IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 Effects of the Zinc o n Activity of Immune System in Male Albino Mice Z. Hussein Departme nt of Biology, Collage of Education Ibn Al-Haitham , Unive rsity of Baghdad Abstract Effect of zinc chloride on the immune functions was st udied in male albino mice aged 6-7 weeks. It was administ rated orally (1ml) in three concentrations (0.5p p m, 1p p m, 2p p m) for 9 days. The results showed that t he first concentration was not effective comp aring with control while the second concentration increased the enhancement of immune sy st em and the cell third one killed the mice 6 hours p ost administ ration, so we can conclude that t he high dose of ZnCl2 could be harmful for all metabolism. Introduction Zinc, an essential trace element, is rich in animal p roducts such as meat (esp ecially beef), p oultry , sea food (esp ecially oy st ers) and dairy foods, which are the best food sources of zinc [1].Cereals, grain p roducts, fruits and vegetables also contain zinc although in smaller amount than in animal p roducts. In addition, zinc, is more readily absorbed from animal than from vegetable sources [2]. It is p resent in all organs, tissues, fluids, and secretions in the body [3].M ore than 100 sp ecific enzy mes require zinc for their cataly tic function.M oreover, under p hy siological conditions, zinc is not subjected to oxido-reductive reactions, so it is relatively non-toxic.These p rop erties make zinc an ideal element to p articip ate in cataly tic, st ructural, and cellular regulatory functions[4].Also it has a variety of effects on the immune and nervous sy st em in vivo and in vitro and these effects mainly depend on the zinc concentration [5].Zinc also p lay s an imp ortant role in sp ecific immune defenses such as humoral and cell- mediated immunity . In mice fed, a low zinc diet for 30 days, 30% to 80% losses in immune defense cap acity occurred [6].Zinc has ot her prop erties that could contribute to its role in lymphocyte functions. It is an antioxidant, p rotecting cell from the damaging effects of oxy gen radicals [7].Besides all these, zinc is requied as a co-factor for numerous metalloenzy mes involved in the continuous p roduction of the immune sy st em cells[8].M any biological st udies indicate that z inc deficiency decreases resist ance to infections diseases.Z inc deficient animals have sup p ressed immune resp onses and are more suscep tible to a diverse range of infections agents including herpes simplex virus [9], bacteria such as salmonella enteritidis [10] and mycobacterium tuberculosis [11],and the protozoan p arasites typanosoma cruzi [12], fasciola hepatica [13],and schistsoma mansoni [14].Zinc sup p lements also have beneficial effects when administ ered during infections, and zinc lozenges were shown to decrease the duration of the common cold [15], and this may be due to the increased zinc concentration in the nasal mucosa, which may alter the conformation of the binding site between the virus and ICAM -1[16]. At last , the very high zinc intakes in adults and children can result in copp er deficiency , anemia, growt h retardation and immunodep ression[17].Thus too much zinc can be as bad as too little, as in most cases; abdominal p ain, anemia, fever and bleeding in the stomach can result[18]. Material and Methods A. Animals: Albino mice were brought from the centre for infertility in al_kadimiy a and, animals were divided into four group s, each group involved 6 mice. The first was a IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 control group and administrated orally (1ml) with distilled water, while the other three group s were administ rated orally (1ml) with ZnCl2 in three concentrations (0.5p p m, 1p p m, 2p p m). One dose p er day for 9 days. B. Prep ration of ZnCl2: ZnCl2 solutions were p repared depending on human needs of zinc (15mg/60kg)[18], and accordingly 4 mg of zinc was dissolved in 100ml of distilled water to y ield a final concentration of 2p p m. C. Tests: 1. White blood cell count: blood taken from mouse tail and examined according to the method of [19]. 2. Diffrential count: blood smear, st ained with leishman st ain and examined the samples under the light microscope by using the oily lens and counted (100) random cells from each slide then find the average p ercentige of each animal according to number of each ty p e of cell. 3. Phagocytic index: p hagocytes were isolated from p eritone and examined according to the method of [20]. 4. M itot ic index: examined according to the method of [21], isolation of cells (bone marrow and sp leen) in the method of [22] 5. Art hurs reaction: Examinted according to the method of [23] t hen examination of delayed ty p e hyp ersensitivity 24 hour. 6. Adenosine deaminase activity in serum: The activity of ADA was determind according to the method of Giusti [24]. Re sults The results in tables 1,2,3,4 and 5 showed that t he first concentration of ZnCl2 (0.5p p m) was associated with normal values and did not show any effect on mice, while the second concentration (1p p m) showed that zinc chloride enhanced immune sy st em in all tests, but it made mice suffered from lassitude and diarrhea. In the third concentration (2p p m), sy mptoms of lassitude, diarrhea, loss of app etite, depression, extremities p alsied and death app eared 6 hour p ost treatment. Discussion The parameters invest igated in tables 1,2,3,4 and 5 showed increased levels with the increase of concentration comp ared with control. Table 1 showed that increased intake of (ZnCl2) st imulated white blood cell count [ total and differential] esp ecially at second concentration, this is may be due to zinc effect on nonsp ecific immunity by its affecting neutrop hil and natural killer cells activity [25].M any st udies showed that very high zinc concentrations could exp ress a microbicidal activity [26].When ZnCl2 was used in concentrations of 10(-4);10(-5);10(-6)M in PFC it was found that the numbers of anti-SRBC antibody -p roducing cells in mice injected with zinc were greater than in the control. This enhancement of PFC was p rop ortional to concentration of zinc. It was determined that ZnCl2 in concentration 10(-4)M activated mouse lymphocytes for migaration inhibitory factors p roduction. This p rove that zinc may enhance the effectiveness of anti-infections immunity [27].Thus more zinc addition in vitro alters the exp ression, function, or both, of lymphocyte surface molecules governing cell-cell interactions [28]. The results in table 2 showed that the increased intake of (ZnCl2) enhanced p hagocytosis .One imp ortant group of leucocy tes is the p hagocytic cells. These cells bind to microorganisms, neutralize them and then kill them [29].M any st udies indicated that when zinc deficient animals infected with p athogenic p arasites, the function of macrophage were imp aired however, p hagocytosis can be restored to normal when the animal receive sufficient zinc (6, 30), Roitt [29] indicates that there were cooperation between T-lymphocyte and p hagocytic cell to kill microbes. Ot her st udies showed that a very high concentration of zinc in vitro inhibited macrophage activity , mobility , p hagocytosis and oxy gen consumption[31].There is much sp eculation regarding the role of zinc in killing p athogens by oxy gen radicals p rodued by macrophages [32]. IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 Thus zinc was not affected on absolute numbers of p erip heral p olymorphonuclear leukocytes, but chemotactic resp onse was impaired and was reversible by in vitro addition of zinc [33].In table 3 the results showed increased levels of mitotic activity comp ared with control .This may be due to carcinogenesis effect of zinc on cell growt h [34].Zinc was demonst rated to inhibit the mutagenic action of some genotoxic carcinogens [35].Furt hermore zinc chloride did not induce mutations at the thy midine kinase locus in L5178Y mouse lymphoma cells [36] The p arameter in table 4 showed increased levels in Art hus reaction and delayed ty p e hy p ersensitivity with the increased level of concentrations in comp arison with control. In Art hus reaction, the animal had app reciable levels of serum antibody , following the injection of antigen reaction develops at the reaction site [29], and zinc p lay s an imp ortant role in sp ecific immune resp onse [5], this exp lains why there is an increase in thickness p ad of injected mice. Our results were similar to the finding reported by[37] Adenosine Deaminase (ADA) is a cytosolic enzy me. ADA p articip ates in the p urine metabolism where it degrades either adenosine or 2’-deoxy adenosine p roducing inosine or 2’- deoxy inosine, resp ectively [38]. The results in table 5 showed the effect of ZnCl2 on ADA activity in animal showed a dose dependent manner because the activity of ADA was elevated when we use a high dose of ZnCl2 in the administration mice. Zinc is a st ructural constituent of a great number of p roteins, including enzy mes belonging to cellular signaling p athways and transcription factors, and it is essential for biological activity [39]. At last this result suggests that ZnCl2 has enhancement immune sy st em but it mightbe deadly imp act when administ rated with high concentration since results showed that third concentration fatal for animals may be due to zinc chloride toxicity . It can p roduce significant lung damage in rate when instilled directly into the lung [40]. Thus oral exp osure to zinc chloride causes reduced growt h rates, reduced body weight and anemia in numbers of rat, the mouse and a sheep st udies, following high oral or dietary intake of zinc [31, 41]. Thus, exp osure to high doses of zinc was associated with p ancreatic atrop hy and histological changes in kidney s, accomp anied by changes in kidney function in rats, mice and sheep [31].Changes in liver, including decreased activities of cytochrome p 450 and liver catalase [42]. 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Table (1): Effects of ZnCl2 on white blood cel l count [Total and Differential] in mal e albino mi ce /Mm 3 b lood and d ifferential count × 10 3 White b lood cells w.b.c count lymph ocytes neutrophils monocytes eosinop hils Dose/ml groups 0.50±7.55 0.02± 4.65 0.06± 2.56 0.01± 0.25 0.01± 0.08 D.W 1 0.10±7.65A 0.06± 4.66B 0.10± 2.62 0.01± 0.26A 0.01± 0.09 0.5 2 1.60±9.91A 0.99± 6.22B 0.57± 3.26 0.04± 0.32A 0.03± 0.11 1 3 - - - - - 2 4 (A) Significant (a<0. 05) (B) Significant (a<0. 01) (C) Significant (a<0. 001) Table (2): Effects of ZnCl2 on phagocytic index i n mal e albino mi ce Phagocytic index (%) dose groups 0.03 ±10.23 D.W 1 0.38 ±10.51C 0.5 2 1.24 ±15.18C 1 3 - 2 4 (C) Significant (p<0.001) Table (3): Effects of ZnCl2 on mi totic index of bone marrow and spleen cells Bone marrow (%) S pleen (%) Dose/ml groups ± 17.00 1.15 ± 11.50 1.29 D.W 1 18.90 ± 2.33 ± 12.36 0.96 0.5 2 ± 23.53A 1.75 14.77A ± 2.31 1 3 - - 2 4 (A) Significant (p<0.05) IBN AL- HAITHAM J. FOR PURE & APPL. S CI VOL.23 (1) 2010 Table (4): Effects of ZnCl2 on hypersensi tivity reactions (Arthus and delayed type) Art hus(M m) Delay ed typ e(M m) Dose/ml group s 0.03 ± 0.54 0.02 ± 0.62 D.W 1 0.88 A ± 0.25 1.63 B ± 1.11 0.5 2 1.13 B ± 0.47 1.88 A ± 0.95 1 3 - - 2 4 (A) Significant (p<0.05) (B) Significant (p<0.01) Table (5): Effect of ZnCl2 on adenosi ne deaminase activity in serum ADA activity / U/mg of p rotein Dose/ml group s 0.01 ± 1.54 D.W 1 ± 1.68 A 0.17 0.5 2 ± 1.86 B 0.29 1 3 - 2 4 (A) Significant (P<0. 05) (B) Significant (P<0. 01) 2010) 1( 23مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد فعالیة الجهاز المناعي في ذكور الفئران البیض فيتأثیر عنصر الزنك زهراء حسین ، جامعة بغدادابن الهیثم - كلیة التربیة ، قسم علوم الحیاة الخالصة ت اسـتعمل .اسـابیع 7-6بعمـر المناعیة على ذكورالفئران البـیضفعالیة في ال ZnCl2 كلورید الزنك دراسة تأثیر تتم 2p( وهـيZnCl2 ثالثـة تراكیـز مـن مـادة p m,1pp m, 0.5p p m ( وبواقـع جرعــة واحـدة فـي الیـوم)1ml ( ایــام9 مــدة. معنویـة مقارنـة بالسـیطرة والجرعـة بینمـا التركیـز الثـاني اظهـر زیـادة ،تـاثیرا معنویـا یـؤثراظهرت النتائج ان التركیـز االول لـم ZnCl2لـذا نسـتنتج ان الجـرع العالیـة مـن ،سـاعات علـى التجریـع االول 6الثالثة كانت ممیتة اذ قتلت الحیوانات بعـد مـرور .داخل الجسم كافة لفعالیات االیضیةلا ضار یكون