Effects of lead on some oxidative stress of the Catfish, Clarias batrachus

This study aimed to determine the effects of sub-lethal concentrations of lead (PbCl2) on some oxidative stress in the catfish Clarias batrachus . Fishes were captured from Ramgarh Lake, Gorakhpur. They were acclimatized and fed with commercial fish diet for one month before starting the experiment. Two experimental groups were exposed to two sublethal concentrations (10% and 30% of LC 50 ) of lead chloride, fishes from different experimental groups were sacrificed after 60 days of exposure. Oxidative stress enzymes such as GSH, CAT, SOD, and MDA were estimated. Group treated with Pb10 % showed significantly lower concentrations of GSH. On the other hand, Pb 30% group showed a significantly higher concentration of CAT, MDA and SOD .

Exposure of the Indian catfish C.batrachus to Pb (NO3)2 caused reduction in GSH, SOD, CAT and MDA compared to control (Saliu and Bawa-Allah, 2012).
The present study aimed to measure the changes of oxidative stress enzymes (malondialdehyde.MDA; Catalase, CAT; glutathione, GSH and superoxide dismutase, SOD) in catfish after exposure to different doses of lead chloride.

MATERIALS AND METHODS
Determination of LC50 One hundred and seventy-two catfish, Clarias batrachus (mean body weight =150±250g) were obtained from Ramgarh lake, Gorakhpur.Fishes, apparently healthy, were transported immediately to the lab and acclimatized for two weeks in glass aquaria (40* 35* 70 cm) with a capacity of 60 L and were fed on a basal fish diet.After acclimation,100 fishes were divided into ten groups (10 fishes/ group) for determination of LC50, each group was exposed to a different concentrations of PbCl2 (100 mg/l, 150 mg/l, 200 mg/l, 250 mg/l, 300 mg/l, 350 mg/l, 400 mg/l, 450 mg/l,) at constant temperature 25ºC.Fishes were kept under observation for 96 hours and numbers of dead fishes were recorded daily.The LC50 was statistically determined by using dose-effect analysis using XLSTAT software.

Experimental fishes model:
After determination of LC50, 72 fishes were divided randomly into equal three groups with 8 fishes in all aquaria, each group was represented by triplets (24 fishes for each treatment).These groups are; 1-Control group, 2-Fish exposed to Pb 10% of LC50 for 60 days 3-Fish exposed to Pb 30% of LC50 for 60 days

Test Compound
Lead as PbCl2 (molecular weight 278.10) manufactured by Oxford Lab Chem, India.

Blood sampling
Fishes fasted for 24 hours before sampling, blood samples were obtained from the caudal vein using 3 ml syringe within less than 3 minutes to minimize handling stress.The collected blood samples were divided into two tubes.Anticoagulant free samples were used for serum preparation by centrifugation for 20 minutes at 1207 g within one hour of sampling.The sera samples were used for determination of MDA, CAT and GSH, while SOD was determined in RBCs lysate later on the same day with commercial kits.Absorbance values of samples and standards were measured using a UV spectrophotometer.

Statistical analysis
Data were expressed as mean ± SEM of different treated groups compared to control ones.Normal distribution of all parameters was tested.The results were analyzed using oneway analysis of variance (ANOVA) followed by Tukey (HSD) test to compare groups with each other and Dennett two-sided test for comparisons with the control group.P<0.05 was considered significant.All statistical analyses were performed using XLSTAT program.

RESULTS AND DISCUSSION
As shown in Table (1), GSH concentration was significantly lower in Pb 10% group compared to control group, while CAT activity was significantly higher in Pb10% and Pb30% group compared to control group.Lipid peroxide product (Malondialdehyde, MDA) was significantly higher in Pb30% group compared to control group, while superoxide 173 Effects of lead on some oxidative stress of the Catfish, Clarias batrachus dismutase (SOD) activity was significantly lower in Pb30% group compared to control group.In the current study, GSH concentration was significantly lower in Pb 10% respectively, compared to the control group.Similarly, GSH level, in the current study, decreased significantly in Pb 10% LC50exposed group.In line to this finding, Catfish Clarias batrachus showed decreased levels of GSH after exposure to 100 μg/l, 300 μg/l and 500 μg/l of lead nitrate (Osman et al., 2007).In another study, GSH level was reduced after 28 days exposure to 5.15, 0.52, 0.052 mg/L of Pb(NO3)2 compared to the controls (Saliu and Bawa-Allah, 2012).The decreased GSH levels in the present study could account for the marked lipid peroxidation tables (1).Glutathione (GSH) serves to protect the cell against oxidative damage as it conjugates with compounds of exogenous and endogenous origin (Liu et al., 2009).Oxidative stress may be initiated by a decline in the antioxidative defense system, GSH loss in fishes reflects the ability of metals in oxidizing sulfhydryl group resulting in inactivation of oxidative enzymes by free radicals (Nwani et al., 2015) and accumulation of reactive oxygen species (ROS) (Osman et al., 2007;Wu et al., 2011).CAT activity increased in 30% of 96 hrs LC50 value -exposed groups compared to control group.Some investigators have suggested that severe oxidative stress may suppress the activity of antioxidant defense enzymes due to oxidative damage and a loss of the compensatory mechanisms (Atli et al., 2006;Liu et al., 2006).Similar results on Clarias batrachus, showed that lead increased CAT activity after 28 days exposure to different concentration (28, 43 and 57 mg/L) of lead nitrate (Loveline et al., 2018).This increase in CAT activity is due to the degradation of H2O2, a potent oxidant at high cellular concentration is affected by CAT due to its induction against increased oxidative stress.MDA, is used as an effective biomarker of toxic pollutants in fish exposed to lead, MDA level, in the current study, increased in Pb30% group compared to the control group.Similarity Maiti et al. (2010) described elevated MDA levels in the catfish Clarias batrachus following a 60-day exposure to lead, MDA increased in common carp (Cyprinus carpio) after exposed to (50 µg/L) of lead for a period of 30 days (Shafiq-ur-Rehman, 2003), the production of MDA is a marker of lipid peroxidation results from the decomposition of polyunsaturated fatty acid due to oxidative stress (Alfanie et al., 2015).SOD activity increased significantly in Pb30% compared to the control group.Similar results were found in Clarias batrachus where SOD activity increased after exposure to 0.1 , 0.01 and 0.001 of 96hrLC50 of Pb(NO3)2 (Saliu and Bawa-Allah, 2012) , these pattern indicate the activation of protective mechanisms required for removing O2-radical in RBCs (Zikić et al., 2001).