Abstract: In this study, for clarifying some possible mechanism of zinc toxicity, the effect of increasing amounts of Zn2+ ion on peroxidase activity was investigated in vitro in serum of cow. The H2O2-mediated oxidation of o-dianisidine was used to assess the peroxidase activity. Results show that after preincubation of serum with 0.2-20 mM Zn2+ concentration for 5 min, peroxidase activity was inhibited compared to the control and decreased rapidly with increasing metal concentrations. The enzyme was completely inhibited after 5 min preincubation in 30 mM Zn2+. When the preincubation of serum and Zn2+ was prolonged to 30 and 60 min, the enzymatic activity decreased more rapidly with increasing metal concentration. Extended exposure of the enzyme to lower concentrations of the metal brought about the same effect as shorter exposure to higher metal concentrations.
INTRODUCTION
Zinc is an essential trace element necessary for sustaining all life. It is required for the action of more than 200 metallo enzymes such as; amino peptidases, alkaline phosphatase, carboxypeptidases, superoxide dismutase, thymidine kinase, carbonic anhydrase and several dehydrogenases (Sousa et al., 2007). Zinc plays an important role in polymeric organization of macromolecules like DNA and RNA, protein synthesis, cell division and metabolism (Smith and Akinbamizo, 2000). In addition, Zinc has been reported to influence the process of spermatogenesis (Ebisch et al., 2003; Wong et al., 2002), controls sperm motility (Wroblewski et al., 2003), stabilizes sperm membrane (Kendall et al., 2000), preserves the ability of sperm nuclear chromatin to undergo decondensation and modulates sperm functions (Suruki et al., 1995). It also required in plants for leaf formation, the synthesis of indole acetic acid (auxin) and anaerobic respiration and is now considered to be neurotransmitters (Hershfinkel et al., 2007; Gadallah, 2000).
Even though zinc is an essential element but it is usually identified as toxic, or even highly toxic, at elevated level (Gazaryan et al., 2007; Dineley et al., 2003; Christopher, 2004; Religa et al., 2006). Several episodes of zinc toxicity in cattle and sheep have been described. Sources of zinc included the accidental addition of zinc oxide to cattle feed, galvanized wire and troughs, heavy use of zinc-containing fertilizers and fungicides, milk replacer containing large amounts of a zinc supplemented home-made molybdenum licks (Sharpe and Livesey, 2005). Excess zinc has been implicated in the formation of osteochondrosis lesions in horses (Jeffcott, 1991), pancreatic injury and lesion in the proventriculus in birds (Samour, 2000), central lobular necrosis of the liver in pigs (Plumlee, 2004), hemolysis and hepatocllular necrosis in canine and feline (Bexfield et al., 2007), hepatic degeneration and lesions in the pancreas in bovine (Radostits et al., 2000). Earlier studies have led to the conclusion that elevated Zn2+ levels promote and/or exacerbate oxidative cell damage by an enhanced production of highly toxic oxygen species (Bishop et al., 2007) and it has been shown that antioxidant defense enzymes play a key role in the protection against metal and heavy metal toxicity (Pillai and Gupta, 2005). Peroxidases are important detoxifying enzymes serving to rid cells of excess H2O2 under normal and stress conditions, including contamination by toxic levels of metals (Richards et al., 1998; Fieldes and Gerhardt, 1998). However, extreme stress conditions may affect the activity of the detoxification enzymes themselves. For clarifying some possible mechanism of zinc toxicity in animals, the effect of increasing amounts of Zn2+ ion on peroxidase activity was investigated in vitro in serum of cow at this study.
MATERIALS AND METHODS
This study was performed during March 2007 to June 2008 in Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran.
Chemicals: O-dianisidine dihydrochloride was obtained from Sigma Chemical Co. (Deisenhofen, Germany) Hydrogen peroxide (30% solution); ZnSO4 and all the other chemicals used in this study were obtained from Merck (Darmstadt, Germany) and were of reagent grade.
Sampling: The blood samples were collected from cows by jugular venepuncture. After 30 min blood was centrifuged at 2000 x g for 10 min at 4°C to separate serum. Serum samples of all subjects were kept at -20°C until analysis (not later than 2 weeks).
Serum peroxidase activity assay: Serum peroxidase activity was measured by following the H2O2-dependent oxidation of o-dianisidine at 460 nm, using an extinction coefficient of 11.3/mM/cm (Worthington, 1988). O-dianisidine stock solutions (2.5 mg mL-1) were prepared by dissolving o-dianisidine in distilled water. H2O2 stock solutions (3 mM) were prepared daily by appropriate dilution of 30% H2O2 in distilled water. Stock solutions of ZnSO4 (1 M) were prepared in distilled water. The assay was performed in 0.1 M citrate buffer, pH 5.0. For assay done in the presence of Zn2+ ions, appropriate amounts of ZnSO4 stock solution were mixed with 0.1 M citrate buffer; the final volume was always 3 mL and the concentration of Zn2+ ions varied from 0.2 to 30 mM. The pH was readjusted to 5.0 whenever required. Serum (50 μL) was added and incubated with the Zn2+ ions for 5, 30 and 60 min before addition of 100 μL o-dianisidine. The reaction was then started by adding 100 μL H2O2 as usual and absorbance was record after an incubation period of 15 min. In order to prepare of Horseradish peroxidase (HRP) standard curve, the same principle as described for the serum peroxidase activity was done with one exception: Instead of serum, 50 μL HRP (110, 135, 780 and 1800 mU mL-1 HRP) was added to the reaction mixture. Serum peroxidase activity was calculated from absorbance readings relating to the HRP standard curve. Results were expressed as milliunits per milliliter. All assays were carried out at room temperature (~22-25°C) using an UNICO 2100 PC spectrophotometer. Results were average of at least three separate experiments.
RESULTS AND DISCUSSION
Figure 1 shows standard curve of HRP activity. Standards
peroxidase kinetic follows a linear order for peroxidase activity.
| Fig. 1: | Standardization of peroxidase activity assay |
| Table 1: | Values for cow serum peroxidase activity after 5, 30 and 60 min preincubation with various Zn2+ concentrations |
| The results are expressed as Mean±SD | |
The effect of Zn2+ on the oxidation of o-dianisidine by cow serum peroxidase in the presence of H2O2 was determined by following the formation of oxidized o-dianisidine at 460 nm, under end point conditions and after 5, 30 and 60 min preincubation of the serum with the metal ion. Incubation of serum with Zn2+ (0.2-20 mM) resulted in inhibition of the serum peroxidase activity. As shown in Table 1, the amount of inhibition of peroxidase activity depended on the length of incubation of the serum with the metal ion and on Zn2+ concentration. Incubation for 5 min at room temperature of serum with up to 0.4 mM Zn2+ not have any effect on the peroxidase activity. In the presence of 1-20 mM Zn2+, serum peroxidase activity was inhibited compared to the control and decreased rapidly with increasing metal concentrations. For example, at 1, 10 and 20 mM Zn2+, serum peroxidase activity decreased from 1713±5 mU mL-1 for the control to 1627±4, 1079±5 and 479±4 mU mL-1, respectively. The enzyme was completely inhibited after 5 min preincubation in 30 mM Zn2+. When the time of preincubation of serum and Zn2+ was increased to 30, 60 min, serum peroxidase activity decreased more rapidly with increasing metal concentration and the enzyme was inhibited at lower metal concentrations. For example, at 0.4 mM Zn2+ after 5 min preincubation serum peroxidase activity was 1713±4 mU mL-1, but after 30 and 60 min preincubation serum peroxidase activity reduced to 1695±8 and 1573±3 mU mL-1, respectively. At 10 mM Zn2+ after 5, 30 and 60 min preincubation serum peroxidase activity was reduced from 1079±3 to 959±7 and 736±3 mU mL-1, respectively. On the other hand, the same value of peroxidase activity was found for various combinations of Zn2+ concentration and incubation time. For example, serum peroxidase activity dropped from 1713±5 mU mL-1 for the control to about 1610±6 mU mL-1 either after a 60 min preincubation in 0.2 mM Zn2+or after a 5 min preincubation in 1 mM Zn2+. Another example shows the value of peroxidase activity dropping to about 1490±5 mU mL-1 either after 60 min preincubation in 1 mM Zn2+ or 5 min preincubation in 2 mM Zn2+. Thus, even though serum peroxidase appeared to have a rather low affinity for Zn2+, extended exposure of the enzyme to lower concentrations of the metal brought about the same effect as shorter exposure to higher metal concentrations.
Earlier studies showed the same results for the effect of cadmium and nickel ions on serum peroxidase and HRP enzymatic activity, respectively. Like Zn2+, Cd2+ and Ni2+ would inhibit peroxidase activity in a time and concentration dependent manner (Tayefi-Nasrabadi et al., 2006, 2008; Keyhani et al., 2005) and extended exposure of the enzyme to lower concentrations of the metals brought about the same effect as shorter exposure to higher metals concentrations. The enzymatic activity remained active over a broader Cd2+ concentration range compared to Zn2+. These results suggest that probably two distinctive binding sites exist for Zn2+ and Cd2+ on serum peroxidase.
Figure 2 shows the remaining activity of cow serum
peroxidase as a function of Zn2+ concentration after 5, 30
and 60 min preincubation of the enzyme with the metal. As shown in Fig.
2 for each preincubation time, the decrease in peroxidase activity
followed a different pattern. Incubation in 1 mM Zn2+ reduced
the peroxidase activity by 5% after 5 min preincubation, by 11% after
30 min preincubation and by 13% after 60 min preincubation. However, incubation
in 10 mM Zn2+ reduced the peroxidase activity by 37% after
5 min preincubation, by 44% after 30 min preincubation and by 57% after
60 min preincubation. Incubation in 20 mM Zn2+ reduced the
peroxidase activity by 72% after 5 min preincubation, by 76% after 30
min preincubation and by 81% after 60 min preincubation. By considering
of long biological half-life of metal in body of animals (Gupta, 2007),
it suggested that the damage caused by exposure to excess zinc ion concentration
is often not only dose-dependent, but also time-dependent. Even though
detoxifying enzymes such as peroxidases may not show any effect after
brief exposure to low concentrations of zinc ion, prolonged incubation
will affect the enzymatic activity, leading eventually to complete inactivation.
| Fig. 2: | Remaining cow serum peroxidase activity as a function of ZnSO4 concentration. Serum and ZnSO4 were preincubated for 5, 30 and 60 min prior to addition of o-dianisidine and H2O2. The activity was measured without Zn2+ was taken as 100%. Each experiment was performed at least three times and data are expressed as average±SD |
As mentioned earlier, excess zinc has been implicated in the formation of various injury and lesions in animals. The mechanism by which zinc toxicosis cause these lesions and injury has not been well understood. Some possible explanations include; inhibition of specific red blood cell enzyme, direct damage to the erythrocytic membrane, mitochondrial dysfunction, susceptibility of erythrocyte to oxidative damage, loss of mitochondrial defenses and interaction between zinc, iron, copper and calcium (Gazaryan et al., 2007; Plumlee, 2004).
Earlier studies have led to the conclusion that elevated Zn2+ levels promote and/or exacerbate oxidative cell damage by an enhanced production of highly toxic oxygen species (Bishop et al., 2007; Pong et al., 2002). The results presented here indicated that zinc ion at elevated level inhibit serum peroxidase activity by time and concentration dependent manner. Inhibition of peroxidases due to zinc ion could be considered as a reason for enhanced production of H2O2 as a highly toxic oxygen species and thereby causing of several pathological processes.
CONCLUSION
In conclusion, present results show that the damage caused by exposure to excess of zinc ion is often not only dose-dependent, but also time-dependent. Even though detoxifying enzymes such as serum peroxidases may not show any effect after brief exposure to low concentrations of zinc ion, extended exposure of the enzyme to lower concentrations of the metal brought about the same effect as shorter exposure to higher metal concentrations. Inhibition of animal`s antioxidant defense enzymes such as peroxidases by elevated level of zinc ion could be considered as an important parameter responsible for enhanced production of highly toxic oxygen species such as H2O2 and causing of several pathological processes in animals.
ACKNOWLEDGMENT
This study was supported by Research Council of University of Tabriz (Project No. 27/49297).