Oxidative Stress Induction by Infectious Pancreatic Necrosis Virus in RTG-2 Cell Line

RTG-2 cell lines were used for IPNV inoculation in this study. RTG-2 cells were cultured into Eagle Minimal Essential Medium (EMEM) and supplemented with 10% fetal calf sera and incubated at 28°C. When the cells reached about 80% confluence, the cells were infected with IPNV (MOI:1) and incubated at 28°C. Also, EMEM was inoculated in negative control. Virus inoculated cell flasks were checked daily under the invert microscope to determine an IPNV cytopathic effect presence. Virus and cell control media were sampled (n=3) for each time period 0, 8, 24, 48, 72 and 96 h and stored at +4°C until analysed for biochemical parameters of oxidative stress. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and malondialdehyde (MDA) concentrations were analysed in all of the medium samples.

MDA in cell culture was determined according to the method reported by Yoshioka et al. (1979). Briefly, 0.5 ml cell medium, 2.5 ml, 20% of trichloroacetic acid and 1 ml of 0.67% thiobarbituric acid were added into test tube. 500 µl trichloroacetic acid was added in blank tube. Tubes were shaken by vortex and heated at 90°C for 30 min. Then, 4.0 ml of n-butanol was added and centrifuged at 1550 g for 10 min. The upper butanol phase was removed and absorbance was recorded at 535 nm at spectrophotometer and MDA concentration was determined according to the standard curve.

The measurement of SOD activity was performed by spectrophotometric method (Sun et al., 1989). Conditioned cell medium (100 µl) was added in a mixture containing 2.0 ml chloroform/ethanole (3:5/v:v) and centrifuged at 4°C, 1550 g for 15 min. Amount of 0.5 ml chloraform phase was aspirated and 2.45 ml working reagent (0.3 mM/l xanthine 0.6 mM/l Na2EDTA, 150 μmol/l NBT, 400 mmol/l Na2CO3, 1g/l BSA) + 0.5 ml bidistilled water + 50 μl xanthine oxidase (167 U/l) was added. 0.5 ml bidistilled water was added in blank tube instead of cell medium. Tubes were placed in water bath and incubated at 25°C for 20 min. Then, reaction was stopped by adding 1.0 ml 0.8 mmol/l of copper (II) chloride and centrifuged at 1550 g for 10 min. The absorbance of supernatants was measured at 560 nm and enzyme activity was calculated.

The activity of GPx was measured by the method of Paglia and Valentine (1967). GPx activity was measured in 20 µL of conditioned cell medium in a mixture containing 100 µl glutathione, 100 µl nicotinamide adenine dinucleotide phosphate (NADPH), 10 µl glutathione reductase, 10 µl sodium azide (NaN3) placed on ice. Tubes were shaken vigorously by vortex. Reaction was started with the addition of 100 ml H2O2 and the mixture was incubated at room temprature for 30 min. After the reaction was stopped, the GPx consumption was measured. Its absorbance at 340 nm was read spectrophotometrically. GPx was calculated according to one unit of GPx activity, which is defined as the amount of enzyme required to oxidize 1 mmol of NADPH /min.

SPSS for Windows (version 22) and R studio for Windows (version 3.0.2) was used for statistical analyses. MDA concentrations, SOD and GPx activities of IPNV and control were analyzed with relation to time by generalized linear models. A value of p<0.001 was regarded to indicate a statistically significant difference between group mean values.

In this study, the repeated measurements (n=3) of MDA concentrations for IPNV cultivated cells media at 0, 8, 24, 48, 72 and 96 h, significantly (p<0.001) increased after the 48th h, SOD concentrations were significantly (p<0.001) decreased after 48th h and GPx concentrations were significantly (p<0.001) decreased after 8th h, compared to control cells (Table I).

Oxidative stress arising from excessive ROS can lead to cell damage and death by supressing the defense mechanisms of the cells and oxidative degradation of the macromolecules (Ryter et al., 2007). Antioxidant enzymes play an important role in protecting cells against oxidative stress by scavenging of excessively produced ROS (Halliwell, 1994). Oxidative stress occurs when the ROS generation is beyond the capacity of antioxidant enzymes (Duracková, 2010; Reed, 1995). Damage due to ROS is inevitable for all creatures that breathe oxygen. Oxidative stress and antioxidant defense mechanisms in

*Statistically significant according to the generalized linear models test P<0.05. **The samples were tested in triplicate, and the data were presented in mean Sig. This experiment was repeated three times with similar results.

fish have been investigated (Bayir et al., 2011), but, the role of oxidative stress in cellular damage caused by IPNV has not been studied before. The results of this study revealed that cell damage of RTG-2 cell line is caused by IPNV related to oxidative stress.

It is already reported that IPNV infection causes economic losses in the production of rainbow trout (Bowers et al., 2008; Espinoza et al., 2005; Albayrak and Ozan, 2010; Candan, 2002). The association of apoptosis and necrotic changes have already been reported with IPNV infection in culture (Hong et al., 1998; Espinoza et al., 2005), but little is known about relationship of this infection with oxidative stress damage. In this study, the effect IPNN iinfection on oxidative stress damage has been studied. At the end of the biochemical analyses, MDA concentrations of IPNV produced cell media were significantly (p<0.001) increased from the 48th h, SOD and GPx activities were significantly (p<0.001) decreased from the 48th and 8th h, respectively, compared to control cell medium.

In conclusion, this study revealed that cell damage caused by IPNV in cell culture is related to oxidative stress for the first time and antioxidants may be an option in treatment of this disease.

This study was supported by Research Fund of Ondokuz Mayis University Samsun/Turkey (Project No. PYO.VET.1904.15.008).

The authors have declared no conflict of interest.