Effects of a Novel Benzenesulfonamide 4-(3-(4-Bromophenyl)-5-(2,4-dimethoxyphenyl)- 4,5-dihydro-1H-pyrazol-1-yl) on Antioxidant Enzymes and Hematological Parameters of Rainbow Trout (Oncorhynchus mykiss)

Dilan Ozmen Ozgun1, Gonca Alak2, Arzu Ucar2, Sinan Bilginer3, Halise Inci Gul3, Mahmut Kocaman2, Cem Yamalı3, Veysel Parlak2, Muhammed Atamanalp2, Ahmad Maslat4 and Telat Yanık2* 1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ibrahim Cecen University of Agriculture, TR-04-100 Agriculture, Turkey 2Ataturk University, Department of Aquaculture, Faculty of Fisheries, TR-25240 Erzurum, Turkey 3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University, TR-25240 Erzurum, Turkey 4Department of Biological Sciences, Faculty of Science, Yarmouk University, Irbid, Jordan Article Information Received 18 November 2019 Revised 20 December 2019 Accepted 08 January 2020 Available online 12 November 2020


O n l i n e F i r s t A r t i c l e
peroxidase (POD), glutathione reductase (GSSG-Rx) and superoxide dismutase (SOD) enzymes have been reported in living organisms (Nimse and Pal, 2015). After normal cellular metabolism, reactive oxygen species (ROS) are produced in living organisms. Enzymatic antioxidants, CAT, glutathione peroxidase (GSH-Px) and SOD is used for the protection of cells from the destructive effects of ROS in the body. Nonenzymatic antioxidants, glutathione, ascorbic acid, α-tocopherol, carotenoids, and phenolic compounds are used also for this purpose (Koruk et al., 2004). Antioxidant defense systems control the negative effects of free radicals in a cell (Gülçin, 2002). Cellular defense enzymes SOD, CAT, and GSH-Px prevent the reduction of oxygen metabolism products (Rahimi et al., 2005). Toxic substances have some adverse effects on the hematology of fishes (Atamanalp and Yanik, 2003). There is, however, very little information about the toxicity of novel pyrazoline derivatives in fish. Therefore, the present study was conducted to test the toxicity of newly synthesized novel pyrazoline derivative, B4, on hematological parameters and enzymatic antioxidants namely SOD, CAT and glutathione peroxidase (GPx) and lipid peroxidation malondialdehyde (MDA) in gill and liver in rainbow trout.

Synthesis of the A4 compound
4-bromoasetophenone and 2,4 dimethoxyasetaldehyde 1:1 mol ratio were dissolved in ethyl alcohol (10 ml), a 20 ml solution of cold NaOH (10%) was added dropwise onto the mixture. The mixture was stirred at room temperature. Progress of the reaction. followed by T.L.C. At the end of the reaction, the balloon contents were poured into ice water and neutralized with enough HCl solution (10%). The purity of the obtained solid was determined by T.L.C. After the second stage of B4 synthesis was used without further purification. The method of synthesis of chalcone is presented in Figure 1.

O n l i n e F i r s t A r t i c l e
Experimental design A set of 6 aquaria with 48 fish (25 ± 2 g) were used for testing the B4 compound at 11 ± 1.5 °C during a 96 h study in the Aquarium Fish Production Unit of Ataturk University. Fish were acclimated for 14 days before the testing experiments. Studied doses were chosen based on the study done by Khedkar et al. (2018). B4 compound was dissolved in dimethyl sulfoxide (DMSO). Exposure doses were 0.00 (control), 1 ml DMSO, 0.25, 0.5, 1 and 2.5 mg / L.
At the end of the experimental period, blood samples were collected and subjected to hematological analyzes by using Prokan PE 6800 blood analyzer device. Liver and gill tissue samples, kept at -86 o C, were used for determination of antioxidant enzyme activities -SOD (Sun et al., 1988), CAT (Aebi, 1984), GPx (Beutler, 1975), total protein (Bradford, 1976) and lipid peroxidation (MDA) (Luo et al., 2006).
The determined data was analyzed statistically by SPSS software. Duncan test was used for the determination of differences between groups at alpha 0.05 level.

RESULTS
Different doses of pyrazoline compound B4; 4-(3-(4-bromophenyl)-5-(2,4-dimethoxyphenyl)-4,5dihydro-1H-pyrazol-1-yl) benzenesulfonamide; caused significant effects on antioxidant enzyme activity in gill (Table I), liver (Table II) and hematology of fish (Table  III). Table I shows decreases of CAT, GPx and SOD levels in gill with increasing concentration of B4 up to 1 mg/L compared to control. MDA levels increased significantly (p<0.05) at 0.5 and 1 mg/L then drop to insignificant level (p>0.05) compared to control. There were no differences between the control and DMSO group (p>0.05) for all tested properties. Table II shows significant reductions in CAT, GPx and SOD values in the liver significantly at 0.5 mg / L of B4 (p<0.05) compared to control. MDA levels did not differ (p>0.05) compared to control except for the dose of 1 mg / L. There were no differences between control and DMSO group (p>0.05) for all tested properties. After exposition to B4 substance, fish mortalities varied 0% in control and DMSO group, 25.5% in 0.25 mg / L group and 25% in 0.5, 1 and 2.5 mg / L group. Table III shows increases in WBC, MCV, MCH and MCHC levels and decreases in RBC Hb, Htc and PLT levels in blood with the increasing concentration of B4 (p<0.05) compared to control. There were no differences between the control and DMSO group (p>0.05) for all tested properties. Table I

DISCUSSION
Considering all enzymes, CAT is the one, which has the highest turnover numbers. It was reported that millions of hydrogen peroxide molecules can be converted into water and oxygen in a second by one-catalase molecule (Koruk et al., 2004;Nimse and Pal, 2015). Mao et al. (1993) hypothesized that the amount of H 2 O 2 should be reduced by CAT and the toxicity of SOD is reduced by catalase at high concentrations. CAT protects cells from oxidative damage by reactive oxygen species (ROS) (Koruk et al., 2004). Winston et al. (2001) reported that SOD, CAT, and glutathione peroxidase (GPx) enzymes can conjugate with antibodies. Therefore, it is thought that having low, SOD and GPx values both in the gill and liver of fish might be due to the negative effects of B4 chemical caused oxidative stress. In the present study, low SOD values may be due to the consumption of the enzyme in converting oxygen to  (Hancock et al., 2001;Forman and Torres, 2002). An increase in the formation of MDA levels shows that there was overwhelming stress in which free radicals suppressed the antioxidant system and caused low antioxidant levels in gill and livers. From the present study, it can be seen that the SOD activity is time-dependent as it was reduced first and then increased again.

O n l i n e F i r s t A r t i c l e
Cellular damage in lipid, DNA, RNA, and proteins is caused by ROS. ROS are produced as a normal product of cellular metabolism. It is mentioned that plasma MDA level was increased by oxidative damage in DNA (8-oxodG, 1, N6-εdA, 1,N2-εdG) (Malavolta and Mocchegiani, 2016). Measurements of higher values in WBC, MCV, MCH, and MCHC, and low values of RBC Hb, Htc and PLT levels compared to the control group shows that fish had great stress after exposition to B4. There are many controversial results in fish hematology after expositions to chemical substances (Atamanalp and Yanik, 2003). Hamed and El-Sayed (2019) reported that exposure to pendimethalin (PM) caused decreases in SOD, CAT, total antioxidant capacity (TAC) and glutathione peroxidase (GSH-Px) levels, and increases in MDA as well as DNA fragmentation in the liver in Nile tilapia, Oreochromis niloticus (L.). Singh et al. (2019) reported that exposure to iron resulted in the accumulation of iron in fish tissues and caused significant fluctuations in erythrocyte and leukocyte counts, hemoglobin, lipid peroxidation, antioxidant enzyme activity (SOD and CAT). It was found that while lipid peroxidation was increasing SOD and CAT was decreasing double in the exposure of excess iron. There were a time and dose-dependent tissue injuries in gills and liver in freshwater fish Labeo rohita (Singh et al., 2019).

CONCLUSION
SOD, CAT, and GPX are the major antioxidant enzymes that protect cells against the harmful effects of ROS to maintain the health of organisms. Activators and inhibitors of these enzymes, therefore, are really of vital importance. The first line of the antioxidant defense system is comprised of the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) (Ighodaro and Akinloye, 2018). Low values of these enzymes show that B4 has negative effects on the total defense system of fish and, therefore, it might be useful testing its microbial properties in further studies before using it as the active substance of medicines.

Statement of conflict of interest
The authors have declared no conflict of interest.