e).

Accumulating evidence of other researchers has shown that Chloramine-T causes

oxidative stress by inducing the generation of reactive oxygen species (ROS)

(Tatsumi 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 oxi-

dation of intracellular sulfhydryls (Tatsumi and Fliss 1994). Sakuma and co-workers

(2009) assessed the influence of monochloramine on the conversion of xanthine de-

hydrogenase into xanthine oxidase in rat liver in vitro. When incubated with the par-

tially purified cytosolic fraction from rat liver, monochloramine (2.5-20 µM) dose-

dependently enhanced xanthine oxidase activity concomitant with a decrease in xan-

thine dehydrogenase activity, implying that monochloramine can convert xanthine

dehydrogenase into the ROS producing form xanthine oxidase. It was found that

monochloramine could increase ROS generation in the cytoplasm of rat primary

243

hepatocyte cultures, and that this increase might be reversed by an xanthine oxidase

inhibitor, allopurinol. These results suggest that monochloramine has the potential to

convert xanthine dehydrogenase into xanthine oxidase in the liver, which in turn

may induce the ROS generation in this region (Sakuma et al. 2009). Moreover, Shim

and co-workers (2013) have identified the subacute inhalation toxicity of chloramine

T under whole-body inhalation exposure conditions. Male and female groups of rats

were exposed to chloramine-T at concentrations of 0.2, 0.9 and 4.0 mg/m³ for

6 hr/day, 5 days/week during 4 weeks. After 28-day repeated inhalation of chlora-

mine-T, there were dose-dependently significant DNA damage in the rat tissues

evaluated and inflammation was histopathologically noted around the terminal airways

of the lung in both genders. As a result of the expression of three types of antioxidant

enzymes (SOD-2, GPx-1, PRX-1) in rat’s lung after exposure, there was no significant

change of all antioxidant enzymes in the male and female rats. The results showed that

no observed adverse effect level (NOAEL) was 0.2 mg/m³ in male rats and 0.9 mg/m³

in female rats under the present experimental condition (Shim et al. 2013).

On the other hand, HOCl and related oxidants such as N-chloramines may damage

DNA (Stanley et al. 2010). There is a strong link between chronic inflammation and the

incidence of many cancers caused by HOCl and related oxidants such as N-chloramines

(Stanley et al. 2010). Stanley and co-workers (2010) examined the ability of HOCl and

various N-chloramines to form chlorinated base products on nucleosides, nucleotides,

DNA, and in cellular systems. Experiments were performed with N-chloramines formed

on Nα-acetyl-histidine (His-C), Nα-acetyl-lysine (Lys-C), glycine (Gly-C), taurine (Tau-

C), and ammonia (Mono-C). Treatment of DNA and related materials with HOCl and

Nα-acetyl-histidine resulted in the formation of 5-chloro-2'-deoxycytidine, 8-chloro-2'-

deoxyadenosine and 8-chloro-2'-deoxyguanosine. Cellular RNA was also a target for

HOCl and His-C, with evidence for the formation of 5-chloro-cytidine. HOCl and the

model N-chloramine, His-C, are able to chlorinate cellular genetic material, which may

play a role in the development of various inflammatory cancers (Stanley et al. 2010).

On the other hand, there are many evidences that chloramine-T could be toxic for

fish. For example, effect of prophylactic chloramine-T treatment on growth perfor-

mance and condition indices of rainbow trout (Oncorhynchus mykiss) have been

studied by Sanchez and co-workers (1997a-c). Using a 24-tank replicate growth as-

say system, rainbow trout (average weight 98 g) were exposed twice weekly to chlo-

ramine-T at 10 mg/l for 1 hour, throughout an 11-week growth trial and compared to

matched controls. Fish were fed ad libitum without feed wastage to assess appetite

and feed conversion. Growth parameters were assessed every 3 weeks, at the end of

weeks 3, 6, 9, and 11. Chloramine-T treatment was not associated with either clinical

disease or mortality. Final weight and specific growth rate were significantly impaired

during the growth trial in the groups of fish treated with chloramine-T compared to

controls. This was attributed to a significant depression of feed conversion efficiency

and to a minor depression in appetite in treated fish (Sanchez et al. 1997b, c). Chlora-

mine-T treatment (at 10 mg/l for 1 h twice weekly for 11 weeks) was not associated

with either clinical disease or mortality. However, by the end of the trial, growth

(based on body weight) of treated fish was significantly suppressed compared with

control fish. Growth suppression was attributed to a significant reduction of feed

conversion efficiency in treated fish. Based on specific growth rates, chloramine-T

244

had an early negative effect on growth. The effect was diminished in later weeks

(although not completely lost), which suggests some degree of compensation by the

fish to the chemical agent (Sanchez et al. 1996).

Sanchez and co-workers (1998) have concluded that although chloramine-T and

formalin may continue to be useful in the aquaculture industry they cause potentially

harmful alterations to fish skin. Repeated treatment, once weekly for 4 weeks,

caused to increase numbers of highly dense vesicles within the apical portions of ep-

ithelial cells. The epidermal mucous cells of chloramine-T-treated fish were signifi-

cantly 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). The use of chloramine-T (10 mg/l, treating twice weekly for

11 weeks with a one-hour static bath) evoked a slight increase in mean lamellar

width, but it did not induce lamellar oedema, lamellar fusion, tissue infiltration, epi-

thelial hyperplasia, chloride cell metaplasia or thrombosis of pillar channels in treat-

ed fish. Treatment caused a trend towards an increased number of mucous cells on

lamellae, associated with a significant shift from neutral mucin to acid mucin pro-

duction based on histochemical characteristics (Sanchez et al. 1997).

The stress response in healthy juvenile rainbow trout after repetitive intermittent

treatment with chloramine-T or formalin also have evaluated by Sanchez and co-

workers (1997b). Tanks of healthy juvenile rainbow trout were exposed to chlora-

mine-T (10 mg/l; 40 fish) or formalin (200 mg/l; 40 fish) for 1 h once per week for

4 weeks. The effect of this treatment on the primary stress response was evaluated

by measuring circulating cortisol levels with a radioimmunoassay technique. Blood

cortisol levels were analyzed at 1, 24, and 96 h after each treatment and compared

with pre-exposure baseline values and with values obtained from sham-treated fish.

At 1 h, fish in all treatment categories had elevated cortisol levels compared with

baseline values, but values in those fish treated with chemicals were no different

from those that were sham treated. Cortisol levels returned to near baseline by 24 h

after treatment. In a second experiment, the effect of twice weekly 1-h exposure to

chloramine-T (10 mg/l) on secondary stress indices of rainbow trout was probed

during an 11-week growth trial by measuring haematocrit, plasma glucose, sodium,

and chloride levels in treated, untreated, and sham-treated fish. No evidence of

a secondary stress response could be detected in fish treated with chloramine-T

when they were compared with either control group. It is concluded that intermittent

exposure to chloramine-T at 10 mg/litre does not elicit a primary or secondary stress

response in rainbow trout and that stress is not the mechanism responsible for

growth deceleration in treated fish (Sanchez et al. 1997).

The effects of repeated intermittent exposure of healthy rainbow trout fingerlings

to sublethal concentrations of chloramine-T (0,5, 10, or 20 mg/l) twice weekly in 1-h

pulses at 11oC for 4 weeks in a replicate-tank facility were examined by Powell and

co-workers (1995). Gills were excised from subsamples of fish before exposure and

at the end of the 4-week experimental period. The gill epithelium from fish treated

with 10 and 20 mg/l chloramine-T appeared swollen and vacuolated, with extensive

intercellular oedema. There was a significant reduction in the number of lamellar

mucous cells and an apparent increase in the numbers of chloride cells. Chloride

cells from both the base of the lamella and the lamellar surface of gills exposed to

245

chloramine-T had an increase in the area of the apical plasmalemma after treatment

with 10 and 20 mg/l, and a reduction in the thickness of the apical plasmalemma-

associated glycocalyx. These morphological changes are consistent with a compen-

satory mechanism for the remedial uptake of ions, suggesting that chloramine-T in-

creased epithelial ion permeability coincident with a possible influx of water leading

to intercellular oedema. Chloride cell proliferation and intercellular oedema may al-

so have affected gas exchange across the branchial epithelium (Powell et al. 1995).

Powell and co-workers (1998) also have examined The physiological effects of

repeated exposure to 9 mg l-1 chloramine-T, a common aquaculture disinfectant for

rainbow trout using a graded hypoxic challenge. Using an extracorporeal circulation,

continuous measurements of blood PO2, PCO2 and pH were made and correlated

with decreasing water PO2. Ventilation amplitude and frequency were also moni-

tored. Following the graded hypoxic challenge, the gills were removed and pro-

cessed for microscopy for morphometric measurements and the determination of the

number of branchial mucous cells. Fish treated with chloramine-T exhibited a higher

arterial PO2 during hypoxia between 10.1 and 11.2 kPa when compared with un-

treated controls; there were no differences in arterial PCO2 or pH between the two

groups. Chloramine-T-treated fish had an elevated pre-hypoxic ventilation frequency

as compared with the controls. However, under the graded hypoxia, control fish ele-

vated their ventilation frequency, whereas chloramine-T-treated fish did not. Both

chloramine-T-treated and control fish increased their ventilation amplitude during

the graded hypoxia and there were no differences between control and chloramine-T-

treated fish. The fish treated with chloramine-T had a reduced thickness of the gill

epithelial blood-to-water diffusion barrier but higher numbers of mucous cells as

compared with controls. We suggest that although there was a mucous cell hyper-

plasia in response to repeated chloramine-T exposure, the thinning of the lamellar

epithelium was sufficient to offset any diffusive limitations, thus ensuring that gas

exchange was not adversely affected (Powell et al. 1998).

Sirri and co-workers (2013) have tested two concentrations of water disinfectants,

chloramine-T and peracetic acid, on Garra rufa to ascertain possible exposure damage

to the epidermis and gills. Fish were exposed to 2 mg/l and 10 mg/l of chloramine-T

and to 15 µl/l and 45 µl/l of peracetic acid in a 40-minute static bath up to six times

a day for one week. The epidermis and gills were checked for histological changes and

the number of epidermal mucous cells, club cells and taste buds were quantified; mu-

cous cells were also characterized histochemically to detect alterations in mucin pro-

duction. No mortality or severe histological changes were found in treated or control

fish. Cell count showed a significant increase (p < 0.05) in mucous cells (mean 49.1 ±

6.7 vs 37.0 ± 13.1 of controls) in animals treated with peracetic acid independently of

the dose. Club cell number showed a significant (p < 0.05) decrease in fish treated

with 2 mg/l of chloramine- (mean 74.3 ± 15.6) and with 45 µl/1 of peracetic acid

(mean 78.17 ± 10.5) compared to controls (mean 107.0 ± 19.2). Histochemical evalua-

tion of mucous cells did not reveal changes in mucin type in fish exposed to the two

disinfectants. The results suggest a good tolerability of Garra rufa to the two disin-

fectants at the concentrations tested (Sirri et al. 2013).

Cellular oxidative stress occurs when pro-oxidant forces overwhelm antioxidant

defenses. These antioxidant defenses comprise enzymatic and non-enzymatic mech-

246

anisms (Xia et al. 2013). SOD is important in the disproportionation of superoxide

anions into hydrogen peroxide (H2O2) and dioxygen (Filipak Neto et al. 2008). The

SOD-CAT system provides the first line of defense against oxygen toxicity and is

usually used as a biomarker of ROS production (Li et al. 2011). Results of the pre-

sent investigation indicated that Chloramine-T significantly decrease the SOD and

CAT activity. The alteration of SOD and CAT activity revealed that muscle tissue

might suffer from oxidative stress. This result is consistent with a previous study

performed with fish exposed to carbamazepine (Li et al. 2011). Similarly, Zhang and

co-workers (2004) found that SOD activity decreased gradually as the concentration

of 2,4-dichlorophenol increased. In addition, the decrease of SOD may be the result

of adaptation or loss in compensatory mechanisms (Jifa et al. 2006).

CAT is mainly located in the peroxisomes and, along with glutathione peroxi-

dase, is responsible for the reduction of H2O2 produced from the metabolism of long

chain fatty acids in peroxisomes (Filipak Neto et al. 2008). CAT has one of the high-

est turnover rates of all enzymes: one molecule of CAT can convert millions of mol-

ecules of hydrogen peroxide to water and oxygen per second (El-Gendy et al. 2009).

In the current study, muscle CAT activity in grayling was significantly inhibited by

Chloramine-T. This reduction demonstrates that Chloramine-T induces peroxidative

damage in the muscle tissue by altering the levels of CAT. Such disruption of antiox-

idant systems would enhance the generation of ROS and produce more serious oxi-

dative damage to tissues (Ferreira et al. 2010).

These enzymes can be induced by reactive oxygen species and they may be use-

ful indicators of oxidative stress. The induction of antioxidants can provide sensitive

early warning signals of incipient oxidative stress. Of the antioxidant enzymes, SOD

and CAT are considered as the first-line defenses against oxidative stress. They have

related functions and are essential for the conversion of ROS to harmless metabo-

lites. To be specific, SOD catalyzes dismutation of superoxide radical anion to H2O

and H2O2, and CAT reduces H2O2 to less toxic H2O and O2. In the present study,

SOD and CAT activities were significantly decreased in chloramine-T-treated

groups. The inhibitive response of the enzymes possibly suggested a failure of the

antioxidant system in keeping the antioxidant defense balance, which could be due

to the excessive ROS production under Chloramine-T exposure, resulting in the ac-

cumulation of the oxidative substances in the cells (Li et al. 2016). Correlative anal-

ysis has revealed positive correlations between oxidative stress biomarkers (aldehy-

dic and ketonic derivatives of oxidatively modified proteins, TBARS as marker of

lipid peroxidation) and CAT activity (r = 0.734, p = 0.016; r = 0.735, p = 0.015)

(Fig. 4).

Following exposure to Chloramine-T, non-significant alterations in glutathione

(GSH)-related enzymes activity were observed in the fish. As the second-line de-

fenses against oxidative damage, GSH and GSH-related enzymes play a major role

in free radical scavenging and reduction of peroxides (Li et al. 2016). GSH is a low-

molecular-weight, non-enzymatic antioxidant that facilitates the removal of oxyradi-

cals from cells by its sulfhydryl group. Oxidative stress caused by xenobiotics can

result in the depletion of GSH, which will reduce the cellular ability to scavenge free

radicals, raising the general oxidative potential in the cells. Variation of GSH level

has been considered as an indicator of the degree of exposure to xenobiotics in fish

247

(Li et al. 2016). Correlative analysis also has revealed positive correlations between

GR activity and TBARS (r = 0.827, p = 0.003) as well as GPx activity (r = 0.725,

p = 0.018) (Fig. 4).

CONCLUSIONS

The current study investigated the biological effects of Chloramine-T on the oxi-

dative stress biomarkers (levels of 2-thiobarbituric acid reactive substances and oxi-

dative modified protein derivatives) and antioxidant defense (superoxide dismutase,

catalase, glutathione reductase, glutathione peroxidase, total antioxidant capacity) in

the muscle tissue of grayling. The present study has revealed that fish developed tis-

sue-specific enzyme responses, such as decrease in superoxide dismutase and cata-

lase activity as well as total antioxidant capacity in muscle tissue with decrease of

lipid peroxidation as response to the Chloramine-T disinfection. This data are com-

patible with lower antioxidant defense. Correlative analysis has revealed positive

correlations between oxidative stress biomarkers (aldehydic and ketonic derivatives

of oxidatively modified proteins, TBARS as marker of lipid peroxidation) and anti-

oxidant defenses. 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 treatment

of grayling. 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 Scien-

tists.

REFERENCES

Booth N.H., MkDonald L.E. 1988. Veterinary Pharmacology and Therapeutics. Iowa State

Press, Ames Iowa: 774-777.

Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quanti-

ties of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254.

Bullock G.L., Herman R.L., Waggy C. 1991. Hatchery efficacy trials with Chloramine-T for

control of bacterial gill disease. J. Aquat. Anim. Heal., 3: 48-50.

Burka J.F., Hammell K.L., Horsberg T.E., Johnson G.R., Rainnie D.J., Speare D.J. 1997.

Drugs in salmonid aquaculture – a review. J. Vet. Pharmacol. Ther., 20(5): 333-349.

Chinabut S., Puttinaowarat S. 2005. The choice of disease control strategies to secure interna-

tional market access for aquaculture products. Dev. Biol. (Basel), 121: 255-261.

Chloramine-T [127-65-1] and Metabolite p-Toluenesulfonamide [70-55-3]. Review of Toxi-

cological Literature. Prepared for Scott Masten, Ph.D., National Institute of Environmen-

tal Health Sciences, Submitted by Karen E. Haneke, M.S. Integrated Laboratory Systems,

2002.

Dubinina E.E., Burmistrov S.O., Khodov D.A., Porotov I.G. 1995. Oxidative modification of

human serum proteins. A method of determining it. Vopr. Med. Khim., 41: 24-26 (in

Russian).

El-Gendy K.S., Radwan M.A., Gad A.F. 2009. In vivo evaluation of oxidative stress bi-

omarkers in the land snail, the a pisana exposed to copper-based pesticides. Chemosph.,

77(3): 339-344.

248

Ferreira D., da Motta A.C., Kreutz L.C., Toni C., Loro V.L., Barcellos L.J. 2010. Assessment of

oxidative stress in Rhamdia quelen exposed to agrichemicals. Chemosph., 79(9): 914-921.

Filipak Neto F., Zanata S.M., Silva de Assis H.C., Nakao L.S., Randi M.A., Oliveira Ribeiro

C.A. 2008. Toxic effects of DDT and methyl mercury on the hepatocytes from Hoplias

malabaricus. Toxicol. In Vitro, 22(7): 1705-1713.

Galaktionova L.P., Molchanov A.V., Elchaninova S.A., Varshavskiy B.Y. 1998. The lipid

peroxidation processes in patients with ulcerous illness of stomach and duodenum. Clin.

Lab. Diagnostics, 6: 10-14 (in Russian).

Glatzle D., Vuilleumier J.P., Weber F., Decker K. 1974. Glutathione reductase test with

whole blood, a convenient procedure for the assessment of the riboflavin status in human.

Experien., 30: 665-667.

Gomes E.C., Silva A.N., Rubino de Oliveira M. 2012. Oxidants, antioxidants and the benefi-

cial roles of exercise-induced production of reactive species. Oxidative Medic. and Cellul.

Longev., 2012: 1-12.

Jifa W., Yu Z., Xiuxian S., You W. 2006. Response of integrated biomarkers of fish

(Lateolabrax japonicus) exposed to benzo[a]pyrene and sodium dodecylbenzene sul-

fonate. Ecotoxicol. Environ. Saf., 65(2): 230-236.

Kamyshnikov V.S. 2004. Reference book on clinic and biochemical researches and laborato-

ry diagnostics. MEDpress-inform, Moscow (in Russian).

Koroliuk M.A., Ivanova L.I., Maĭorova I.G., Tokarev V.E. 1988. A method of determining

catalase activity. Laborotor. Delo, 1: 16-19 (in Russian).

Kostiuk V.A., Potapovich A.I., Kovaleva Zh.V. 1990. A simple and sensitive method of de-

termination of superoxide dismutase activity based on the reaction of quercetin oxidation.

Vopr. Meditsin. Khimii, 36: 88-91 (in Russian, abstract in English).

LeChevallier M.W., Cawthon C.D., Lee R.G. 1988. Inactivation of biofilm bacteria. Appl.

Environ. Microbiol., 54(10): 2492-2499.

Levine R.L., Garland D., Oliver C.N., Amici A., Climent I., Lenz A.-G., Ahn B.-W., Shaltiel

S., Stadtman E.R. 1990. Determination of carbonyl content in oxidatively modified pro-

teins. Methods Enzymol., 186: 465-478.

Li C., Qin L., Qu R., Sun P., Wang Z. 2016. Responses of antioxidant defense system to

polyfluorinated dibenzo-p-dioxins (PFDDs) exposure in liver of freshwater fish Carassius

auratus. Ecotoxicol. Environ. Saf., 126: 170-176.

Li Z.H., Zlabek V., Velisek J., Grabic R., Machova J., Kolarova J., Li P., Randak T. 2011.

Acute toxicity of carbamazepine to juvenile rainbow trout (Oncorhynchus mykiss): effects

on antioxidant responses, hematological parameters and hepatic EROD. Ecotoxicol. Envi-

ron. Saf., 74(3): 319-327.

Moin V.M. 1986. A simple and specific method for determining glutathione peroxidase activ-

ity in erythrocytes. Labarator. Delo, 12: 724-727 (in Russian, abstract in English).

Nunes-Silva A., Freitas-Lima L.G. 2015. The association between physical exercise and reac-

tive oxygen species (ROS) production. J. Sports Med. Doping Stud., 5: 1.

Powell M., Harris J. 2004. Influence of oxygen on the toxicity of Chloramine-T to Atlantic

salmon smolts in freshwater and seawater. J. of Aquat. Anim. Health, 16(2): 83-92. Powell M.D., Perry S.F. 1996. Respiratory and acid-base disturbances in rainbow trout (On-

corhynchus mykiss) blood during exposure to chloramine-T, paratoluenesulfonamide, and hypochlorite. Can. J. Fish Aquat. Sci., 53: 701-708.

Powell M.D., Speare D.J., MacNair N. 1994. Effects of intermittent Chloramine-T exposure

on growth, serum biochemistry, and fin condition of juvenile rainbow trout (Oncorhyn-

chus mykiss). Can. J. Fish. Aquat. Sci., 51(8): 1728-1736.

Powell M.D., Haman F., Wright G.M., Perry S.F. 1998. Response of rainbow trout (On-

corhynchus mykiss) to a graded hypoxia following repeated intermittent exposure to chlo-

ramine-T. Aquacult., 165(1): 27-39.

249

Powell M.D., Wright G.M., Speare D.J. 1995. Morphological changes in rainbow trout (On-

corhynchus mykiss) gill epithelia following repeated intermittent exposure to chloramine-

T. Canad. J. Of Zool., 73(1): 154-165.

Principles and practice of disinfection, preservation, and sterilization. A.P. Fraise, J.-Y. Mail-

lard, S.A. Sattar (eds.). Rev. ed. of Russell, Hugo & Ayliffe’s principles and practice of

disinfection, preservation & sterilization, 2004.

Rach J.J., Bills T.B., Marking L.L. 1988. Effects of physical and chemical factors on the tox-

icity of Chloramine-T. United States fish and Wildlife Service Information Bulletin,

Gdańsk: 69-88.

Sakuma S., Miyoshi E., Sadatoku N., Fujita J., Negoro M., Arakawa Y., Fujimoto Y. 2009.

Monochloramine produces reactive oxygen species in liver by converting xanthine dehy-

drogenase into xanthine oxidase. Toxicol. Appl. Pharmacol., 239(3): 268-272.

Sanchez J.G., Speare D.J., Johnson G.J. 1997a. Morphometric and histochemical assessment

of the branchial tissue response of rainbow trout, Oncorhynchus mykiss (Walbaum), asso-

ciated with chloramine-T treatment. J. Of Fish Diseas., 20(5): 375-381.

Sanchez J.G., Speare D.J., Johnson G.J., Horney B.S. 1997b. Evaluation of the stress response

in healthy juvenile rainbow trout after repetitive intermittent treatment with chloramine-T

or formalin. J. Of Aquat. Anim. Health, 9(4): 301-308.

Sanchez J.G., Speare D.J., MacNair N., Johnson G.R., Aquaculture Assoc. of Canada, S.A.,

N.B. [C.], 1997c. Effect of prophylactic chloramine-T treatment on growth performance

and condition indices of rainbow trout (Oncorhynchus mykiss). In: Aquaculture Associa-

tion of Canada Special Publications. M.D.B. Burt & S.L. Waddy (eds.), no. 2: 51-57. De-

partment Pathology & Microbiology, Atlantic Veterinary College, 550 University Ave.,

Charlottetown, Canada: Aquaculture Association of Canada.

Sanchez J.G., Speare D.J., MacNair N., Johnson G.R. 1996. Effects of a prophylactic chlora-

mine-T treatment on growth performance and condition indices of rainbow trout. J. of

Aquat. Anim. Health, 8(4): 278-284.

Sanchez J.G., Speare D.J., Sims D.E., Johnson G.J. 1998. Morphometric assessment of epi-

dermal and mucous-biofilm changes caused by exposure of trout to chloramine-T or for-

malin treatment. J. Of Comparat. Pathol., 118(1): 81-87.

Shao Z.J. 2001. Aquaculture pharmaceuticals and biologicals: current perspectives and future

possibilities. Adv. Drug Deliv. Rev., 50(3): 229-243.

Shim I., Seo G.B., Oh E., Lee M., Kwon J.T., Sul D., Lee B.W., Yoon B.I., Kim P., Choi K.,

Kim H.M. 2013. Inhalation exposure to chloramine T induces DNA damage and inflam-

mation in lung of Sprague-Dawley rats. J. Toxicol. Sci., 38(6): 937-946.

Sirri R., Zaccaroni A., Di Biase A., Mordenti O., Stancampiano L., Sarli G., Mandrioli L. 2013.

Effects of two water disinfectants (chloramine-T and peracetic acid) on the epidermis and

gills of Garra rufa used in human ichthyotherapy. Pol. J. Vet. Sci., 16(3): 453-461.

Stanley N.R., Pattison D.I., Hawkins C.L. 2010. Ability of hypochlorous acid and N-chloramines

to chlorinate DNA and its constituents. Chem. Res. Toxicol., 23(7): 1293-1302.

Tatsumi T., Fliss H. 1994. Hypochlorous acid and chloramines increase endothelial permea-

bility: possible involvement of cellular zinc. Am. J. Physiol., 267(4 Pt 2): H1597-1607.

Thorburn M.A., Moccia R.D. 1993. Use of chemotherapeutics on trout farms in Ontario.

J. Aquat. Anim. Health, 5: 85-91.

Tkachenko G.M., Grudniewska J. 2015. Tissue-specific response of protein oxidation in the

grayling (Thymallus thymallus L.) disinfected by chloramine-T. Scient. Medic. Bulletin,

1(1): 76-82.

Tkachenko H., Grudniewska J. 2016a. Biochemical changes in the muscle tissue of rainbow

trout (Oncorhynchus mykiss Walbaum) disinfected by Chloramine-T. Balt. Coastal Zone

– J. of Ecol. and Protect. of the Coastline, 20: 101-116.

250

Tkachenko H., Grudniewska J. 2016b. Effect of chloramine-T disinfection on oxidative stress

biomarkers in the gill tissue of grayling (Thymallus thymallus). Trudy VNIRO (Труды

ВНИРО), 162: 150-160.

Tkachenko H., Grudniewska J. 2016c. Influence of chloramine-T on oxidative stress

biomarkers in the cardiac tissue of grayling (Thymallus thymallus Linn.). In: Globalisa-

tion and regional environment protection. Technique, technology, ecology. T. Noch, W.

Mikołajczewska, A. Wesołowska (eds.). Gdańsk High School Publ., Gdańsk: 213-234.

Tkachenko H., Grudniewska J. 2016d. Influence of chloramine-T on oxidative stress bi-

omarkers in the muscle tissue of grayling (Thymallus thymallus). Scientific journal «Ka-

liningrad State Technical University News», 42: 49-58.

Tkachenko H., Grudniewska J. 2016e. Lipid and protein oxidation in the muscle tissue of

grayling (Thymallus thymallus Linn.) after Chloramine-T disinfection. Materials of the

International Forum “The Current State and Prospects for the Development of Aquacul-

ture in the Caspian Region”, dedicated to the 85th anniversary of Dagestan State Univer-

sity and the 75th anniversary of Professor F. Magomayev. F. Magomayev, S. Chalayeva,

S. Kurbanova, A. Shakhnazova (eds.). (Makhachkala, 17-19 October, 2016) – Makhach-

ka-la, Printing house IPE RD: 168-175.

Tkachenko H., Kurhaluk N., Grudniewska J. 2012. Effects of Chloramine-T exposure on ox-

idative stress biomarkers and liver biochemistry of rainbow trout, Oncorhynchus mykiss

(Walbaum), brown trout, Salmo trutta (L.), and grayling, Thymallus thymallus. Arch. Pol.

Fish., 21: 41-51.

Xia J., Zhao H.Z., Lu G.H. 2013. Effects of selected metal oxide nanoparticles on multiple

biomarkers in Carassius auratus. Biomed. Environ. Sci., 26(9): 742-749.

Zar J.H. 1999. Biostatistical Analysis. Prentice-Hall Inc. Englewood Cliffs, New Jersey.

Zhang J., Shen H., Wang X., Wu J., Xue Y. 2004. Effects of chronic exposure of 2,4-

dichlorophenol on the antioxidant system in liver of freshwater fish Carassius auratus.

Chemosph., 55(2): 167-174.

SUMMARY

Chloramine-T, as an anti-microbial agent, has had widespread use in a broad

range of practices. In aquaculture, as a therapeutic agent, it is used as an effective

treatment of bacterial gill disease in freshwater or marine aquaria, garden ponds, or

other aquatic systems at concentrations ranging from 6.5 to 10.0 mg/l [23.1 to 35.5

µM] and as a preventative, prophylactic, and disinfectant treatment in many fresh

water hatcheries. It is effective for the control of proliferative and bacterial gill dis-

ease as well as flexibacteriosis in freshwater, and more recently attention has turned

to its use in seawater. However, despite the wide use of chloramine-T, only few stud-

ies have examined its toxicity to fish (Powell and Harris 2004). Therefore, the aim

of the current study was to examine the effects of disinfection by Chloramine-T on

the muscle tissue of grayling (Thymallus thymallus Linck) using oxidative stress bi-

omarkers [levels of 2-thiobarbituric acid reactive substances (TBARS) and oxidative

modified protein (OMP) derivatives] and antioxidant defense [superoxide dismutase

(SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx),

total antioxidant capacity (TAC)] to observe the its toxic effects. The endpoints ob-

tained from this study will be useful to monitor the effects of disinfectant bathing

with Chloramine-T for this species of fish. Twenty clinically healthy grayling were

used in the experiments. In the disinfectant exposure, grayling (n = 10) were ex-

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posed to Chloramine-T in final concentration 9 mg per l. Control group of grayling

(n = 10) 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. Significantly lower TBARS level (by 39%, p = 0.004) in

grayling disinfected by chloramine-T compared to control group was observed. Al-

dehydic and ketonic derivatives of oxidatively modified proteins in the muscle tissue

of grayling disinfected by chloramine-T were non-significantly lower compared to

controls. The CAT activity was lower by 72% (p = 0.001) after Chloramine-T influ-

ence. The SOD activity in the muscle tissue of grayling disinfected by Chloramine-T

was also decreased by 15% (p = 0.001) compared to the controls. Significant de-

crease of TAC level (by 13%, p = 0.026) in the muscle tissue of grayling as a conse-

quence of bathing with chloramine-T was found. Correlative analysis has revealed

positive correlations between oxidative stress biomarkers (aldehydic and ketonic de-

rivatives of oxidatively modified proteins, TBARS as marker of lipid peroxidation)

and antioxidant defenses. Muscle aldehydic derivatives of OMP correlated positively

with TBARS (r = 0.854, p = 0.002) and catalase activity (r = 0.734, p = 0.016), ke-

tonic derivatives of OMP correlated positively with TBARS (r = 0.852, p = 0.002)

and aldehydic derivatives of OMP (r = 0.965, p = 0.000), catalase activity correlated

positively with TBARS (r = 0.735, p = 0.015) and GR activity (r = 0.714, p = 0.020),

as well as GR activity correlated positively with TBARS (r = 0.827, p = 0.003) and

GPx activity (r = 0.725, p = 0.018). Our studies indicated that Chloramine-T in dose

9 mg per l could at least partly attenuate oxidative stress and can be used for prophy-

lactic treatment of grayling. However, more detailed studies on using of these specif-

ic biomarkers to monitor the disinfectant treatment in aquaculture are needed.

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