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Dietary zinc deficiency induced-changes in the activity of enzymes and the levels of free radicals, lipids and protein electrophoretic behavior in growing rats
Toxicology 175 (2002) 223– 234 www.elsevier.com/locate/toxicol
Dietary zinc deficiency induced-changes in the activity of enzymes and the levels of free radicals, lipids and protein electrophoretic behavior in growing rats M.I. Yousef a,*, H.A. El Hendy b, F.M. El-Demerdash a, E.I. Elagamy c a
Department of En6ironmental Studies, Institute of Graduate Studies and Research, Alexandria Uni6ersity, 163 Horreya A6enue, P.O. Box 832, Alexandria 21526, Egypt b Department of Home Economics, Faculty of Agriculture, Alexandria Uni6ersity, Alexandria, Egypt c Department of Dairy Science, Faculty of Agriculture, Alexandria Uni6ersity, Alexandria, Egypt Received 19 May 2001; accepted 31 January 2002
Abstract Zinc (Zn) is an essential nutrient that is required in humans and animals for many physiological functions, including immune and antioxidant function, growth and reproduction. The present study was conducted to investigate the effects of adequate Zn level (38 mg/kg diet, as a control) and two low levels that create Zn deficiencies (19 mg/kg diet, 1/2 of the control and 3.8 mg/kg diet, 1/10 of the control) in growing male and female rats for 10 weeks. To evaluate the effects of these levels, the concentrations of thiobarbituric acid-reactive substances (TBARS), biochemical parameters and protein pattern were studied. Lipid peroxidation in liver, brain and testes of rats fed Zn-deficient diet was indicated by increased TBARS. Serum, liver, brain and testes glutathione S-transferase (GST) activities were significantly (PB 0.05) increased in Zn-deficient rats, the effect was pronounced in rats fed the lowest level of Zn (1/10 of control). The activity of lactate dehydrogenase (LDH) was significantly (P B 0.05) increased in liver, brain and testes, but decreased in serum in a dose-dependent manner. Zinc deficiency increased (P B 0.05) liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in a dose-dependent manner, while there was no effect on the activity of these enzymes in testes. Zinc deficiency resulted in a significant (P B0.05) decrease in the activity of alkaline phosphatase (AlP) in serum and liver in a dose-dependent manner, but no effect in testes was found. The activity of acid phosphatase (AcP) was not affected in serum, liver and testes. Zn-deficient rats had higher liver concentrations of total lipids (TL), cholesterol, triglyceride (TG), and low density lipoprotein (LDL), while high density lipoprotein (HDL) was significantly (PB 0.05) declined in a dose-dependent manner. Brain and serum acetylcholinesterase (AChE) activities were, however, not affected (PB 0.05) by Zn deficiency. Protein content in liver, brain and testes showed a significant (PB 0.05) decrease in rats fed the lowest level of Zn (1/10 of control). Polyacrylamide gel electrophoresis (native-PAGE) of serum proteins revealed that the intensity of immunoglobulins, serum albumin as well as several peptide bands were decreased in rats fed 1/2 or 1/10 of Zn adequate, i.e. their synthesis was affected and it was pronounced with the lowest level of Zn deficiency (1/10 of control). However, no clear effect on the transferrin was observed in both cases compared to controls. From the results of this
* Corresponding author. 0300-483X/02/$ - see front matter © 2002 Published by Elsevier Science Ireland Ltd. PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 0 4 9 - 5
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study it can be concluded that Zn deficiency exerts numerous alterations in the studied biochemical parameters, protein pattern, and increased lipid peroxidation. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Zinc deficiency; Rats; Free radicals; Enzymes; Lipids; Lipoprotein; Gel electrophoresis; Protein
1. Introduction Zinc (Zn) is involved in many biochemical processes supporting life. The most important of these processes are cellular respiration, cellular utilization of oxygen, DNA and RNA, reproduction, maintenance of cell membrane integrity, and sequestration of free radicals (Chan et al., 1998). Zinc participates in the regulation of cell proliferation in several ways; it is essential to enzyme systems that influence cell division and proliferation. Removing Zn from the extracellular milieu results in decreased activity of deoxythymidine kinase and reduced levels of adenosine(5%) tetraphosphate(5%)-adenosine. Hence, Zn may directly regulate DNA synthesis through these systems (MacDonald, 2000). Zinc is essential to the structure and function of myriad proteins, including regulatory, structural and enzymatic. It is estimated that up to 1% of the human genome codes for Zn finger proteins (Frederickson et al., 2000). Zinc requirements in total diet of beef cattle, dairy cattle, swine, horses, sheep, goats, chickens, and turkeys are 30, 40, 50, 40, 20– 33, 40 – 75, 40–50, and 40– 75 ppm, respectively (NRC, 1978a,b, 1981, 1988). It is required for the activity of the antioxidant enzyme superoxide dismutase (Odeh, 1992). Also, Zn is involved with enzymes which govern DNA function (McClain et al., 1992; Varela et al., 1992). Filipe et al. (1995) found that Zn had an inhibitory effect on the spontaneous lipid peroxidation in rat brain. The results supported the effect of Zn in antioxidant properties, which may be potentially relevant to the protection of human serum constituents, competing with the transition metals for redox reactions. Zinc deficiency affects many systems because of Zn’s essential roles in many aspects of metabolism (Vallee and Falchuk, 1993), including the activity of more than 300 enzymes, the structure of many proteins, and control of genetic expression. Zinc status affects
aging through its requirement for synthesis and repair of DNA (Chesters et al., 1990), RNA (Blanchard and Cousins, 1996) and protein (Hicks and Wallwork, 1987). Zinc deficiency causes alterations in the activities of some enzymes such as alkaline phosphatase (AlP), acid phosphatase (AcP), lactic dehydrogenase, xanthine oxidase and NADPH oxidase (Eltohamy and Younis, 1991; Afanas’ev et al., 1995). Zinc is essential for human health but many adults and children may not be getting enough Zn in their diets (Xia et al., 1995). Because there is not enough studies on the effects of severe Zn deficiency in growing animals, therefore, the objective of this study was carried out to investigate the effects of two low levels that create Zn deficiencies on enzyme activities, lipid metabolism, protein pattern and lipid peroxidation in growing rats.
2. Materials and methods
2.1. Animals and treatments Forty-two weaned male and female albino rats (1-month-old and weighing 42.790.54 g) were used. Animals were caged in groups and given food and water ad libitum throughout the 10week experimental period. After the period of acclimation, animals were divided into three equal groups each of 14 animals (seven males and seven females). The first group (control) was fed the requirement of Zn, 38 mg Zn/kg basis diet (Ruz et al., 1999), the second group (1/2 of control) was fed a Zn-deficient diet (19 mg Zn/kg basis diet) and the third group (1/10 of control) was fed a very low Zn-deficient diet (3.8 mg Zn/kg basis diet). Rats received a purified diet (Table 1) with dried skim milk being the protein source. Rats fed Zn-deficient diets gained less (PB 0.05) than the control groups. Male and female rats fed 1/2 and 1/l0 of Zn adequate weighed 39, 42, 70 and 69%
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less than the control rats, respectively, which was previously mentioned by El Hendy et al. (2001).
2.2. Enzyme assessments At the end of the experimental period (10 weeks), male and female rats were sacrificed by cervical decapitation. Brain, liver and testes from male, and brain and liver from female were immediately removed weighed and washed using chilled saline solution. Brain, liver and testes were minced and homogenized (10% w/v) in icecold 1.15% KCl–0.01 M sodium, potassium phosphate buffer (pH 7.4) in a Potter– Elvehjem type homogenizer. The homogenate was centrifuged at 10,000×g for 20 min at 4 °C, and the resultant supernatant was used for different enzyme assays. Liver and testes alanine aminotransferase (ALT; EC 2.6.1.2) and aspartate aminotransferase (AST; EC 2.6.1.1) activities were assayed by the method of Reitman and Frankel (1957). Serum, brain, liver and testes lactate dehydrogenase (LDH, EC 1.1.1.27) activity was determined by the method of Cabaud and Wroblewski (1958). AlP (EC 3.1.3.1) activity was measured at 405 nm by the formation of paranitrophenol from para-nitrophenylphosphate as a substrate (Principato et al., 1985). For assaying AcP (EC 3.1.3.2) activity, the method of Moss (1984) was used. Glutathione S-transferase (GST; EC 2.5.1.18) activity was determined according to Habig et al. (1974), using P-nitrobenzylchloride as a substrate. Acetylcholinesterase (AChE; EC 3.1.1.7) activity was estimated using acetylcholine iodide as a substrate according to the method of Ellman et al. (1961). Thiobarbi-
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turic acid-reactive substances (TBARS) were measured in the homogenate of brain and liver by using the method of Tappel and Zalkin (1959) with slight modification. Protein concentration was assayed by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
2.3. Lipids and lipoprotein parameters Liver concentration of total lipids (TL), cholesterol, and triglyceride (TG) were determined according to the methods of Knight et al. (1972), Watson (1960), Fossati and Principe (1982), respectively. High density lipoprotein (HDL) and low density lipoprotein (LDL) were determined according to the methods of Warnick et al. (1983), Bergmenyer (1985), respectively. Very low density lipoprotein (VLDL) was calculated by dividing TG by 5.
2.4. Gel electrophoresis of serum proteins Vertical polyacrylamide gel slab electrophoresis at pH 8.6 (alkaline-PAGE) was carried out with Mini-Protean II cell (Bio-Rad) at 120 V and room temperature for 2 h. The gel used (0.75 mm thick) consisted of 4.5% T staking gel and 10% T separation gel (T% is an expression represent the concentration of acrylamide plus bisacrylamide in the gel). The electrode and migration buffers consisted of 0.19 M-glycine and 0.024 M-Tris at pH 8.6. After electrophoresis, proteins were localized in the gel using Coomassie blue 0.1% (Hames and Rickwood, 1990).
2.5. Statistical analysis
Table 1 Composition of the experimental diet Ingredient
Amount (g/kg)
Dried skim milk Corn oil Corn starch Sucrose Cellulose Vitamin mixture Mineral mixture
375 90 300 225 5 1 4
Data were analyzed by least-squares analysis. Male and female data were analyzed separately using completely randomized design (Steel and Torrie, 1980). The analyses were conducted according to the General Linear Model procedure of Statistical Analysis System (SAS) (1995). Difference between each two dose means within each sex was compared by LSD at 0.05 significant level (Steel and Torrie, 1980).
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3. Results and discussion
3.1. Effect of Zn deficiency on TBARS and GST Zinc has a wide spectrum of biological activities and its deficiency has been related to various tissue dysfunction and alterations of normal cell metabolism. Zinc also plays an important role in the antioxidant cellular defences being a structural element of the nonmitochondrial form of the enzyme superoxide dismutase (CuZnSOD) (Virgili et al., 1999). Free radicals are highly reactive species that have been implicated in the pathogenesis of many diseases. Reactive oxygen species (ROS) can initiate lipid peroxidation and DNA damage leading to mutagenesis, carcinogenesis and cell death, if the antioxidant system is impaired (Devi et al., 2000). DiSilvestro (2000) reported that Zn could exert a number of indirect antioxidant functions. In the present study, lipid peroxidation in liver, brain and testes was detected by significantly (PB 0.05) increased TBARS formation in both male and female rats fed 1/2 and 1/10 of Zn adequate (Table 2). These results are in agreement with the findings of Cao and Chen (1991), Shaheen and El-Fattah (1995), Chen and Young (1998) reported that Zn deficiency induced an increased free radical generation and lipid peroxidation in blood and liver of mice and rats, and decreased superoxide dismutase activity in the liver of mice. Also, Zn deficiency increased lipid peroxidation in mitochondria and microsomes from maternal and fetal liver, maternal kidney, maternal lung microsomes, and fetal lung mitochondria (Gunther and Hollriegl, 1989). Henning et al. (1999) reported that oxidative stress was increased in Zn-deficient endothelial cells. They suggested that Zn may in part be antiathergenic by inhibiting oxidative stress-responsive events in endothelial cell dysfunction. DiSilvestro (2000) reported that mild Zn deficiency in rats produces low resistance to chemically induced liver oxidant injury, and it produces high vulnerability of lipoproteins to oxidation. Chan et al. (1998) demonstrated that Zn is involved in destruction of free radicals through cascading enzyme systems. Superoxide radicals are reduced to hydrogen peroxide by superoxide dismutases in the presence of
Table 2 Serum, liver, testes and brain TBARS and GST in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9SE) Parameter Control
1/2 Zn
1/10 Zn
Male TBARS* Liver Brain Testes
31.83 90.63b 29.65 92.57b 33.85 90.36b
34.83 9 0.78b 33.46 90.78b 34.88 90.28b
GST** Serum Liver Brain Testes Female TBARS Liver Brain GST Serum Liver Brain
26.48 91.84a 21.50 91.11a 28.47 91.66a 0.53 9 0.037a 0.39 9 0.008a 0.49 9 0.008a 0.31 9 0.003a
25.77 9 1.31a 22.38 91.22a 0.56 9 0.016a 0.37 9 0.004a 0.48 9 0.009a
0.67 90.008b 0.42 9 0.008a 0.57 90.024ab 0.38 9 0.022b
32.13 90.60b 29.56 91.36b 0.68 9 0.009b 0.43 9 0.020b 0.53 9 0.011a
0.739 0.008b 0.52 9 0.024b 0.65 9 0.041b 0.3 90.014b
34.31 90.68b 31.74 90.66b 0.74 9 0.009c 0.49 9 0.018c 0.64 9 0.060b
Values are the means of seven rats. abcWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * TBARS is expressed as nmol per gram tissue. ** GST specific activity: mmol per hour per mg protein.
Zn cofactor. Hydrogen peroxide is then reduced to water by the selenium–glutathione peroxidase couple. Efficient removal of these superoxide free radicals maintains the integrity of membranes, reduces the risk of cancer, and slows the aging process (Chan et al., 1998). The generation of ROS is a steady-state cellular event in respiring cells. Their production can be grossly amplified in response to a variety of pathophysiological conditions such as inflammation, immunologic disorders, hypoxia, hyperoxia, metabolism of drug or alcohol, exposure to UV or therapeutic radiation, and deficiency in antioxidant vitamins. Uncontrolled production of ROS often leads to damage of cellular macromolecules (DNA, protein, and lipids) and other small antioxidant molecules (Chan et al., 1999). In general, the mechanism of antioxidation by Zn can be divided into acute and chronic effects.
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Chronic effects involve exposure of an organism to Zn on a long-term basis, resulting in induction of some other substance that is the ultimate antioxidant, such as the metallothioneins. Chronic Zn deprivation generally results in increased sensitivity to some oxidative stress (Powell, 2000). The acute effects involve two mechanisms: protection of protein sulfhydryls or reduction of OH formation from H2O2 through the antagonism of redoxactivate transition metals, such as iron and copper. Protection of protein sulfhydryl groups is thought to involve reduction of sulfhydryl reactivity through one of three mechanisms: (1) direct binding of Zn to the sulfhydryl; (2) steric hindrance as a result of binding to some other protein site in close proximity to the sulfhydryl group; or (3) a conformational change from binding to some other site on the protein. Antagonism of redox-active, transition metal-catalyzed, sitespecific reactions has led to the theory that Zn may be capable of reducing cellular injury that might have a component of site-specific oxidative damage, such as postischemic tissue damage. Zn is capable of reducing postischemic injury to a variety of tissues and organs through a mechanism that might involve the antagonism of copper reactivity. Although the evidence for the antioxidant properties of Zn is compelling, the mechanisms are still unclear (Powell, 2000). The GSTs form a group of multi-gene isoenzymes involved in the cellular detoxification of both xenobiotic and endobiotic compounds (Salinas and Wong, 1999). Glutathione and glutathione-related enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as ROS (Knapen et al., 1999). The present data showed a significant increase (PB 0.05) in GST activities in serum, liver, brain and testes of male rats and in serum, liver and brain of female rats fed 1/2 and1/10 of Zn adequate (Table 2). In agreement with the present data, it has found that rats fed Zn-deficient diet had higher concentrations of TBARS, GST activity, lower concentration of glutathione and glutathione peroxidase as well as lower activity of superoxide dismutase in plasma (Kraus et al., 1997).On the other hand, Jagadeesan (1989) reported that the activity of GST,
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a key enzyme in conjugation reaction, was significantly lowered in Zn deficiency. A number of major cellular defense mechanisms exist to neutralize and combat the damaging effects of these reactive substances. The enzymatic system functions by direct or sequential removal of ROS (superoxide dismutase, catalase, and glutathione peroxidase), thereby terminating their activities. Metal binding proteins, targeted to bind iron and copper ions, ensure that these Fenton metals are cryptic. Nonenzymic defense consists of scavenging molecules that are endogenously produced (GSH, ubiquinols, uric acid) or those derived from the diet (vitamins C and E, lipoic acid, selenium, riboflavin, Zn, and the carotenoids) (Chan et al., 1999). Oteiza et al. (1996) found that the activities of copper-Zn superoxide dismutase (CuZn SOD) and glutathione reductase (GRed) were significantly higher (the oxidant defense system) in the testes of Zn-deficient rats. They reported that Zn deficiency can cause an oxidative stress situation in testes, for which cells tend to compensate by increasing select components of the oxidant defense system. This finding is agreement with our results which GST activity increased in Zn-deficient rats (Table 2).
3.2. Effect of Zn deficiency on enzyme acti6ities AST and ALT are imported and critical enzymes in the biological processes. These enzymes are involved in the breakdown of amino acids into 8 Keto acid which are routed for complete metabolism through the Kreb’s cycle and electron transport chain. Consequently, they are considered as a specific indicator for hepatic dysfunction and damage (Osuna et al., 1977; Shakoori et al., 1994). AcP is a marker enzyme for lysosomes, thus related to general cell metabolism. AlP enzyme is a sensitive biomarker to metallic salts since it is a membrane bound enzyme related to the transport of various metabolites (Lakshmi et al., 1991). AlP was the first Zn enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme (Coleman,
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1992). The increase of AlP in serum could be a result of damage of liver cells and bile duct obstruction due to proliferation of its cells and/or related to the progressive liver necrosis (Tietz, 1976). LDH is one of the metabolic requirements of tissue and involved in energy production. LDH activity indicates the switching over of anaerobic glycolysis to aerobic respiration. Also, it can be used as an indicator for cellular damage, clinical practice and cytotoxicity of toxic agents (Bagchi et al., 1995). The increment of the activities of AST, ALT, LDH and AlP in plasma is mainly due to the leakage of a these enzymes from the liver cytosol into the blood stream (Navarro et al., 1993), which indicated liver damage and disruption of normal liver function (Shakoori et al., 1994). Acetyleholinesterase (AChE), an enzyme that is responsible for hydrolysing and so deactivating acetylcholine in the nervous system (Forget et al., 1999). The inhibited AChE could decrease cellular metabolism, induce deformities of cell membrane, and disturbed metabolic and nervous activity (Suresh et al., 1992). Also, the decrease in AChE activity could lead to ionic refluxes and differential membrane permeability (Tolosa et al., 1996). The present data indicated an increase (P B 0.05) in the activity of liver AST, ALT and LDH of both sexes fed 1/2 and 1/10 of Zn adequate (Table 3). The activity of AlP, however was decreased (PB 0.05) by 17 and 31% of 1/2 and 1/10 of Zn adequate in males, and 12 and 29% in females, respectively. On the other hand, the activity of AcP did not change (Table 3). The present results showed that brain LDH was markedly inhibited (P B0.05) in male and female rats fed 1/2 and 1/10 of Zn adequate by 23, 57, 26 and 52%, respectively (Table 4). The effects of Zn deficiency on testis enzymes in rats are presented in Table 5. These data showed a significant decline (PB0.05) in AcP activity, while LDH was significantly increased of male rats that fed 1/10 of Zn adequate. Testes AST, ALT and AlP activities were not affected by reducing Zn level in the diet. Male and female rats fed the lowest Zn level (1/10 of control) showed a reduction (P B 0.05) in serum AlP and LDH, and the magnitude of reduction was 27 and 40% for males, 23 and 38%
for female compared to control, respectively (Table 6). These results are in agreement with Alayash (1989), who reported that serum levels of AlP and LDH, two Zn dependent enzymes in these patients, in North American Blacks with sickle cell anemia, who are known to have Zn deficiency coupled with a decrease in the activity of these enzymes. Also, Wan et al. (1993) found that Zn deficiency was produced a significant reductions in plasma AlP activity (48%) of rats. Tables 4 and 6 show that serum and brain AChE activities were not affected by reducing Zn level in the diet. The changes in the activities of tested enzymes in Zn-deficient animals might be due to that Zn deficiency affects many systems because it has essential roles in many aspects of metabolism including the activity of more than 300 enzymes (Vallee and Falchuk, 1993; Prasad, 1996). Zinc is a component of many enzymes, including AlP, lactate and glutamate dehydrogenase, carbonic Table 3 Assay of liver enzyme activities and protein levels in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9SE) Parameters
Control
1/2 Zn
1/10 Zn
Male AST (IU/mg)* 106 91.1a ALT (IU/mg) 67 9 0.7a AlP (IU/mg) 1131 931a AcP (IU/mg) 697 92.6a LDH (IU/g)** 2690 9140a Protein (mg/g) 237 916.0a
118 94.0b 81 9 3.0b 942 918b 686 96.3a 3407 9118b 221 95.7ab
130 92.0c 97 9 6.1c 781 9 51c 685 92.4a 4023 9181b 181 912.0b
Female 101 91.7a AST (IU/mg) ALT (IU/mg) 71 9 2.7a AlP(IU/mg) 1055 920a AcP (IU/mg) 695 91.2a LDH (IU/g) 2413 978a Protein (mg/g) 255 98.5a
116 93.4b 8493.4b 925 927b 686 97.1a 3089 9176b 234 99.2a
128 92.1c 98 9 4.9c 746 940c 673 92.6a 3740 9170c 199 99.1b
Values are the means of seven rats. abcWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * IU/mg: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substrate per minute per mg protein. ** IU/g: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substratesubstrate per minute per gram tissue.
M.I. Yousef et al. / Toxicology 175 (2002) 223–234 Table 4 Assay of brain AChE, LDH activities and protein levels in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9 SE) Parameter
Control
1/2 Zn
1/10 Zn
Male AChE* LDH** Protein (mg/g)
4.4590.14ab 4.70 90.18a 4.079 0.23b 1553 9 69.2a 19139 140.5b 24449 57.5c 78.19 2.62ab 68.39 5.87b 85.3 92.34a
Female AchE LDH Protein (mg/g)
4.04 90.47a 1500 9 88.0a 80.1 94.14a
3.72 90.28a 3.739 0.15a 18969 182.8b 22859 54.6c 73.69 5.37ab 61.19 4.21b
Values are the means of seven rats. abcWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * AChE activity: mmol substrate hydrolyzed per minute per mg protein. ** IU/g: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substrate per minute per gram tissue.
anhydrase, DNA polymerase and some peptidases (Guyton, 1981; Martin, 1983). In agreement with the present results, Eltohamy and Younis (1991) found that there was significant reduction in the levels of AlP, AcP and lactic dehydrogenase of both testes and epididymis of Zn-deficient rabbits. Also, Zn deficiency led to a reduction of serum and testis AlP activity (Reeves, 1990; Naber et al., 1996; Kraus et al., 1997). Prasad (1983) reported that the activities of many Zn-dependent enzymes have been shown to be affected adversely in Zndeficient tissues. Three enzymes, AlP, carboxypeptidase and thymidine kinase, appear to be most sensitive to Zn restriction in that their activities are affected adversely within 3–6 days of institution of a Zn-deficient diet to experimental animals.
3.3. Effect of Zn deficiency on protein content Zinc is essential for synthesis of coenzymes that mediate biogenic-amine synthesis and metabolism (Sandstead et al., 2000). Male and female rats fed the lowest Zn level (1/10 of control) showed a
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reduction (PB 0.05) in protein, and the magnitude of reduction was 24, 20 and 28% in liver, brain and testes of males, 22 and 24% in liver and brain of female compared to control, respectively (Tables 3–5). In agreement with the present results, Eltohamy and Younis (1991) reported that there was significant reduction in the levels of protein of both testes and epididymis of Zn-deficient rabbits. Prasad (1996) found that Zn deficiency influence DNA synthesis, cell division and protein synthesis. In addition, Oteiza et al. (1996) demonstrated that Zn deficiency could be associated with high rates of oxidative damage to proteins and DNA in male rats. The observed decrease in protein content might be attributed in part to the concomitant induction in lipid peroxidation (Table 2) in both male and female rats fed 1/2 and 1/10 of Zn adequate.
3.4. Effect of Zn deficiency on the concentrations of lipids and lipoproteins Table 7 shows the changes in liver TL, cholesterol, TG, HDL, LDL and VLDL of growing Table 5 Assay of testes enzyme activities and protein levels in male rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9 SE) Parameter
Control
1/2 Zn
1/10 Zn
50.4 9 1.77a AST 55.0 9 1.85a 52.4 9 2.58a (IU/mg)* ALT 14.81 94.48a 15.05 98.90a 12.60 910.73a (IU/mg) AlP 1050 948.4a 987 920.1a 975 919.0a (IU/mg) 517 9 13.02b AcP 581 98.28a 560 99.99ab (IU/mg) LDH 2108 9147.1a 2380 9118.7ab 2741 9255.1b (IU/g)** Protein 31.8 90.87a 30.7 90.82a 22.9 9 1.626b (mg/g) Values are the means of seven rats. abWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * IU/mg: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substrate per minute per mg protein. ** IU/g: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substrate per minute per gram tissue.
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Table 6 Assay of serum enzyme activities in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9SE) Parameter
Control
1/2 Zn
Male AChE* AlP** AcP** LDH**
1.419 0.21a 1.29 90.02a 236.4 98.65a 226.29 12.43a 5.91 90.22a 5.6590.06a 2212 9 106.9a 1946 9 132.8a
Female AChE AlP** AcP** LDH**
1.18 90.03 1.099 0.13 260.1 98.89a 241.89 16.90ab 6.61 90.18a 6.05 9 0.42a 2245 9 222.9a 1627 9 122.9b a
1/10 Zn
a
1.3290.02a 172.99 21.47b 5.4290.54a 13319 176.7b a
1.1390.05 200.09 15.67b 5.9090.46a 13989 111.9b
Values are the means of seven rats. abWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * AChE activity: mmol substrate hydrolyzed per ml serum per minute. ** (IU/l)
male and female rats fed Zn deficient diets (1/2 or 1/10 of control). Liver HDL was decreased (P B 0.05) in male and female rats that fed 1/10 of Zn adequate. Liver TL, cholesterol, TG, LDL and VLDL, however were increased (P B0.05) in Zndeficient rats in a dose-dependent manner (Table 7). In agreement with the present data, it has found that Zn deficiency was associated with lower HDL levels (Schneeman et al., 1986). On the other hand, Neggers et al. (2001) found that Zn supplements caused an increase in HDL value in an adult African– American community. Eder and Kirchgessner (1995) reported that Zn-deficient rats characterized by elevated levels of triglyceride. Also, Eltohamy and Younis (1991) found that there was significant increase in the levels of cholesterol in testes of Zn-deficient rabbit. In addition, lipid composition and essential fatty acids concentration are altered in the brain of Zn-deficient animals (Essman, 1987). On the other hand, Paul et al. (2001) found that Zn supplementation caused a significant reduction in plasma cholesterol and TG of mice. Cunnane (1988) discussed the role of Zn in the metabolism of lipids and the role of it in membranes as well as
Zn’s known effects on receptors. He reported that Zn intimately affects many aspects of lipid metabolism through established enzymes but also has modulatory effects whose mechanism is not obvious or established.
3.5. Gel electrophoresis of serum proteins Fig. 1 shows the electrophoretic patterns of serum proteins of male and female rats fed Zn adequate, 1/2 and 1/10 of Zn adequate. The patterns showed several peptide bands differ in migration positions and intensities. Five major peptide bands, in addition other minor bands were recognized in each pattern. The major bands belong to immunoglobulins, transferrin, serum albumin and another two unknown fractions (F1 and F2). It is obvious that there were no differences between male and female control serum samples in bands number or intensity. However, the intensity of immunoglobulins, serum albumin, F1, F2 as well as the minor peptide bands was decreased in rats fed 1/2 of Zn adequate compare
Table 7 Liver TL, cholesterol, TG, HDL, LDL and VLDL in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9 SE) Parameter (mg/g)
Control
1/2 Zn
1/10 Zn
Male TL Cholesterol TG HDL LDL VLDL
34.8 9 4.07a 3.25 90.19a 11.0 90.93a 0.35 9 0.03a 1.63 90.10a 2.21 9 0.19a
40.0 9 2.77a 3.96 90.31b 13.9 9 1.70ab 0.29 9 0.01ab 2.15 90.20b 2.77 9 0.34ab
52.0 9 13.35b 4.24 9 0.58b 14.8 9 0.44b 0.24 9 0.02b 2.58 9 0.21b 2.95 9 0.09b
Female TL Cholesterol TG HDL LDL VLDL
35.78 91.89a 3.44 9 0.25a 11.5 90.70a 0.36 90.03a 1.64 9 0.11a 2.30 90.14a
37.9 9 5.73a 4.29 90.44b 14.0 9 0.56b 0.32 9 0.01ab 2.01 9 0.29b 2.80 9 0.11b
52.6 9 4.72b 4.69 9 0.28b 15.5 9 0.82b 0.29 9 0.02b 2.47 9 0.35b 3.1190.17b
Values are the means of seven rats. abWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05).
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Fig. 1. Alkaline-polyacrylamide gel electrophoresis (alkaline-PAGE) at pH 8.6 of rat serum proteins. Lanes 1 and 2: control serum samples of untreated male and female rats, respectively; lanes 3 and 4: serum samples of male and female rats fed 1/2 of Zn adequate; lanes 5 and 6: serum samples of male and female rats fed the lowest level of Zn (1/10 of control). Each lane represents the pooled sample of seven animals for each group. Anode is toward bottom of photo.
to controls. This means that the synthesis of such peptides was affected. Prasad (1998) reported that Zn plays an important role in the immune system and Zn deficient subjects may experience increased susceptibility to a variety of pathogens. It is clear that Zn affects multiple aspects of the immune system, from the barrier of the skin to gene regulation within lymphocytes. Zinc is crucial for normal development and function of cells mediating nonspecific immunity such as neutrophils and natural killer cells (Shankar and Prasad, 1998). Zinc plays an important role in immune function, helping increase resistance to infection and tumor growth. Low levels of Zn result in a decrease in helper T-cells and thymic hormone, which may adversely affect immune functioning (Eby et al., 1984). No clear effect on transferrin was noticed in serum samples of rats fed 1/2 and 1/10 of Zn adequate. At the lowest level of Zn (1/10 of control) the effect on minor peptides was pronounced specially F7, F8 and F9. These fractions may represent some enzymes or hormones. From the present results it can be concluded that the synthesis of several proteins is affected in rats fed 1/2 and 1/10 of Zn adequate. Also, Prasad (1996) reported that approximately 300 enzymes are known to require Zn for their activities. Zinc is
required for each step of cell cycle in microorganism and is essential for DNA synthesis, cell division and protein synthesis. They reported that the effect of Zn on protein synthesis may be attributable to its vital role in nucleic acid metabolism. In addition, Tate et al. (1995) found that protein synthesis in low Zn medium (0.25 mM Zn) of human retinal pigment epithelium was depressed compared to the control (11 mM Zn). Giugliano and Millward (1987) found that protein synthesis and RNA concentrations were reduced in rats fed on a Zn-deficient diet. They also reported that Zn deficiency impairs growth by a combination of reduced food intake, a reduced anabolic response to food due to a reduced capacity for protein synthesis and reduced activation of protein synthesis. The present results concluded that dietary Zn deficiency exerts alterations in the enzyme activities in serum, liver, brain and testes. Also, Zn deficiency caused a significant increase in the levels of free radicals and GST activity in all tested tissues. The levels TL, cholesterol, TG, and LDL were significantly increased in the liver of Zn-deficient rats, while HDL concentrations were decreased. Protein content in liver, brain and testes showed a significant (P B 0.05) decrease in rats fed the lowest level of Zn (1/10 of control). Con-
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cerning the protein patterns, the intensity of immunoglobulins, serum albumin, as well as several peptides bands were decreased in rats fed 1/2 of Zn adequate. All of these alterations were pronounced in rats fed the lowest level of Zn (1/10 of control). To be sure, structural studies of Zn in biology will continue to be a fruitful source of bioinorganic advances, as well as surprises, in the future.
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Dietary zinc deficiency induced-changes in the activity of enzymes and the levels of free radicals, lipids and protein electrophoretic behavior in growing rats M.I. Yousef a,*, H.A. El Hendy b, F.M. El-Demerdash a, E.I. Elagamy c a
Department of En6ironmental Studies, Institute of Graduate Studies and Research, Alexandria Uni6ersity, 163 Horreya A6enue, P.O. Box 832, Alexandria 21526, Egypt b Department of Home Economics, Faculty of Agriculture, Alexandria Uni6ersity, Alexandria, Egypt c Department of Dairy Science, Faculty of Agriculture, Alexandria Uni6ersity, Alexandria, Egypt Received 19 May 2001; accepted 31 January 2002
Abstract Zinc (Zn) is an essential nutrient that is required in humans and animals for many physiological functions, including immune and antioxidant function, growth and reproduction. The present study was conducted to investigate the effects of adequate Zn level (38 mg/kg diet, as a control) and two low levels that create Zn deficiencies (19 mg/kg diet, 1/2 of the control and 3.8 mg/kg diet, 1/10 of the control) in growing male and female rats for 10 weeks. To evaluate the effects of these levels, the concentrations of thiobarbituric acid-reactive substances (TBARS), biochemical parameters and protein pattern were studied. Lipid peroxidation in liver, brain and testes of rats fed Zn-deficient diet was indicated by increased TBARS. Serum, liver, brain and testes glutathione S-transferase (GST) activities were significantly (PB 0.05) increased in Zn-deficient rats, the effect was pronounced in rats fed the lowest level of Zn (1/10 of control). The activity of lactate dehydrogenase (LDH) was significantly (P B 0.05) increased in liver, brain and testes, but decreased in serum in a dose-dependent manner. Zinc deficiency increased (P B 0.05) liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in a dose-dependent manner, while there was no effect on the activity of these enzymes in testes. Zinc deficiency resulted in a significant (P B0.05) decrease in the activity of alkaline phosphatase (AlP) in serum and liver in a dose-dependent manner, but no effect in testes was found. The activity of acid phosphatase (AcP) was not affected in serum, liver and testes. Zn-deficient rats had higher liver concentrations of total lipids (TL), cholesterol, triglyceride (TG), and low density lipoprotein (LDL), while high density lipoprotein (HDL) was significantly (PB 0.05) declined in a dose-dependent manner. Brain and serum acetylcholinesterase (AChE) activities were, however, not affected (PB 0.05) by Zn deficiency. Protein content in liver, brain and testes showed a significant (PB 0.05) decrease in rats fed the lowest level of Zn (1/10 of control). Polyacrylamide gel electrophoresis (native-PAGE) of serum proteins revealed that the intensity of immunoglobulins, serum albumin as well as several peptide bands were decreased in rats fed 1/2 or 1/10 of Zn adequate, i.e. their synthesis was affected and it was pronounced with the lowest level of Zn deficiency (1/10 of control). However, no clear effect on the transferrin was observed in both cases compared to controls. From the results of this
* Corresponding author. 0300-483X/02/$ - see front matter © 2002 Published by Elsevier Science Ireland Ltd. PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 0 4 9 - 5
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study it can be concluded that Zn deficiency exerts numerous alterations in the studied biochemical parameters, protein pattern, and increased lipid peroxidation. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Zinc deficiency; Rats; Free radicals; Enzymes; Lipids; Lipoprotein; Gel electrophoresis; Protein
1. Introduction Zinc (Zn) is involved in many biochemical processes supporting life. The most important of these processes are cellular respiration, cellular utilization of oxygen, DNA and RNA, reproduction, maintenance of cell membrane integrity, and sequestration of free radicals (Chan et al., 1998). Zinc participates in the regulation of cell proliferation in several ways; it is essential to enzyme systems that influence cell division and proliferation. Removing Zn from the extracellular milieu results in decreased activity of deoxythymidine kinase and reduced levels of adenosine(5%) tetraphosphate(5%)-adenosine. Hence, Zn may directly regulate DNA synthesis through these systems (MacDonald, 2000). Zinc is essential to the structure and function of myriad proteins, including regulatory, structural and enzymatic. It is estimated that up to 1% of the human genome codes for Zn finger proteins (Frederickson et al., 2000). Zinc requirements in total diet of beef cattle, dairy cattle, swine, horses, sheep, goats, chickens, and turkeys are 30, 40, 50, 40, 20– 33, 40 – 75, 40–50, and 40– 75 ppm, respectively (NRC, 1978a,b, 1981, 1988). It is required for the activity of the antioxidant enzyme superoxide dismutase (Odeh, 1992). Also, Zn is involved with enzymes which govern DNA function (McClain et al., 1992; Varela et al., 1992). Filipe et al. (1995) found that Zn had an inhibitory effect on the spontaneous lipid peroxidation in rat brain. The results supported the effect of Zn in antioxidant properties, which may be potentially relevant to the protection of human serum constituents, competing with the transition metals for redox reactions. Zinc deficiency affects many systems because of Zn’s essential roles in many aspects of metabolism (Vallee and Falchuk, 1993), including the activity of more than 300 enzymes, the structure of many proteins, and control of genetic expression. Zinc status affects
aging through its requirement for synthesis and repair of DNA (Chesters et al., 1990), RNA (Blanchard and Cousins, 1996) and protein (Hicks and Wallwork, 1987). Zinc deficiency causes alterations in the activities of some enzymes such as alkaline phosphatase (AlP), acid phosphatase (AcP), lactic dehydrogenase, xanthine oxidase and NADPH oxidase (Eltohamy and Younis, 1991; Afanas’ev et al., 1995). Zinc is essential for human health but many adults and children may not be getting enough Zn in their diets (Xia et al., 1995). Because there is not enough studies on the effects of severe Zn deficiency in growing animals, therefore, the objective of this study was carried out to investigate the effects of two low levels that create Zn deficiencies on enzyme activities, lipid metabolism, protein pattern and lipid peroxidation in growing rats.
2. Materials and methods
2.1. Animals and treatments Forty-two weaned male and female albino rats (1-month-old and weighing 42.790.54 g) were used. Animals were caged in groups and given food and water ad libitum throughout the 10week experimental period. After the period of acclimation, animals were divided into three equal groups each of 14 animals (seven males and seven females). The first group (control) was fed the requirement of Zn, 38 mg Zn/kg basis diet (Ruz et al., 1999), the second group (1/2 of control) was fed a Zn-deficient diet (19 mg Zn/kg basis diet) and the third group (1/10 of control) was fed a very low Zn-deficient diet (3.8 mg Zn/kg basis diet). Rats received a purified diet (Table 1) with dried skim milk being the protein source. Rats fed Zn-deficient diets gained less (PB 0.05) than the control groups. Male and female rats fed 1/2 and 1/l0 of Zn adequate weighed 39, 42, 70 and 69%
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less than the control rats, respectively, which was previously mentioned by El Hendy et al. (2001).
2.2. Enzyme assessments At the end of the experimental period (10 weeks), male and female rats were sacrificed by cervical decapitation. Brain, liver and testes from male, and brain and liver from female were immediately removed weighed and washed using chilled saline solution. Brain, liver and testes were minced and homogenized (10% w/v) in icecold 1.15% KCl–0.01 M sodium, potassium phosphate buffer (pH 7.4) in a Potter– Elvehjem type homogenizer. The homogenate was centrifuged at 10,000×g for 20 min at 4 °C, and the resultant supernatant was used for different enzyme assays. Liver and testes alanine aminotransferase (ALT; EC 2.6.1.2) and aspartate aminotransferase (AST; EC 2.6.1.1) activities were assayed by the method of Reitman and Frankel (1957). Serum, brain, liver and testes lactate dehydrogenase (LDH, EC 1.1.1.27) activity was determined by the method of Cabaud and Wroblewski (1958). AlP (EC 3.1.3.1) activity was measured at 405 nm by the formation of paranitrophenol from para-nitrophenylphosphate as a substrate (Principato et al., 1985). For assaying AcP (EC 3.1.3.2) activity, the method of Moss (1984) was used. Glutathione S-transferase (GST; EC 2.5.1.18) activity was determined according to Habig et al. (1974), using P-nitrobenzylchloride as a substrate. Acetylcholinesterase (AChE; EC 3.1.1.7) activity was estimated using acetylcholine iodide as a substrate according to the method of Ellman et al. (1961). Thiobarbi-
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turic acid-reactive substances (TBARS) were measured in the homogenate of brain and liver by using the method of Tappel and Zalkin (1959) with slight modification. Protein concentration was assayed by the method of Lowry et al. (1951) using bovine serum albumin as a standard.
2.3. Lipids and lipoprotein parameters Liver concentration of total lipids (TL), cholesterol, and triglyceride (TG) were determined according to the methods of Knight et al. (1972), Watson (1960), Fossati and Principe (1982), respectively. High density lipoprotein (HDL) and low density lipoprotein (LDL) were determined according to the methods of Warnick et al. (1983), Bergmenyer (1985), respectively. Very low density lipoprotein (VLDL) was calculated by dividing TG by 5.
2.4. Gel electrophoresis of serum proteins Vertical polyacrylamide gel slab electrophoresis at pH 8.6 (alkaline-PAGE) was carried out with Mini-Protean II cell (Bio-Rad) at 120 V and room temperature for 2 h. The gel used (0.75 mm thick) consisted of 4.5% T staking gel and 10% T separation gel (T% is an expression represent the concentration of acrylamide plus bisacrylamide in the gel). The electrode and migration buffers consisted of 0.19 M-glycine and 0.024 M-Tris at pH 8.6. After electrophoresis, proteins were localized in the gel using Coomassie blue 0.1% (Hames and Rickwood, 1990).
2.5. Statistical analysis
Table 1 Composition of the experimental diet Ingredient
Amount (g/kg)
Dried skim milk Corn oil Corn starch Sucrose Cellulose Vitamin mixture Mineral mixture
375 90 300 225 5 1 4
Data were analyzed by least-squares analysis. Male and female data were analyzed separately using completely randomized design (Steel and Torrie, 1980). The analyses were conducted according to the General Linear Model procedure of Statistical Analysis System (SAS) (1995). Difference between each two dose means within each sex was compared by LSD at 0.05 significant level (Steel and Torrie, 1980).
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3. Results and discussion
3.1. Effect of Zn deficiency on TBARS and GST Zinc has a wide spectrum of biological activities and its deficiency has been related to various tissue dysfunction and alterations of normal cell metabolism. Zinc also plays an important role in the antioxidant cellular defences being a structural element of the nonmitochondrial form of the enzyme superoxide dismutase (CuZnSOD) (Virgili et al., 1999). Free radicals are highly reactive species that have been implicated in the pathogenesis of many diseases. Reactive oxygen species (ROS) can initiate lipid peroxidation and DNA damage leading to mutagenesis, carcinogenesis and cell death, if the antioxidant system is impaired (Devi et al., 2000). DiSilvestro (2000) reported that Zn could exert a number of indirect antioxidant functions. In the present study, lipid peroxidation in liver, brain and testes was detected by significantly (PB 0.05) increased TBARS formation in both male and female rats fed 1/2 and 1/10 of Zn adequate (Table 2). These results are in agreement with the findings of Cao and Chen (1991), Shaheen and El-Fattah (1995), Chen and Young (1998) reported that Zn deficiency induced an increased free radical generation and lipid peroxidation in blood and liver of mice and rats, and decreased superoxide dismutase activity in the liver of mice. Also, Zn deficiency increased lipid peroxidation in mitochondria and microsomes from maternal and fetal liver, maternal kidney, maternal lung microsomes, and fetal lung mitochondria (Gunther and Hollriegl, 1989). Henning et al. (1999) reported that oxidative stress was increased in Zn-deficient endothelial cells. They suggested that Zn may in part be antiathergenic by inhibiting oxidative stress-responsive events in endothelial cell dysfunction. DiSilvestro (2000) reported that mild Zn deficiency in rats produces low resistance to chemically induced liver oxidant injury, and it produces high vulnerability of lipoproteins to oxidation. Chan et al. (1998) demonstrated that Zn is involved in destruction of free radicals through cascading enzyme systems. Superoxide radicals are reduced to hydrogen peroxide by superoxide dismutases in the presence of
Table 2 Serum, liver, testes and brain TBARS and GST in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9SE) Parameter Control
1/2 Zn
1/10 Zn
Male TBARS* Liver Brain Testes
31.83 90.63b 29.65 92.57b 33.85 90.36b
34.83 9 0.78b 33.46 90.78b 34.88 90.28b
GST** Serum Liver Brain Testes Female TBARS Liver Brain GST Serum Liver Brain
26.48 91.84a 21.50 91.11a 28.47 91.66a 0.53 9 0.037a 0.39 9 0.008a 0.49 9 0.008a 0.31 9 0.003a
25.77 9 1.31a 22.38 91.22a 0.56 9 0.016a 0.37 9 0.004a 0.48 9 0.009a
0.67 90.008b 0.42 9 0.008a 0.57 90.024ab 0.38 9 0.022b
32.13 90.60b 29.56 91.36b 0.68 9 0.009b 0.43 9 0.020b 0.53 9 0.011a
0.739 0.008b 0.52 9 0.024b 0.65 9 0.041b 0.3 90.014b
34.31 90.68b 31.74 90.66b 0.74 9 0.009c 0.49 9 0.018c 0.64 9 0.060b
Values are the means of seven rats. abcWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * TBARS is expressed as nmol per gram tissue. ** GST specific activity: mmol per hour per mg protein.
Zn cofactor. Hydrogen peroxide is then reduced to water by the selenium–glutathione peroxidase couple. Efficient removal of these superoxide free radicals maintains the integrity of membranes, reduces the risk of cancer, and slows the aging process (Chan et al., 1998). The generation of ROS is a steady-state cellular event in respiring cells. Their production can be grossly amplified in response to a variety of pathophysiological conditions such as inflammation, immunologic disorders, hypoxia, hyperoxia, metabolism of drug or alcohol, exposure to UV or therapeutic radiation, and deficiency in antioxidant vitamins. Uncontrolled production of ROS often leads to damage of cellular macromolecules (DNA, protein, and lipids) and other small antioxidant molecules (Chan et al., 1999). In general, the mechanism of antioxidation by Zn can be divided into acute and chronic effects.
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Chronic effects involve exposure of an organism to Zn on a long-term basis, resulting in induction of some other substance that is the ultimate antioxidant, such as the metallothioneins. Chronic Zn deprivation generally results in increased sensitivity to some oxidative stress (Powell, 2000). The acute effects involve two mechanisms: protection of protein sulfhydryls or reduction of OH formation from H2O2 through the antagonism of redoxactivate transition metals, such as iron and copper. Protection of protein sulfhydryl groups is thought to involve reduction of sulfhydryl reactivity through one of three mechanisms: (1) direct binding of Zn to the sulfhydryl; (2) steric hindrance as a result of binding to some other protein site in close proximity to the sulfhydryl group; or (3) a conformational change from binding to some other site on the protein. Antagonism of redox-active, transition metal-catalyzed, sitespecific reactions has led to the theory that Zn may be capable of reducing cellular injury that might have a component of site-specific oxidative damage, such as postischemic tissue damage. Zn is capable of reducing postischemic injury to a variety of tissues and organs through a mechanism that might involve the antagonism of copper reactivity. Although the evidence for the antioxidant properties of Zn is compelling, the mechanisms are still unclear (Powell, 2000). The GSTs form a group of multi-gene isoenzymes involved in the cellular detoxification of both xenobiotic and endobiotic compounds (Salinas and Wong, 1999). Glutathione and glutathione-related enzymes are involved in the metabolism and detoxification of cytotoxic and carcinogenic compounds as well as ROS (Knapen et al., 1999). The present data showed a significant increase (PB 0.05) in GST activities in serum, liver, brain and testes of male rats and in serum, liver and brain of female rats fed 1/2 and1/10 of Zn adequate (Table 2). In agreement with the present data, it has found that rats fed Zn-deficient diet had higher concentrations of TBARS, GST activity, lower concentration of glutathione and glutathione peroxidase as well as lower activity of superoxide dismutase in plasma (Kraus et al., 1997).On the other hand, Jagadeesan (1989) reported that the activity of GST,
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a key enzyme in conjugation reaction, was significantly lowered in Zn deficiency. A number of major cellular defense mechanisms exist to neutralize and combat the damaging effects of these reactive substances. The enzymatic system functions by direct or sequential removal of ROS (superoxide dismutase, catalase, and glutathione peroxidase), thereby terminating their activities. Metal binding proteins, targeted to bind iron and copper ions, ensure that these Fenton metals are cryptic. Nonenzymic defense consists of scavenging molecules that are endogenously produced (GSH, ubiquinols, uric acid) or those derived from the diet (vitamins C and E, lipoic acid, selenium, riboflavin, Zn, and the carotenoids) (Chan et al., 1999). Oteiza et al. (1996) found that the activities of copper-Zn superoxide dismutase (CuZn SOD) and glutathione reductase (GRed) were significantly higher (the oxidant defense system) in the testes of Zn-deficient rats. They reported that Zn deficiency can cause an oxidative stress situation in testes, for which cells tend to compensate by increasing select components of the oxidant defense system. This finding is agreement with our results which GST activity increased in Zn-deficient rats (Table 2).
3.2. Effect of Zn deficiency on enzyme acti6ities AST and ALT are imported and critical enzymes in the biological processes. These enzymes are involved in the breakdown of amino acids into 8 Keto acid which are routed for complete metabolism through the Kreb’s cycle and electron transport chain. Consequently, they are considered as a specific indicator for hepatic dysfunction and damage (Osuna et al., 1977; Shakoori et al., 1994). AcP is a marker enzyme for lysosomes, thus related to general cell metabolism. AlP enzyme is a sensitive biomarker to metallic salts since it is a membrane bound enzyme related to the transport of various metabolites (Lakshmi et al., 1991). AlP was the first Zn enzyme to be discovered in which three closely spaced metal ions (two Zn ions and one Mg ion) are present at the active center. Zn ions at all three sites also produce a maximally active enzyme (Coleman,
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1992). The increase of AlP in serum could be a result of damage of liver cells and bile duct obstruction due to proliferation of its cells and/or related to the progressive liver necrosis (Tietz, 1976). LDH is one of the metabolic requirements of tissue and involved in energy production. LDH activity indicates the switching over of anaerobic glycolysis to aerobic respiration. Also, it can be used as an indicator for cellular damage, clinical practice and cytotoxicity of toxic agents (Bagchi et al., 1995). The increment of the activities of AST, ALT, LDH and AlP in plasma is mainly due to the leakage of a these enzymes from the liver cytosol into the blood stream (Navarro et al., 1993), which indicated liver damage and disruption of normal liver function (Shakoori et al., 1994). Acetyleholinesterase (AChE), an enzyme that is responsible for hydrolysing and so deactivating acetylcholine in the nervous system (Forget et al., 1999). The inhibited AChE could decrease cellular metabolism, induce deformities of cell membrane, and disturbed metabolic and nervous activity (Suresh et al., 1992). Also, the decrease in AChE activity could lead to ionic refluxes and differential membrane permeability (Tolosa et al., 1996). The present data indicated an increase (P B 0.05) in the activity of liver AST, ALT and LDH of both sexes fed 1/2 and 1/10 of Zn adequate (Table 3). The activity of AlP, however was decreased (PB 0.05) by 17 and 31% of 1/2 and 1/10 of Zn adequate in males, and 12 and 29% in females, respectively. On the other hand, the activity of AcP did not change (Table 3). The present results showed that brain LDH was markedly inhibited (P B0.05) in male and female rats fed 1/2 and 1/10 of Zn adequate by 23, 57, 26 and 52%, respectively (Table 4). The effects of Zn deficiency on testis enzymes in rats are presented in Table 5. These data showed a significant decline (PB0.05) in AcP activity, while LDH was significantly increased of male rats that fed 1/10 of Zn adequate. Testes AST, ALT and AlP activities were not affected by reducing Zn level in the diet. Male and female rats fed the lowest Zn level (1/10 of control) showed a reduction (P B 0.05) in serum AlP and LDH, and the magnitude of reduction was 27 and 40% for males, 23 and 38%
for female compared to control, respectively (Table 6). These results are in agreement with Alayash (1989), who reported that serum levels of AlP and LDH, two Zn dependent enzymes in these patients, in North American Blacks with sickle cell anemia, who are known to have Zn deficiency coupled with a decrease in the activity of these enzymes. Also, Wan et al. (1993) found that Zn deficiency was produced a significant reductions in plasma AlP activity (48%) of rats. Tables 4 and 6 show that serum and brain AChE activities were not affected by reducing Zn level in the diet. The changes in the activities of tested enzymes in Zn-deficient animals might be due to that Zn deficiency affects many systems because it has essential roles in many aspects of metabolism including the activity of more than 300 enzymes (Vallee and Falchuk, 1993; Prasad, 1996). Zinc is a component of many enzymes, including AlP, lactate and glutamate dehydrogenase, carbonic Table 3 Assay of liver enzyme activities and protein levels in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9SE) Parameters
Control
1/2 Zn
1/10 Zn
Male AST (IU/mg)* 106 91.1a ALT (IU/mg) 67 9 0.7a AlP (IU/mg) 1131 931a AcP (IU/mg) 697 92.6a LDH (IU/g)** 2690 9140a Protein (mg/g) 237 916.0a
118 94.0b 81 9 3.0b 942 918b 686 96.3a 3407 9118b 221 95.7ab
130 92.0c 97 9 6.1c 781 9 51c 685 92.4a 4023 9181b 181 912.0b
Female 101 91.7a AST (IU/mg) ALT (IU/mg) 71 9 2.7a AlP(IU/mg) 1055 920a AcP (IU/mg) 695 91.2a LDH (IU/g) 2413 978a Protein (mg/g) 255 98.5a
116 93.4b 8493.4b 925 927b 686 97.1a 3089 9176b 234 99.2a
128 92.1c 98 9 4.9c 746 940c 673 92.6a 3740 9170c 199 99.1b
Values are the means of seven rats. abcWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * IU/mg: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substrate per minute per mg protein. ** IU/g: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substratesubstrate per minute per gram tissue.
M.I. Yousef et al. / Toxicology 175 (2002) 223–234 Table 4 Assay of brain AChE, LDH activities and protein levels in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9 SE) Parameter
Control
1/2 Zn
1/10 Zn
Male AChE* LDH** Protein (mg/g)
4.4590.14ab 4.70 90.18a 4.079 0.23b 1553 9 69.2a 19139 140.5b 24449 57.5c 78.19 2.62ab 68.39 5.87b 85.3 92.34a
Female AchE LDH Protein (mg/g)
4.04 90.47a 1500 9 88.0a 80.1 94.14a
3.72 90.28a 3.739 0.15a 18969 182.8b 22859 54.6c 73.69 5.37ab 61.19 4.21b
Values are the means of seven rats. abcWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * AChE activity: mmol substrate hydrolyzed per minute per mg protein. ** IU/g: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substrate per minute per gram tissue.
anhydrase, DNA polymerase and some peptidases (Guyton, 1981; Martin, 1983). In agreement with the present results, Eltohamy and Younis (1991) found that there was significant reduction in the levels of AlP, AcP and lactic dehydrogenase of both testes and epididymis of Zn-deficient rabbits. Also, Zn deficiency led to a reduction of serum and testis AlP activity (Reeves, 1990; Naber et al., 1996; Kraus et al., 1997). Prasad (1983) reported that the activities of many Zn-dependent enzymes have been shown to be affected adversely in Zndeficient tissues. Three enzymes, AlP, carboxypeptidase and thymidine kinase, appear to be most sensitive to Zn restriction in that their activities are affected adversely within 3–6 days of institution of a Zn-deficient diet to experimental animals.
3.3. Effect of Zn deficiency on protein content Zinc is essential for synthesis of coenzymes that mediate biogenic-amine synthesis and metabolism (Sandstead et al., 2000). Male and female rats fed the lowest Zn level (1/10 of control) showed a
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reduction (PB 0.05) in protein, and the magnitude of reduction was 24, 20 and 28% in liver, brain and testes of males, 22 and 24% in liver and brain of female compared to control, respectively (Tables 3–5). In agreement with the present results, Eltohamy and Younis (1991) reported that there was significant reduction in the levels of protein of both testes and epididymis of Zn-deficient rabbits. Prasad (1996) found that Zn deficiency influence DNA synthesis, cell division and protein synthesis. In addition, Oteiza et al. (1996) demonstrated that Zn deficiency could be associated with high rates of oxidative damage to proteins and DNA in male rats. The observed decrease in protein content might be attributed in part to the concomitant induction in lipid peroxidation (Table 2) in both male and female rats fed 1/2 and 1/10 of Zn adequate.
3.4. Effect of Zn deficiency on the concentrations of lipids and lipoproteins Table 7 shows the changes in liver TL, cholesterol, TG, HDL, LDL and VLDL of growing Table 5 Assay of testes enzyme activities and protein levels in male rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9 SE) Parameter
Control
1/2 Zn
1/10 Zn
50.4 9 1.77a AST 55.0 9 1.85a 52.4 9 2.58a (IU/mg)* ALT 14.81 94.48a 15.05 98.90a 12.60 910.73a (IU/mg) AlP 1050 948.4a 987 920.1a 975 919.0a (IU/mg) 517 9 13.02b AcP 581 98.28a 560 99.99ab (IU/mg) LDH 2108 9147.1a 2380 9118.7ab 2741 9255.1b (IU/g)** Protein 31.8 90.87a 30.7 90.82a 22.9 9 1.626b (mg/g) Values are the means of seven rats. abWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * IU/mg: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substrate per minute per mg protein. ** IU/g: international unit, the amount of the enzyme that under defined assay conditions will catalyze 1 mol of substrate per minute per gram tissue.
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Table 6 Assay of serum enzyme activities in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9SE) Parameter
Control
1/2 Zn
Male AChE* AlP** AcP** LDH**
1.419 0.21a 1.29 90.02a 236.4 98.65a 226.29 12.43a 5.91 90.22a 5.6590.06a 2212 9 106.9a 1946 9 132.8a
Female AChE AlP** AcP** LDH**
1.18 90.03 1.099 0.13 260.1 98.89a 241.89 16.90ab 6.61 90.18a 6.05 9 0.42a 2245 9 222.9a 1627 9 122.9b a
1/10 Zn
a
1.3290.02a 172.99 21.47b 5.4290.54a 13319 176.7b a
1.1390.05 200.09 15.67b 5.9090.46a 13989 111.9b
Values are the means of seven rats. abWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05). * AChE activity: mmol substrate hydrolyzed per ml serum per minute. ** (IU/l)
male and female rats fed Zn deficient diets (1/2 or 1/10 of control). Liver HDL was decreased (P B 0.05) in male and female rats that fed 1/10 of Zn adequate. Liver TL, cholesterol, TG, LDL and VLDL, however were increased (P B0.05) in Zndeficient rats in a dose-dependent manner (Table 7). In agreement with the present data, it has found that Zn deficiency was associated with lower HDL levels (Schneeman et al., 1986). On the other hand, Neggers et al. (2001) found that Zn supplements caused an increase in HDL value in an adult African– American community. Eder and Kirchgessner (1995) reported that Zn-deficient rats characterized by elevated levels of triglyceride. Also, Eltohamy and Younis (1991) found that there was significant increase in the levels of cholesterol in testes of Zn-deficient rabbit. In addition, lipid composition and essential fatty acids concentration are altered in the brain of Zn-deficient animals (Essman, 1987). On the other hand, Paul et al. (2001) found that Zn supplementation caused a significant reduction in plasma cholesterol and TG of mice. Cunnane (1988) discussed the role of Zn in the metabolism of lipids and the role of it in membranes as well as
Zn’s known effects on receptors. He reported that Zn intimately affects many aspects of lipid metabolism through established enzymes but also has modulatory effects whose mechanism is not obvious or established.
3.5. Gel electrophoresis of serum proteins Fig. 1 shows the electrophoretic patterns of serum proteins of male and female rats fed Zn adequate, 1/2 and 1/10 of Zn adequate. The patterns showed several peptide bands differ in migration positions and intensities. Five major peptide bands, in addition other minor bands were recognized in each pattern. The major bands belong to immunoglobulins, transferrin, serum albumin and another two unknown fractions (F1 and F2). It is obvious that there were no differences between male and female control serum samples in bands number or intensity. However, the intensity of immunoglobulins, serum albumin, F1, F2 as well as the minor peptide bands was decreased in rats fed 1/2 of Zn adequate compare
Table 7 Liver TL, cholesterol, TG, HDL, LDL and VLDL in male and female rats fed Zn deficiency (1/2 and 1/10 of Zn adequate) (mean 9 SE) Parameter (mg/g)
Control
1/2 Zn
1/10 Zn
Male TL Cholesterol TG HDL LDL VLDL
34.8 9 4.07a 3.25 90.19a 11.0 90.93a 0.35 9 0.03a 1.63 90.10a 2.21 9 0.19a
40.0 9 2.77a 3.96 90.31b 13.9 9 1.70ab 0.29 9 0.01ab 2.15 90.20b 2.77 9 0.34ab
52.0 9 13.35b 4.24 9 0.58b 14.8 9 0.44b 0.24 9 0.02b 2.58 9 0.21b 2.95 9 0.09b
Female TL Cholesterol TG HDL LDL VLDL
35.78 91.89a 3.44 9 0.25a 11.5 90.70a 0.36 90.03a 1.64 9 0.11a 2.30 90.14a
37.9 9 5.73a 4.29 90.44b 14.0 9 0.56b 0.32 9 0.01ab 2.01 9 0.29b 2.80 9 0.11b
52.6 9 4.72b 4.69 9 0.28b 15.5 9 0.82b 0.29 9 0.02b 2.47 9 0.35b 3.1190.17b
Values are the means of seven rats. abWithin rows, between control and treated animals, means with different superscript letters differ significantly (PB0.05).
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Fig. 1. Alkaline-polyacrylamide gel electrophoresis (alkaline-PAGE) at pH 8.6 of rat serum proteins. Lanes 1 and 2: control serum samples of untreated male and female rats, respectively; lanes 3 and 4: serum samples of male and female rats fed 1/2 of Zn adequate; lanes 5 and 6: serum samples of male and female rats fed the lowest level of Zn (1/10 of control). Each lane represents the pooled sample of seven animals for each group. Anode is toward bottom of photo.
to controls. This means that the synthesis of such peptides was affected. Prasad (1998) reported that Zn plays an important role in the immune system and Zn deficient subjects may experience increased susceptibility to a variety of pathogens. It is clear that Zn affects multiple aspects of the immune system, from the barrier of the skin to gene regulation within lymphocytes. Zinc is crucial for normal development and function of cells mediating nonspecific immunity such as neutrophils and natural killer cells (Shankar and Prasad, 1998). Zinc plays an important role in immune function, helping increase resistance to infection and tumor growth. Low levels of Zn result in a decrease in helper T-cells and thymic hormone, which may adversely affect immune functioning (Eby et al., 1984). No clear effect on transferrin was noticed in serum samples of rats fed 1/2 and 1/10 of Zn adequate. At the lowest level of Zn (1/10 of control) the effect on minor peptides was pronounced specially F7, F8 and F9. These fractions may represent some enzymes or hormones. From the present results it can be concluded that the synthesis of several proteins is affected in rats fed 1/2 and 1/10 of Zn adequate. Also, Prasad (1996) reported that approximately 300 enzymes are known to require Zn for their activities. Zinc is
required for each step of cell cycle in microorganism and is essential for DNA synthesis, cell division and protein synthesis. They reported that the effect of Zn on protein synthesis may be attributable to its vital role in nucleic acid metabolism. In addition, Tate et al. (1995) found that protein synthesis in low Zn medium (0.25 mM Zn) of human retinal pigment epithelium was depressed compared to the control (11 mM Zn). Giugliano and Millward (1987) found that protein synthesis and RNA concentrations were reduced in rats fed on a Zn-deficient diet. They also reported that Zn deficiency impairs growth by a combination of reduced food intake, a reduced anabolic response to food due to a reduced capacity for protein synthesis and reduced activation of protein synthesis. The present results concluded that dietary Zn deficiency exerts alterations in the enzyme activities in serum, liver, brain and testes. Also, Zn deficiency caused a significant increase in the levels of free radicals and GST activity in all tested tissues. The levels TL, cholesterol, TG, and LDL were significantly increased in the liver of Zn-deficient rats, while HDL concentrations were decreased. Protein content in liver, brain and testes showed a significant (P B 0.05) decrease in rats fed the lowest level of Zn (1/10 of control). Con-
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cerning the protein patterns, the intensity of immunoglobulins, serum albumin, as well as several peptides bands were decreased in rats fed 1/2 of Zn adequate. All of these alterations were pronounced in rats fed the lowest level of Zn (1/10 of control). To be sure, structural studies of Zn in biology will continue to be a fruitful source of bioinorganic advances, as well as surprises, in the future.
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