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Catalase Enzyme Response to Chronic Pb+Cd Metal Mixture Exposure, Its Purification and Partial Characterization from the Kidney of Freshwater Fish, Oreochromis niloticus.

Byline: Tanveer Ahmed, Sajid Abdullah, Khalid Abbas and Muhammad Anjum Zia

ABSTRACT

In the present study, kidney tissues were selected to measure the catalase (CAT) enzyme activity due to their responsibility in the elimination of the compounds generating reactive oxygen species (ROS). Variation in Oreochromis niloticus kidney CAT activity (114.33+-0.33 to 159.5+-0.03) was observed throughout the study period. During first week of experimental trial, higher CAT activity (159.5+-0.03) was noted in Pb+Cd metal mixture stressed fish kidney in comparison to control fish (128.8+-0.06). Similarly, lower kidney CAT activity (114.33+-0.33) was recorded in second week of experimental trial in metal mixture exposed fish as compared to control group. Significant differences (pthe weight of organ for 15 min with the help of a homogenizer, filtered and centrifuged at 10, 000 rpm for 15 min. Both the pellets and supernatants were separated for further analyses.

CAT enzyme assay

The activity of CAT was determined by measuring its ability to decompose H2O2 at 240nm by following the methods of Chance and Mehaly (1977) with some modifications. A 50mM phosphate buffer (pH 7.0) and 10mM hydrogen peroxide (H2O2) were prepared to make buffer substrate solution. The reaction mixture (2mL) contained 1.95mL buffered substrate solution and 0.05mL enzymes extract. The buffered substrate solution was used as blank.

Protein content estimation

Biuret method of Gornall et al. (1949) was used for the estimation of protein contents with the help of DC Protein Assay Kit (Bio-Rad Laboratories, USA) by using BSA (bovine serum albumin) as standard.

Purification of kidney CAT

Purification of kidney catalase was carried out by using methods of Nakamura et al. (2000) with some modifications. All purification steps were carried out at 4degC.

Ammonium sulfate precipitations

Crude extract of CAT was saturated with 25% ammonium sulfate by dissolving 17.5g ammonium sulfate in 100mL. After 6 hours incubation, it was centrifuge at 13,000 rpm for 15 min at 4degC. The supernatant that was obtained from salting in procedure was subjected to salting out method by adjusting the saturation upto 50%. It was incubated at 4degC for overnight and then centrifuged at 13,000 rpm for 15 min at 4degC. Residues obtained from salting out were re-suspended and subjected to desalting with the help of dialysis bag in phosphate buffer (1.5mM; pH 7.4).

Ion exchange chromatography

The column of DEAE-cellulose (diethyl amino ethyl-cellulose) was prepared (1x20cm) for the purification of kidney CAT. Slurry was prepared and an amount of 250uL desalted sample was applied on column. The sample was eluted out with the help of 10mM phosphate buffer (pH 7.4) while the drop rate was kept constant (1 mLmin-1). A total of 50 fractions with 2 mL of elution were collected. All the fractions optical density were noted at 280 nm against blank (buffer). Fractions showing higher absorbance were selected for protein content estimation and enzyme assay.

Gel filtration chromatography

Column (1x20cm) of sephadex G-150 was prepared in phosphate buffer (10mM; pH 7.0). An amount of 250uL of sample (with highest specific activity after ion exchange chromatography) was applied and 50 fractions with 2 mL were collected. Fractions showing higher absorbance at 280nm were selected for protein content estimation and enzyme assay.

Partial characterization of purified kidney CAT

Optimum pH, temperature and buffers concentration was determined by assaying the purified kidney CAT enzyme from both control and Pb+Cd metal mixture exposed Tilapia by following the methods of Nakamura et al. (2000) and Al-Bar (2012).

Statistical analysis

Data obtained in this study were analyzed by Mean Standard Deviation (Mean+-SD). ANOVA was calculated to measure statistical difference in CAT activity among both metal stressed and control fish at p<0.05 (Steel et al., 1997). Multiple comparison test was also performed by applying LSD.

RESULTS

The present research work was performed to study the response of CAT against Pb+Cd metal mixture in the kidney tissues of O. niloticus. To purify and partially characterize the CAT from the kidney tissues of O. niloticus both from control and metal mixture stressed fish was also the objective of this study.

CAT activity in control and Pb+Cd metal mixture exposed O. niloticus

No fish mortality was observed in first week of experimental trial, however, at the end of second week, fish mortality was observed in metal mixture treated aquarium. After the administration of chronic Pb+Cd metal mixture concentration, CAT showed higher activity in kidney tissues of metal mixture stressed O. niloticus in comparison to control fish. However, during the second week of experimental trial, lower CAT activity was observed in metal mixture stressed fish as compared to its opponent i.e. control (Fig. 1). Significant differences were observed at p<0.05 when both control and metal mixture treated fish kidney CAT activity was compared statistically. Multiple comparison test after analysis of variance revealed that all means were significantly different from each other.

Significant statistical differences for purification inferences (from homogenate to Sephadex G-150 resins) were observed at p<0.05 among both fishes. Highest specific activity (1314.9) was observed in control fish compared to metal stressed fish (1011.84). Fold purification was measured 18.95 and 15.66 in this study for control and metal mixture stressed fish, respectively (Table I).

Partial characterization of purified kidney CAT

CAT enzyme purified from control and metal stressed O. niloticus kidney was partially characterized. The effect of different pH on purified CAT both from control and metal stressed O. niloticus kidney was studied. From both control and metal mixture stressed fish, the pH at which purified CAT revealed maximum activity was measured 7 (Fig. 2A).

By keeping pH at optimum level i.e. 7, effect of different temperatures on purified kidney CAT was studied for measuring the optimum temperature. The temperature at which purified kidney CAT showed maximum activity was observed 25degC both for control and metal stressed fish (Fig. 2B).

By keeping the pH and temperature at optimum level i.e. 7.0 and 25degC, optimum substrate concentration was determined both for control and metal stressed fish purified kidney CAT. The substrate concentration at which purified kidney CAT showed maximum activity was observed 50 mM both for control and metal stressed fish (Fig. 2C).

Vmax value was measured 2.02 UmL-1 for control fish while, 2.09 UmL-1 for Pb+Cd metal mixture stressed fish kidney purified CAT. Low value of Vmax indicates CAT stronger ability to bind with H2O2 (Fig. 3A).

Km value was noted 7.59 mM H2O2mL-1 and 1.17 mM H2O2mL-1for control and Pb+Cd metal mixture stressed O. niloticus, respectively (Fig. 3B).

Partial characterization of purified kidney CAT from control and metal stressed O. niloticus

In Table II, all the characterization parameters measured in this study are compared between both control and metal stressed O. niloticus. Although the values of optimum pH, temperature and substrate concentration are same for both control and metal stressed fish, but the specific activity was recorded lower on the average basis in metal mixture stressed fish as compared to control one. When both control and metal mixture stressed fish kidney CAT activities were compared after measuring its optimum pH, temperature and substrate concentration, significant differences (p<0.05) were observed statistically.

Table I.-Comparative kidney CAT purification results from control and metal stressed O. niloticus.

###Control O. niloticus kidney###Metal stressed O. niloticus kidney

###Specific###Specific

###Enrichment###Enrichment

Steps###Activity###Yield (%)###Activity###Yield (%)

###(fold)###(fold)

###(U/mg)###(U/mg)

Crude enzyme###69.29###100###1.0###64.59###100###1.0

(NH4)2SO4

###86.42###65.23###1.24###72.43###65.88###1.12

precipitation

Desalted###89.42###57.14###1.29###74.35###60.64###1.15

DEAE-Cellulose###524.24###46.75###7.56###413.33###54.22###6.40

Sephadex G-150###1314.9###44.67###18.95###1011.84###53.05###15.66

Table II.-Comparative partial characterization of purified kidney CAT from control and metal stressed O. niloticus.

Parameters###Metal

###Control###stressed

###kidney CAT###kidney CAT

Specific activity (U/mg)###1314.9###1011.84

Fold Purification###18.95###15.66

Optimum pH###7.0###7.0

Optimum temperature (degC)###25###25

Optimum phosphate buffer (mM)###50###50

Km (mM H2O2mL-1)###7.59###1.17

Vmax (mM H2O2mL-1)###2.02###2.09

DISCUSSION

The present study was conducted to assess the impact of chronic metal mixture on the CAT activity in the O. niloticus kidney tissues due to their responsibility in the elimination of the compounds generating reactive oxygen species (ROS). Partial characterization of purified kidney CAT both from Pb+Cd metal mixture stressed and control O. niloticus was also performed in this study.

A wealth of information is present in literature in which individual effect of different heavy metals on fish antioxidant systems are studied. However, very little literature was found in which metal mixture effect on fish antioxidant systems studied although in aquatic ecosystem different metals disturb fish physiology jointly not individually.

Aquatic environments are the ultimate destination for most of the metals released from natural and man-made sources. Fish liver and kidney tissues are highly endowed with antioxidant enzymes including catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), glutathione S-transferase (GST) and glutathione reductase (GR) to protect them from oxidative stress.

Variation in O. niloticus kidney CAT activity was recorded in this study period. During the first week of experimental trial, higher CAT activity was noted in metal mixture stressed fish kidney compared to control fish group. Lower kidney CAT activity was recorded in second week of experimental trial in metal treated fish compared to control group of fish. Significant differences were observed at p<0.05 when compared kidney CAT activity among control and metal mixture stressed O. niloticus. Variation in responses of the antioxidant enzymes to metal exposures, depending upon body tissues, metals and exposure types (lethal or sub-lethal).

Elevated renal CAT activity in first week of experimental trial in this study is associated with increased production of ROS or oxidative stress by metal mixture. Further, redox active (Cu, Cr and Fe) and redox-inactive metals (Pb, Cd and Hg) can cause significant increases in rate of ROS production and followed by a situation known as oxidative stress that becomes the reason of several dysfunctions in DNA, proteins and lipids (Ercal et al., 2001; Pinto et al., 2003).

Enhance kidney CAT activity in first week of experimental trial are according to the findings of Palace and Klaverkamp (1993) in the liver tissues of rainbow trout (Oncorhyncus mykiss), Avci et al. (2005) in muscle and hepatic tissues of Silurus glanis, Hansen et al. (2006) in brown trout (Salmo trutta), Atli et al. (2006) in brain, gills, liver, kidney and skin of O. niloticus, Atli and Canli (2008) in gills, liver and muscle tissues of O. niloticus, Lin et al. (2011) in gills and liver tissue of genetically improved farmed tilapia (O. niloticus).

At the end of experimental trial, decrease in renal

CAT activity was observed in metal mixture stressed fish in comparison to control fish which might be associated with direct binding of Pb+Cd to the CAT thiol (-SH) group that transferred active CAT to inactive. Lower CAT activity in various tissues of fish is associated with the direct effect of different metal exposures and increased generation of ROS (Radi and Matkovics, 1988; Basha and Rani, 2003; Dautremepuits et al., 2004; Ahmad et al., 2005; Atli et al., 2006). In the killifish, Fundulus heteroclitus, inhibited hepatic CAT activity was found by Pruell and Engelhardt (1980) both in vivo and in vitro exposure to dissolved Cd2+.

Lower kidney CAT activity at the end of experimental trial are according to the findings of Palace et al. (1992) who exposed rainbow trout to cadmium and reported lower CAT activity in the hepatic tissues and concluded that reduction in CAT level is due to direct binding of metals that alter its structure. Bainy et al. (1996) observed lower CAT activity in erythrocytes, gills, kidney and liver tissues of O. niloticus collected from metal polluted areas and are according to the findings of present study inferences. Similarly, Romeo et al. (2000) measured lower CAT activity in the kidney tissues of the sea bass, Dicentrarchus labrax kept under Cd stress compared to control fish. Lower CAT and glutathione peroxidase activity in hepatic tissues of Cyprinus carpio captured from polluted areas compared to non-polluted areas of Karakaya Dam Lake was also observed by Yilmazi et al. (2006).

Lower CAT activity was observed in hepatic, gills, cardiac and renal tissues of African catfish kept under cadmium (Cd) and copper (Cu) stressed by Farombi et al. (2007). Firat and Kargin (2010) noted lower CAT activity in red blood cells of O. niloticus kept under Zn+Cd metal mixture compared to individual metal effect in which higher CAT activity was measured.

CAT activity show variation in different aquatic animals when exposed to metals which depends upon exposed duration, environmental factors, divergence and compounds of heavy metals used for stress (Atli and Canli, 2010). As a result of oxidative stress, fish adapted to either increase or decrease antioxidants level (Firat and Kargin, 2010).

A great improvements on purification and characterization of CAT have been realized in superior organism principally in mammals but less in fish species. From both control and metal mixture stressed fish, the pH at which purified CAT revealed maximum activity was measured 7 and are according to the findings of Peterson and Salin (1995) in Halobacterium halobium; Nakamura et al. (2000) in beagle dog; Aydemir and Kuru (2003) in chicken erythrocytes; Yasseen and Jadallah (2009) in bovine liver; Zeng et al. (2010) in Serratia marcescens; Arabaci and Usluoglu (2012) in Malva sylvestris; Al-Bar (2012) in liver of Camelus dromedaries; Tariq et al. (2014) in Cirrhinus mrigala liver and Sarwar et al. (2014) in liver of Ctenopharyngodon idella.

Similarly, temperature at which purified kidney CAT shown highest activity was recorded 25degC and are according to the inferences of Yasseen and Jadallah (2009) in bovine liver, Arabaci and Usluoglu (2012) in Malva sylvestris, Al-Bar (2012) in liver of Camelus dromedaries, Sarwar et al. (2014) in liver of grass carp (Ctenopharyngodon idella) and Tariq et al. (2014) in liver of Cirrhinus mrigala.

Optimum substrate concentration was measured 50mM for purified kidney CAT and are according to findings of Peterson and Salin (1995) in Halobacterium halobium, Aydemir and Kuru (2003) in chicken erythrocytes, Arabaci and Usluoglu (2012) in Malva sylvestris, Tariq et al. (2014) in liver of Cirrhinus mrigala and Sarwar et al. (2014) in liver of grass carp (Ctenopharyngodon idella).

For control and metal mixture stressed fish, Km values were measured 7.59 and 1.17 mM H2O2 mL-1, respectively while, Vmax values were recorded 2.02 and 2.09 UmL-1for control and stressed fish, respectively in this study. However, no data about Km and Vmax values are available in literature about fish for comparison. Al-Bar (2012) measured Km value for purified CAT 22.7 mM H2O2 mL-1 and Vmax value for purified catalase was found 7.9 UmL-1 in the liver of Camelus dromedaries.

The Km value for purified liver catalase was noted 6 mM H2O2 in Ctenopharyngodon idella by Sarwar et al. (2014).

The present study suggested that the enzymes which are antioxidant in function are highly sensitive to metal pollution as their activities change significantly, suggesting they could be helpful in predicting sub-lethal metal toxicity and useful as an early warning tool in bio-monitoring studies.

CONCLUSION

On the basis of this study and previous studies, it is concluded that antioxidant enzymes are helpful in preventing the harmful effects of metals. Moreover, they are cautionary indicators for severe damage to organisms living in aquatic environment. Consequences of existing research work further reveals that CAT is a susceptible bio-indicator of an organism antioxidant defense system. However, it is still essential to study further antioxidant system enzymes in different aquatic animal models to understand better.

ACKNOWLEDGEMENTS

This research work was completed under the technical guidance of Samreen Rasul (PhD Scholar), Enzyme Biotechnology Lab. (EBL), Dept. of Biochemistry, University of Agriculture, Faisalabad, Pakistan and my beloved elder brother Shakeel Ahmed who helped me financially and spiritually.

Statement of conflict of interest

Authors have declared no conflict of interest.

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Author:Ahmed, Tanveer; Abdullah, Sajid; Abbas, Khalid; Zia, Muhammad Anjum
Publication:Pakistan Journal of Zoology
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Date:Dec 31, 2016
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