Impact of Zinc toxic levels on Hepatic Heme Biosynthesis in Chick Embryos

Introduction

Zinc an essential metal is also known to be an environmental pollutant [1] . The homeostatic mechanism evidently fails at higher levels of zinc intake leading to zinc intoxication [2] . Zinc toxicity causes dehydration, electrolyte imbalance, stomach pain, nausea, lethargy and muscular incoordination [3] . Zinc functions as a stabilizer of bio membranes and biostructures [4] . Zinc is required for porphyrin biosynthesis and its potential effect on cellular heme metabolism has been demonstrated [5] . Haemoglobin liberated into plasma from disrupted erythrocytes is dangerous, because it can accelerate peroxidation of lipids in the presence of H2O2 [6,7] . Hence alterations or defects in enzyme activities of heme biosynthetic pathway result in profound change in these functions [8] . Most of the investigations conducted on effects of zinc have been concerned with metal mixtures [9] . Zinc supplementation has improved heme biosynthesis in rats exposed to lead has been examined extensively [10] . Whereas, very little attention was given to studies on embryonic system. Hence the present work was carried out to study the effect of toxic doses of zinc on heme biosynthesis in chick embryos.

Materials and Methods

Freshly laid Bobcock strain zero-day old eggs were procured from the Balaji hatcheries, Chittoor dt. Andhra Pradesh. They were placed in an egg incubator, the temperature of which was maintained at 37C with 65% relative humidity. The eggs were rotated manually once in a day during incubation. Injections of zinc as ZnSO4 (25 and 75 moles/kg egg wt.) dissolved in distilled water were given in a total volume of 10l to 14-day old chick embryos. Control embryos were injected with the same amount of distilled water. A minimum of 25 embryos were used in each group. Embryos were sacrificed on 20th day to collect hepatic tissues, because heme is predominantly synthesized in liver cells. Hepatic ALA levels were measured according to the method of Mauzerall and Granick [11] . ALA-d activity was assayed by the method of Granick et al. [12] , ALA-S activity was carried out according to the method of Lien and Beattie [13] . Heme synthetase was determined according to the method of Porra and Jones [14] . Heme was extracted according to Gerber and Maes [15] and estimated by the method of Christian de duve [16] . The protein content was determined by using bovine serum albumin as a standard [17] . Statistical evaluation was done as per Snedecor [8] .

Results and Discussion

A major site for regulation of hepatic heme biosynthesis was proposed at the level of ALA formation, hence the primary focus appears was on ALA-S enzyme. The mechanism involved in the regulation of the activity of ALA-S is of main interest, because perturbations in its activity are linked to several inherited and acquired disorders collectively known as porphyrias in broiler chicken [19] . The ALA-S activity inhibited in a dose dependent manner with 25 and 75 µ moles of zinc concentration. (Table . 1). Similar results were reported with sodium sulphide treatment where ALA-S activity was decreased [20] . ALA-d plays an important role in the supply of blood pigment precursor in chick embryo liver and it is a key step in heme biosynthesis [21] . Hence a major site for control of hepatic heme biosynthesis is at the level of ALA formation. Zinc treatment has not altered ALA levels (Table 1) and ALA-d activity (Table 2) in chick embryos, which may be due to the requirement of zinc metal for enzyme activation [22] . Though Zinc acts as a cofactor for AlA-d enzyme, Zinc toxic levels could not increase the enzyme activity further beyond its physiological levels, this suggests that the enzyme ALA-d has been saturated with Zinc at its physiological levels and could not increase enzyme activity further with increase in zinc levels to their toxic levels. Hepatic heme synthetase activity and heme levels increased with zinc treatment (Table 3). Though ALA levels has not increased, increased activity of heme synthetase may be responsible for elevated levels of heme production [23] . Further, elevated levels of hepatic heme with zinc treatment could be attributed to the decrease in ALA-S activity by means of feedback inhibition, because heme regulates its own production by down regulating the heme biosynthesis at the level of rate limiting enzyme ALA-S. To elucidate further, the excess heme produced due to increased activity of heme synthetase with zinc treatment has inhibited the first enzyme, ALA-S because both the enzymes ALA-S and heme synthetase are located in the mitochondrial membrane [24] . Further, many studies suggest that the intracellular heme presumed to be in the form of free heme pool is considered as ultimate mediator of heme biosynthesis control through its effect on ALA production and transport [25] . However, excess free heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation thus leading to membrane injury and ultimately apoptosis [26] . Therefore, excess heme produced and released could be reason for zinc toxicity in neurons mediated mainly by oxidative stress and zinc induced cell death may be associated with increased levels of ROS in neuron [27] . These findings were endorsed by one of our previous works where Zinc treatment to chick embryos has increased TBARS and SOD activity [28] . In contrast, few other studies have reported that heme synthetase activity measured in terms of metalloporphyrin's formed are serving as SOD mimetics and stated they could combat oxidative stress [29] .

Conclusion

Zinc is an essential mineral and is required for normal function of many enzymes. Zinc at its toxic levels has affected the embryonic heme biosynthesis by increasing the levels of free heme, by increasing the activity of heme synthetase. Though it acts as cofactor for ALA-d enzyme, zinc toxic levels couldn't enhance this enzyme activity in embryos. Zinc by increasing the heme levels and it being the end product of heme biosynthesis, has indirectly been the cause of decreased ALA-S activity, a regulatory enzyme of this pathway. Further this study suggests that the toxic levels of zinc has mediated its deleterious effects of increased ROS production through high free heme levels, which is a ROS generator through its peroxidase activity.

Acknowledgement

Acknowledgements: Authors are thankful to SVVU and SVU for providing funding and facilities for conduct of the research.

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