Long Term Excessive Zn Supplementation Induced Oxidative Stress in Wistar Rats Fed on Semi-Synthetic Diet

The use of Zn as micronutrient in husbandry and agriculture practices and also in baby foods and multivitamin supplements have been increased during the last two decades. However, the effect of long term increasing Zn load on the oxidative stress in the body has not been worked so far. The supplementation of pharmacological dosage of Zn in otherwise Zn adequate diet was investigated with the aim if excess Zn in the diet triggers oxidative stress and its conesquence on enzymes of antioxidant defense system. In this study, three groups of rats were fed on semi-synthetic diet containing 20 mg Zn/kg (control, group-I), 40 mg Zn/kg (group-II) and 80 mg Zn/kg (group-III) diet respectively for a period of 6 months. Blood lipid profile in the serum, lipid peroxidation status and the activities of antioxidant enzymes and trace minerals level were estimated both in the liver and kidney of three groups of rats. The study revealed that the gain in body weight increased in rats in Zn concentration dependent manner. The blood lipid profile displayed a significant rise in serum glucose, total lipids, cholesterol, triglycerides, LDL-cholesterol, VLDL-cholesterol whereas HDL-cholesterol showed a reduction in their levels in group-II and III than their control counter parts. The lipid peroxidation products were higher and the enzyme activities of superoxide dismutase, catalase, glutathione-s-transferase, glutathione reductase, glutathione (reduced) and glucose-6-phosphate dehydrogenase were significantly lowered in liver and kidney of group-II and group-III. Their mineral status revealed a higher Zn concentration and lower Cu, Mg and Mn both in liver and kidney. This data suggest that Zn in excess in diet when fed for longer periods of time induces oxidative stress by altering the status of minerals.


Introduction
Oxidative stress is the set of intracellular or extracellular conditions that leads to the chemical or metabolic generation of the reactive oxygen species (ROS), among which are highly reactive intermediates free radicals, such as superoxide anion ( 2O ), hydrogen peroxide (H 2 O 2 ), hydroxyl free radical (OH), lipid peroxides [1].It has become increasingly clear that oxidative stress plays a major role in the pathogenesis of a number of human diseases such as atherosclerosis, chronic renal failure, ischemia/reperfusion injury, neurodegenerative diseases, hypertension, cancer and diabetic micro and macro diseases [2].In mammalian cells, there are several mechanisms in which organisms defend itself against oxidative stress.Among them, there are antioxidant scavenging enzymes such as cellular Cu/Zn superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx),  Glutathione-s-transferase (G-s-T), glutathione reductase (GRD), glucose-6-phosphate dehydrogenase (Glu-6-PD) and small molecular antioxidants such as reduced glutathione (GSH) [3].As antioxidant enzymes have an important role in the protection against free radicals damage, a decreased in the activities or expression of these enzymes may predispose tissues to free radical damage.It has been reported that deficiency and excess of metals also promotes oxidative stress and lipid peroxidation [4].The activities of Cu and Zn containing antioxidant enzymes including superoxide dismutase (SOD), cytochrome-C oxidase (CCO), catalase (CAT) and glutathione peroxidase (GPx) have been reported to diminish under Zn or Cu-deficiency or due to alteration in Cu-Zn ratio [5].Moreover, activities of many enzymes are also influenced by Mg [6] and Mn status [7] and deficiencies or impairment in their metabolism may results an increased lipid peroxidation which possibly may be a con-tributory factor resulting in oxidative stress.Zn is an essential trace element and acts as indirect antioxidants via stabilization of cell membranes and the inhibition of free radicals production [8].The importance of Zn is systematically considered for its role in Cu/Zn SOD.Coudray et al. [9] found that experimental Zn deficiency causes a slight decreased in SOD activity accompanied by the increased production of the peroxidation lipids.On the contrary, there are increasing evidences that excessive Zn in diet induces obesity, diabetes, dyslipidemia and hypertension in experimental animals [10].Higher concentration of Zn and lower concentration of Cu, Mg and Mn in the tissues of human and some populations have been reported to link the ionic imbalance of nutritionally important elements to the etiology of diseases [11].Moreover, excessive Zn intake by supplementation can impair immunologic function, interfere with metabolism of other essential minerals and other lipid indices [12].Inspite of the fact that Zn is an essential micronutrient, the use of Zn supplement has been discourage by some health professional because excessive intake of Zn has been reported to induce Cu, Mg, Mn and Fe and Se deficiencies due their antagonistic interactions [13] and these minerals are required as cofactor for optimum catalytic activities and effective antioxidant defense mechanism of the body.During the past two decades, there has been a rise in the consumption of higher Zn either from Zn fortified foods as in the USA [14] or from vegetables (40 mg/kg or more in above ground vegetables and 120 mg/kg above in the underground vegetables) and meat foodstuffs (102 -165 mg/kg) as in some States of India [15,16] and this will further increase over time.As the population in these parts of the countries have been consuming excess amount of Zn through diet since a long period of time, the effect of such increasing Zn load in the body needs to be investigated by employing long term period of excess Zn supplementation just like in the real situation.It is believed that human individuals susceptible to many diseases are genetically predisposed to absorb or retain more Zn than their control counterparts like those of db/db mice and Zucker rats and on spontaneous development of diseases they start losing Zn in their urine.They are associated with weak endogenous defense mechanisms to protect against harmful radical induced cell damage.This led us to investigate the real situation of the long term effect of increasing Zn load in the antioxidant enzymes status of the body.Therefore, the supplementation of pharmacological dosage of Zn in otherwise Zn adequate diet was investigated with the aim if excess Zn in the diet triggers oxidative stress and its consequence on enzymes of antioxidant defense system in rats which are not genetically predisposed to any diseases.The results of the present study are given in this communication.

Materials and Methods
For the present investigations a semi-synthetic basal diet rich in fat and refined sucrose was preferred over the standard pellet rat diet consisting of the natural ingredients to keep the consistency of the composition of diet particularly of fat, sucrose and micronutrients through out of the experiment and to rule out the possibility of Zn-interaction with fibers and phytates [17] which are known to reduced bioavailability of Zn by binding it in digestive tract and may take longer time duration of feeding to manifest the impact excessive Zn in diet.Accordingly, isocaloric semi-synthetic basal diet for the rats was prepared following modified Orgebin-crist et al. [18].

Divisions of Diet
The basal diet was further divided into 2 parts: control diet consisting of basal diet containing 20 mg Zn/kg diet (per-se) for Group-I and Zn-supplement-diet-I containing 40 mg Zn/Kg diet for Group-II and Zn supplementeddiet-II containing 80 mg/Zn diet for Group-III by accordingly increasing ZnSO 4 •7H 2 O in basal diet.

Preparation of Diet
For each diet, the mineral & water-soluble vitamins were ground in sucrose and fat-soluble vitamins were dissolved in corn oil.Agar, which served as a binder and was dissolved in 25 ml of triple distilled, deionized warm water (60˚C).On cooling to 40˚C, the contents of each diet were thoroughly mixed in agar solution in separate containers.The dough so formed was put in petridishes and solidified in refrigerator.The solidified diet was cut into small pieces of 2 × 2 × 2 cm size and stored in the container at the temperature > −4˚C.

Experimental Design
Male wistar rats (30), aged 6 weeks, weighing 60 -70 g was procured from Central Animal House, Panjab University, Chandigarh.They were maintained in plastic cages with stainless steel top grill at room temperature (25˚C -28˚C) with 10:14 hr L:D cycles of 70% -80% RH as per guide lines of Institutional Animals Ethics Committee.They were fed on standard pellet rat feed for one week to acclimatize.Thereafter, the rats were divided into 3 groups-I, II and II and in such a way that their mean initial body weights remained almost similar in each group.The animals were fed on their respective diets ad libitium and triple distilled deionised water was made freely available to them for 180 days.The body weights were recorded at the beginning of the dietary treatment and thereafter every week.After the end of the dietary treatment of 180 days, the male rats of each group were sacrificed using diethyl ether as anesthesia.

Assessment of Blood Lipid Profile
The blood samples were collected by puncturing the heart and blood serum was prepared by centrifuging blood at 2500 rpm for 15 minutes.The freshly prepared serum was analyzed for cholesterol [19] triglycerides [20], HDL-cholesterol [21] (all by using commercially available kits-Reckon Diagnostic Pvt, Ltd., Baroda, India and ERBA diagnostic Mannheim GmbH, Mannheim, Germany, supplied through Transasia Bio-Medicals LTD, Daman) and total lipids [22].The LDL and VLDL-cholesterol was calculated by Friedwald's equation [23].

Assessments of Lipid Peroxidation and Antioxidant Enzymes Status in the Liver and Kidney
The liver and kidney of three groups of rats were removed for the study of enzyme activities.For this purpose, their homogenates were prepared in 50 mM Tris-HCL buffer (pH 7.4) and then centrifuged at 1000 g for 10 minute at 4˚C to remove nuclie and debris.Supernatant was again centrifuged at 1000 g for 30 minute to obtain post mitochondrial supernatant (PMS).The levels of lipid peroxidation (LPO) products were evaluated by the method of Beuge and Aust [24] and glutathione (reduced) (GSH) by the method of Ellman [25] in their PMS fraction.Activities of superoxide dismutase (SOD) [26] catalase (CAT) [27], glutathione-s-transferase (Glus-T) [28] glutathione reductase (GRD) [29] and glucose-6-phosphate dehydrogenase (G-6-PD) [30] were estimated in PMS of liver and kidney.Protein was evaluated as per of Lowry et al. [31].

Estimation of Minerals in Liver and Kidney
Zn, Cu, Mg and Mn were estimated on atomic absorption spectrophotometer (Electronic Corporation of India Lim-ited, Hyderabad-AAS 4139) using hollow cathode lamps (213.9 nm, 324.8 nm, 285.2 nm and 279.5 nm for Zn, Cu, Mg and Mn respectively).Samples of liver and kidney are digested separately in 3:1 v/v nitric acids and perchloric acids on a sand bath until a white ash formed.The ash was dissolved in 6 ml of 10 mM HNO 3 and filtered through ash free filter paper before analysis.Standards of Zn, Cu, Mg and Mn from Sigma Chemical Co., USA were prepared by dilution in triple distilled deionised water (TDW).

Statistical Analysis
The values are presented as means ± standard error of the mean (SEM).One way ANOVA was used to determine whether treatment groups differed significantly with control group.Differences were considered significant when P < 0.05.

Changes in Body Weight
The results of this study revealed a significant rise in monthly body weights (P < 0.001) in group-II and group-III up to a dietary treatment of 150 days (5 months).After that a significant decreased in body weights was observed in group-II and group-III after the next 30 days of dietary treatment (Table 1).

Changes in Blood Profile
After six month of the dietary treatment, a significant rise in serum glucose, total lipids, cholesterol, triglycerides, VLDL-c, LDL-c (P < 0.001) was observed with a significant decreased (P < 0.001) in serum HDL-c in group-II and group-III revealing alteration in blood lipid metabolisms in excess Zn supplemented groups (Table 2).

Lipid Peroxidation and Antioxidant Enzymes Status
The LPO products in group-II and III were found to be significantly higher (P < 0.001) in liver and kidney of group-II and III than the control group-I showing weak antioxidant defense system in former than the latter group.Their higher levels in group-II and III coincided with enzymes of antioxidant defense system i.e.SOD, CAT, Glu-s-T, GRD, GSH and Glu-6-PD whose activities were evaluated significantly less (P < 0.001; P < 0.05) in these groups after six months of dietary treatment (Tables 3 and 4).

Changes in Minerals Status
The difference in the activities of the enzymes of antioxidant system and lipid peroxidation coincided with difference in mineral status in different groups.Zn concentration was higher (P < 0.001) and Cu, Mg and Mn concentrations were significantly lowered (P < 0.001) in the liver and kidney of the group-II and III than the control group-I showing ionic imbalances in them (Table 5).

Discussion
The increase in body weight in three groups of rats varied significantly with the increase in Zn concentration in diet during the first 150 days of dietary treatment and thereafter it fell in group-II and III during the next 30 days with respect to their weight at 150 days than their control group-I at day 180 of the experiment.It suggests that Zn is highly potent nutrient which initially promotes the gain in body weight in concentration dependent manner but reduced body weights when such intakes are continued for a prolonged period as revealed in group-II   and group-III in the present investigations (Table 1).This observation is consistent as observed by Taneja et al. [32] who had discussed at length the mechanism of induction of this disorder in the rats induced by Zn and also reported by others [33].It also lends support to the report that an increase of Zn in diet to double the amount of recommended dietary allowances (RDA) increases the portion of body fat during 25 weeks period of dietary treatment in healthy children [34].The blood lipid profile of treated rats revealed significant rise of total lipids, cholesterol, triglycerides, VLDL-c and LDL-c and decreased HDL-c after 180 days of Zn supplementation in group-II and group-III as compared to the control group-I (Table 2).These observations are in agreement with previous reports from both human and animal studies wherein supplementation of Zn increased blood cholesterol (both LDL and VLDL-c) and decreased in HDLcholesterol level [35,36].This alterations in blood lipid profiles in the Zn treated group-II and III in the present investigations may be resulted from deficiencies of Cu, Mg and Mn in the tissues as a consequence of high Zn supplementation.The assessment of the metal status in the present study showed that Cu, Mg and Mn were approximately up to 30% less in group-II and up to 50% less in group-III rats both in liver and kidney indicating the induction of the Cu, Mg and Mn deficiencies or rise in ratios of Zn to Cu/Mg/Mn in the tissues.The observed increase of plasma cholesterol by increasing dietary Zn at all levels of Cu suggests that dietary Zn seems to suppress the plasma cholesterol lowering effect of dietary Cu [37] as a result of the increased in the activity of liver enzyme, 3-hydroxy-3 methylglutamate coenzyme A re-ductase [38], the rate limiting enzyme in cholesterol biosynthesis and decrease in the lipoprotein lipase, hepatic lipase and lecithin cholesterol acyltransferase (ICAT) activity [39].Experimental Mg deficiency has also been shown to increase triglycerides, cholesterol, VLDL, LDL rich lipoproteins and reduced HDL-c [40], almost similar to Cu deficiency.In Mg deficient animals, hypertriglyceridemia could have arisen as a consequence of increased synthesis of triglycerides in the liver, decreased removal of lipid from the blood or combination of both.An increased in the HDL-c concentration is closely associated with triglyceride metabolism and defective triglyceride removal from the plasma compartment might explain low HDL-c level as found in the present investigations.One more possible mechanism for the alteration of lipid levels is due to Mn-deficient in these animals which resulted an increased of lipid peroxidation in the endoplasmic reticulum, which is the site of lipoprotein synthesis.Bell and Hurley [41], observed swollen and irregular endoplasmic reticulum in tissues of Mn-deficient mice.This suggests that the observed rise in level of cholesterol, triglycerides, VLDL-c and LDL-c and reduction in HDLc in the group-II and III rats in the present investigations resulted due to Cu deficiency couple with Mg and Mn deficiencies in tissues caused by excessive Zn intake in diet.The LPO products was found to be higher in the liver and kidney of group-II and III rats than those of the control group-I showing weak antioxidant defense system in the former Zn supplemented groups in the present study.The production of lipid peroxidation products depends on activities of antioxidant enzymes such as SOD, CAT, Glu-s-T, GRD, GSH and Glu-6-PD which registered a significant reduction in liver and kidney of group-II and group-III rats (Tables 3 and 4).Since the group-II and group-III rats are displaying alteration in blood lipid metabolisms, as expected the lipid peroxidation products were higher in them than those of control rats indicating that the rats are under the oxidative stress.
To control lipid peroxidation, there is defensive system consisting of antioxidant enzymes that play an important role in scavenging reactive oxygen species.The organism's susceptibility to free radical stress and peroxidation stress is related to balance between free radical load and adequacy of antioxidant defenses.The decrease in SOD and CAT activities in the group-II and III rats in the present study may be attributed to exhaustion/inactivation possibly due to Cu and Mn deficiencies induced by high Zn intake in diet.Our results are in agreement with Cousins [42] and Strain [43] who reported that increased Zn intake reduces the activities of important antioxidant like SOD and CAT by limiting the bioavailability of Cu and increased LPO products in tissue [44].SOD and CAT constitute principal component of antioxidant enzymes system and a decreased in the activities/expression of these enzymes can result in oxidative stress by increasing LPO products [2] as found in the Zn treated group-II and III rats in the present investigations.Decreased activity of Glu-s-T in the present study suggests a reduction in availability of the substrate GSH which was recorded also in low level in the Zn treated group-II and III rats than the control group-I.The decreased GSH level in group-II and group-III rats could be due to decreased synthesis, increased consumption or increased loss through efflux during oxidative stress.In conformity of our results, other studies also has been reported depletion of GSH level due to inhibition of oxidized glutathione (GSSG) reductase by excess Zn [45].As a result of oxidative stress in group-II and III, the hexose monophosphate shunt (HMS) might have been impaired, so NADPH availability has been reduced and the ability to recycled GSSG to GSH by GRD might have been decreased.GRD catalyzes NADPH dependent reduction of oxidized glutathione serving to maintain intracellular glutathione stores and a favorable redox status [46].Therefore, these changes in the redox status could be considered particular sensitive indicative of oxidative stress in the Zn supplemented group-II and III found in the present investigations.The difference in the activities of the of antioxidant defense system and LPO products coincided with the difference in mineral status in different groups of rats.The assessment of mineral status in group-II and III revealed an ionic imbalance wherein Zn concentration in liver and kidney were higher and that of Cu, Mg and Mn were lowered compared to their control counterpart (Table 5).As a consequence of this, the activities of all the enzymes investigated were decreased in the group-II and III rats which in turn led to an increase of the LPO products in their respective groups.This ionic imbalance may be attributed to the overexpression of Zn-metallothieonien during high Zn treatment period.As a result, they absorbed and retained greater amount of Zn than the control counterpart leading to the deficiencies of Cu, Mg and Mn due to their negative interactions as found in the present study [47].The present data of minerals status indicated that Zn in excess even in pharmacological doses in diet over a period of time replaces Cu, Mg and Mn leading to their leaching and sloughing and excretion in the urine even if these metals are adequate in diet.And the deficiencies/imbalance of these elements are link to subsequent development of dyslipidemia and oxidative stress as observed in present study since these metallic ions being integral components of enzymes such as SOD, CAT, and GPx.

Conclusion
The data of the study showed that the depletion of the antioxidant defence system may be compensatory mecha-nisms against oxidative stress induced by high Zn intake in diet in treated rats which causes ionic imbalance of metals required for proper functioning of antioxidant enzymes.The results of the present study thus provide strong evidences that excessive Zn in diet even in pharmacological doses induced oxidative stress.Since oxidative stress play a major role in the pathogenesis of number of human diseases like atherosclerosis, diabetic micro and macro diseases, hypertension, cancer and obesity related diseases etc., the rising prevalence of diseases per se during past two decades may not essentially be due to changes in the lifestyle but also may be associated with the genetic predisposition from excessive Zn absorption or food intake through food chain due to its liberal use as ZnSO 4 in agriculture and animal husbandry practices employed by the farmers, Zn rich dietary supplements, Zn fortified foods and low amount of Zn binding protein such as fibres and phytates in diet.Thus, there appear to be oxidative stress related consequences of such pharmacological doses of Zn particularly when such intakes are continued for a prolonged period.

Table 1 . Month wise body weight of male rats of group-I, group-II and group-III during 180 days of dietary treat- ment [Values are mean ± SE of 6 observation each].
Units: gram; P values: a <0.001(values of group-II and group-III were compared with group-I).

Table 2 . Blood lipid profile of male rats of group-I, group- II and group-III during 180 days of dietary treatment [Val- ues are mean ± SE of 6 observation each].
a <0.001 (values of group-II and group-III were compared with group-I).

Table 3 . Mean lipid peroxidation and enzymes activities in liver of male rats of group-I, group-II and group-III during 180 days of dietary treatment [Values are mean ± SE of 6 observation each].
b <0.05 (values of group-II and group-III were compared with group-I).

Table 4 . Mean lipid peroxidation and enzymes activities in kidney of male rats of group-I, group-II and group-III dur- ing 180 days of dietary treatment [Values are mean ± SE of 6 observation each].
a <0.001; b <0.05 (values of group-II and group-III were compared with group-I).