IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 The Effect of Mercuric Exposure on Oxidative Stress and Enzymatic Antioxidant Defense System I.A.Mohammed , M.I .AL-Jobouri , W.F.AL-Taie Departme nt of Chemistry ,College of Education Ibn AL-Haitham, Unive rsity of Baghdad Abstract Throughout the centuries, several incidents of mercury toxicity have been reported. M ercury is found in many industries such as batt ery, thermometer and barometer manufacturing, in the agricultural industry is used in fungicides and in medicine, mercury is used in dental amalgams. An imp ortant mechanism involved in cellular injury is induced by exp osure to different forms of mercury involves in the induction of oxidative st ress. This st udy was conducted on non-smoker, male working in a chloroalkali p lant for different p eriods, all workers were not suffering from chronic disease. Healthy non-smoker males that are not exp osed, matched age were used as controls(C), workers aged (22-61) y ears, t hey were divided into t hree group s: G1: workers with exp osed p eriod less than 10 years, G1 < 10 y ears. G2: workers with exp osed p eriod (10-19) y ears . G3: included workers with exp osed p eriod more than 19 years, G3 > 19 y ears. The result we had through examining the different p arameters led us to add another group which included individuals with high mercury levels regardless the occup ation p eriod, in this group we found high significant changes in the defense sy st em p arameters that we measured. This st udy showed an elevation in M DA levels in all workers group , sp ecially those with high level mercury , which were 9.31, 12.78, 12.99, 14.73, and 18.11nmol/dl for C, G1, G2 , G3 ,and high level mercury workers resp ectively . No alteration was found in SOD activity in ery throcyte (0.67, 0.73, 0.72, and 0.77 U/g.Hb) for C, G1, G2 and G3 resp ectively. There were highly significant decrease in catalase activity (P < 0.001) in erythrocyte of all worker group s comp ared to normal control. The values were (1.5, 0.8, 0.88, 0.815 and 0.45 U/gm/Hb) for C, G1, G2, G3 and G4 resp ectively. While there were high elevation in glutathione S-transferase activity in worker group s comp ared to control (P < 0.001) and values were (1.85, 2.589, 2.441, 2.776 and 3.2 U/gm.Hb) for C, G1, G2 and G3 resp ectively. Introduction Free radical induced oxidative damage has been imp licated in the toxic action of many chemicals and environmental agents and in the p athogenesis of a number of diseases [1]. M ercury may occur in the elemental form or as inorganic and organic comp ounds [2]. From biochemical p oint of view, no other metal bett er illustrates the diversity of effects caused by different chemical sp ecies than does mercury [3,4] . Each of the three forms of mercury has characterist ic toxicokinetics and health effects [5] . In as much as mercury is ubiquitous in the environment, it is nearly imp ossible for most humans to avoid exp osure to some form or forms of mercury on a regular basis[6]. All forms of mercury cause toxic effects in a number of tissues and organs, depending on the chemical form of mercury, the level of exp osure and the duration of exp osure[4,7,8] ____________________________________ This work is ap art of a PhD. Thesis of the first co-author IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Normally the body burden of mercury in humans is p redominantly caused by the diet(3,4) and the dental amalgam [9] Ot her sources such as air or drinking water contribute, substantially to the tot al burden only in the case of local mercury contamination . The high vapour p ressure of metallic mercury is the main reason for the occup ational burden where ever mercury is mined, melted, refined, treated or recycled [4] Free radical induced lip id p er-oxidation may be involved in M eHg-induced cell damage[10,11]. This hy p othesis is sup p orted by findings in which M eHg exp osure in vivo elevates ROS in brain regions sensitive to M eHg [12,13]. Living organisms have devised several antioxidant defense mechanisms. These mechanisms consist of a variety of metallo-enzy mes and several ty p es of antioxidant molecules [14] . Sup eroxide dismutase SOD is a metalloprotein that is p resent in all aerobic organisms[15]. It cataly zes the conversion of two sup eroxide anions into hy drogen-peroxide and molecular oxy gen [16] .  2O +  2O H2O2 + O2 The hy drogen p eroxide formed by sup eroxide dismutase, and by the uncataly zed reaction of hy drop eroxy radicals O2  2O 2OH  H2O2 Area scaven ged by catalase [17] which is an ubiquitous heme p rotein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxy gen [18]. Glutathione – S – transferases (GSTs) constitut e a family of enzy mes that catalyze the conju gation of glutathione to electrop hilic comp ounds includin g metabolites of several mutagens and carcinogens, and major class of p hase – II enzy mes involved in xenobiotic biotransformation and detoxification. Methods Samp ling was conducted in a chloroalkali p lant, that used mercury electrodes to p roduce chlorine and soda, during the p eriod Feb. 2001 – M ay 2002. Fort y one non-smoker males working in the p lant for different p eriods were included in the st udy . All workers were not suffering from chronic diseases.Sixteen healthy non-smoker males, who are not occup ationally exp osed, matched age, were used as a control group (C) . Workers aged 22-61 y ears, t hey are divided into three group s: G1: included workers with exp osed p eriod less than 10 years, G1 < 10 y ears. G2: included workers with exp osed p eriod 10-19 y ears, G2 10-19 y ears. G3: included workers with exp osed p eriod more than 19 y ears, G3> 19 y ears . About 10 mls of venous blood were drawn by using disp osable needles and sy ringes. Samp les were collected between 9.00-12.00 a.m. with no regard to meals. e - H+ O2 2OH  Superoxide anion Hydroperoxyl radical Hydrogen peroxide 2H + SOD IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Plasma samples were used to measure M DA content .The anticoagulant was added to the blood sample, centrifuged for 15 minutes at 3000 g then p lasma was sep arated from red cells. Red cells samples were used to determine catalase, SOD and GST activities. M ercury was analyzed by atomic absorp tion sp ectrometric technique uses sodium borohy dride as a reducing agent, which is cap able of reducing all the mercury in the samp le to elemental mercury vapour [19] . Lip id p eroxidation was determined by using the thiobarbituric acid method [20]. In this method malondialdehyde (M DA), formed from the breakdown of p olyunsaturated fatt y acids was identified as the p roduct of lip id p eroxidation that reacts with thiobarbituric acid (TBA) to give a red chromop hore absorbing at 532 nm. M alondialdehy de concentrations were calculated using molar absorp tivity coefficient of 1.56  10 5 L.M ol 1 .cm 1 . Sup eroxide dismutase (SOD) was assay ed according to Winterbourn [21].This assay depends on the enzy me ability to inhibit the reduction of nitro blue tetrazolium (NBT ) by (  2 O ) anions that were generated subsequent to the reduction of riboflavin by illumination in the presence of methionin which is an oxidizable comp ound. Catalase activity was determined by using assay method that depends on it’s ability to decompose H2O2 to give H2O and O2. This assay was based on the reduction in the absorbance of hy drogen p eroxide H2O2 at 240 nm. The difference in absorp tion (A240) p er unit t ime is a measure of catalase activity [22]. Glutathione-S-transferase GST cataly zes the conjugation of GSH with 1-Chloro-2,4- dinitrobenzene (CDNB), as co-substrate, to form 2,4-dinitrop henyl glutathione which absorbs light at 340 nm, and the determination of the rate of it’s formation reflects enzy me activity in the sy st em. Re sult and Discussion Fig. (1) shows the concentrations of mercury in the st udy group s, male workers, in chloroalkali p lant and occup ationally unexp osed control group . The concentrations of mercury in serum were significantly higher in the Hg-group s comp ared to control group .There were no significant differences between the Hg-group s them-selves (p > 0.05). Though there were some individual cases within the Hg-group s which showed values more than the accep table level (3 g/dl) [23]. Four cases from G3, three from G2 and four cases from G1 showed values of Hg concentration in serum above(6 g/dl) M ercury in blood of chloroalkali- workers correlated significantly with current ty p e work, but not with length of exp osure[24,25]. Lip id peroxidation products measured as malondialdehyde (M DA) content were detectable in a significantly higher concentration in all mercury exp osed group s comp ared to the normal control group (p < 0.001) as shown in Fig. (II) and Table (1). M ean M DA levels were increased 37%, 39% and 58% over the mean control level for G1,G2 and G3 resp ectively. Fig.(III) correlates M DA p roduction in workers and control with the increase of Hg- concentration, as we can see that levels of serum Hg-concentration when became higher than 3 g/dl the M DA levels increased rapidly (r = 0.911). Workers from different occup ation p eriods having mercury levels less than (3 g/dl) have serum M DA concentration within normal range of that for control. M DA p roduction of high mercury level workers (Hg > 3 g/dl) was increased (172%) over normal control. A number of st udies have p rop osed that elevated blood level of lip id p eroxidation might be involved in cellular dy sfunction [26,27].M oreover, it is well known that mercury increases the level of oxy gen reactive intermediates in tissue cells [28] .The data in table (1) indicates that a st ate of oxidative st ress and increase in process of lip id p eroxidation in workers is comp atible with the observation of Queiroz et al., 1998 [29], these authors reported that the average of lip id p eroxidation in serum is higher in workers exp osed to mercury than in normal control.The highly significant IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 increase of M DA level in the third group of workers (58%) over control level could be due to age as it is known that oxidative st ress is age related [30]. Table (2) shows activity of SOD in mercury exp osed workers and control as it is noticed there are no significant differences between the three group s of workers and normal control (p > 0.05). Our results are in agreement with reported values for SOD where the authors found no significant differences in mercury exp osed workers in SOD sy st ems. SOD p lay s a significant role in erythrocyte defense against oxidation so the balance between antioxidant and oxidative damage maintains equilibrium between oxidative injury and defense sy st em (29). On the other hand some authors reported that SOD activity in ery throcytes of workers occup ationally exp osed to mercury was significantly lower than control group as it can be exp lained that workers in elemental mercury leads to increase lip id p eroxidation in erythrocytes, and they p ost ulated that this exp osure leads to decrease activity of SOD in erythrocytes (1). In mercury exp osed group s SOD activity was incompatible and no alte- ration was found by Barregared, 1990 [31], who described a normal concentration of SOD in p lasma and blood of mercury exp osed workers. On the other hand, Perrin-Nadif., 1996 [32]observed increased SOD activity in workers with higher urinary mercury concentrations Our results showed a highly significant decrease (p < 0.001) in catalase activity in mercury exp osed workers comp ared to normal control group . 50% decrease was found in the enzy me activity in each worker group relative to the normal control group , while no significant differences (p > 0.05) were noticed within the worker group s themselves as shown in Fig. (IV) .Fig. (V) showed the levels of catalase activity in mercury exp osed workers as a function of Hg-concentration, a highly negative correlation between the two p arameters (p < 0.001) is shown r =  0.8517. In this figure only levels of mercury over 3 g/dl are represented for the workers. Catalase enzy matically removes hy drogen p eroxides formed from enzy matic removal of sup eroxide. Changes in catalase activity occuring after chronic exp osure to high levels of mercury were invest igated in the red cell sy st em and an increase in catalse activities in worker group s was reported, they reported also that no alteration is detected in red cell antioxidant sy st em [29].In a recent review of the influence of antioxidant sy st ems in the erythrocyte survival Kurata[33] reported in all mammals, a p ositive correlation between the red cell life-sp an and the intracellular levels of SOD and GSH. However, no correlation was observed for catalase. The authors suggested that t he relative strengths of the effectiveness of the cellular antioxidant sy st ems and oxy gen radical formation are p otential candidates in governing the ageing p rocess and in the determining red cell life sp an in mammalian sp ecies. Catalase activity was rep orted to be unaltered in oxidant injury and oxidant resp onse induced by mercury [34,25]. Catalase activity was reduced in the renal cortex of rats exp osed to mercury[35]. GST activities in all exp osed group s were significantly higher than normal control levels as shown in Fig. (VI), there is 39%, 31% and 50% increase in GSTs activities in group s G1, G2 and G3 resp ectively. Fig. (VII) showed a highly p ositive relation (r = 0.76, p < 0.001) between mercury and GST activity in high mercury levels workers (Hg > 3 g/dl) regardless the occup ation p eriod. Fig. (VIII) shows the p ositive relationship between M DA levels for workers (Hg > 3 g/dl) and GST activity (r = 0.54, p < 0.001) compared to normal control. GST activity in high Hg-level workers (average 3.18 U/gm.Hb) was increased by 172% over control levels.T he elevation in activity of the enzy me for mercury exp osed subjects in our st udy is due to the detoxification effect of the enzy me against lip id p eroxidation and xenobiotics [36]. Our result is comp atible with researches p erformed on laboratories animals exp osed to inorganic mercury which confirmed the p rotection effect of GST enzy me as a general mechanism against a wide variety of p ulluent including heavy metals and carcinogenic aromatic comp ounds [37]. IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Re ferences 1. Nikolic, J.; Kocic, G. and Jevtovic-Stojmenov,T. (2006), Pharmacology online 3: 669- 675. 2. Wallace Hay es, A. (2001) Princip les and M ethods of Toxicology 4th ed., Taylor & Francis; Philadelp hia. 3.WHO (1990)b IPCS Environmental Health Criteria 101, M ethy lmercury . 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Pharmacol.,112: 161-165. 14.M cKee, T. and M cKee, J.R.(1996) Biochemist ry ”, The M cGraw Hill Companies Inc., 246. 15.M cCord, J.M . and Fridorich, I.(1969), J. Biol. Chem., 244: 6049. 16.Clarkson, P.M .and Tomp son, H.S.(2000), Am. J. Clin. Nutr., 72: 6375-6465. 17.Stry er, L.Biochemistry ,(1997), 4th ed., Freeman and Comp any, New York. 18.Chance, B.; Sies, H. and Boveris, A.(1979) Phy siol. Rev., 59: 527-605. 19.Sharma, D.C. and Davis, P.S.(1979), Clin. Chem., 25: 769-772. 20.Fong, K. L.; M cCay , P.B. and Poy er, J.L. (1973) J. Biol.Chem., 248: 7792-7797. 21.Winterbourn ,C.C.; Hawkings, R.E.; Brain, M . and Carrel, R.W. (1975), J. Jab. Clin. M ed., Febr.: 337-341. 22.Acbi Hugo(1974), M ethods of Enzy matic analysis, 2: 684. 23.World Health Organization;(1976), Environmental Health Criteria I; M ercury ; Geneva. 24.Queiroz, M .L.; Bicoletto, C.; Quadros, M .R. and DeCap itani, E.M . (1999),Immunipharmacology and Immunotoxicology , 21 (1): 141. 25.Langwort h, S.(1990), “Early Effect of Occup ational and Environmental Exp osure to Inorganic M ercury ”; Karolinska Inst itut e, Sweden, M EDDR Degree. 26.Wali, R.K.; Jafe, S.; Kumar, D.; Sorgenete, N. and Karla, V.K.(1987), J. Cell. Phy siol.; 133: 25-36. 27.Giugliano, D.; Ceriello, A. and Paolisso, G.(1955) M etabolism; 44: 363-386. 28.Dacie, N. and Lewis, S.M .( 1975), “Practical Hematology ”, 5th edition, Churchill and Livingst one, 79-80. 29.DeSouza Queiroz ,M .L.; Pena, S.C.; Ide Salles, T.S.; De Cap itani, E.M . and Olalla Saad, S.T.(1998), Human and Exp erimental Toxicology , 17: 225-230. 30.M eydani ,M .(1990), M ech. Ageing. Dev., 11 (2-3): 123-123. 31.Berregard, L.; Thomassen, Y.; Schutz , A. and M arkfund ,S.L(1990) Sci. Total Environ., 99: 37-47. 32.Perrin-Nadif ,R.(1996), J. T oxicol. Environ. Health, 48: 107-119. 33.Kurata, M .; Suzuki, M . and Agart, N.S.(1993), Compar. Biochem. and Phy siol., 106 B: 477-487. IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 34.Nath, K.A.;Croatt, A.J.; Likely, S.; Behrens, T.W. and Warden, D. (19960 ,Kidney Int., 50 (3): 1032-1043. 35.Lovasova, E.; Sipulova, A.; Racz, O. and Nist iar, F.(2002) Sep 17-19, 14 th Congress of Pathological and Clinical Phy siology , Hradec Kralove,Czech Republic. 36.Hay s, J.D. and Pulford, D.J.(1995), Crit. Rev. Biochem. M ol. Biol., 30: 445-600. 37.Shireen, K.F.; M ahboob, M . and Khan, A.T .(2004), Toxicoloy International, 11(1):1- 7 . Table (1): MDA product levels in se ra of the four studie d groups Groups No. MDA level g/dl M ean  S.D. T-test C 16 9.31  0.79  G1 15 12.78  0.53 P < 0.005 G2 14 12.99  3.94 P < 0.001 G3 13 14.74  5.5 P < 0.001 Table (2): S hows the mean values and the standard deviation of S OD activities for the four studied groups. Groups No. U / g.Hb mean  S.D. T-test C 16 0.674  0.109  G1 14 0.736  0.187 P = 0.26 G2 12 0.72  0.149 P = 0.3 G3 13 0.77  0.289 P = 0.22 IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Fig. (1): Concentrations of mercury in the study groups (worke rs and normal control Wher e  represents C group (normal control group )  represents G1 group (workers with exp osed p eriod less than 10 y ears)  represents G2 group (workers with exp osed p eriod 10-19 y ears)  represents G3 group (workers with exp osed p eriod more than 20 y ears) Fig. (2): Levels of MDA products i n control and worker groups. 0 1 2 3 4 5 6 7 8 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Studied group s C G1 G2 G3 H g C o n c.  g /d l 0.158 2.949 2.64 2.65 5 1 0 1 5 2 0 2 5 3 0 0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 Studied group s C G1 G2 G3 M D A 9.31 12.78 12.996 14.739 IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Fig. (3): Rel ationship between MDA production and Hg concentration in worke rs and control. Fig. (4) :Erythrocyte catalase activity for the studied groups 0 1 2 3 4 5 6 7 8 9 0 5 10 15 20 25 30 MDA nmol/dl H g C o n c. r = + 0. 911 P < 0.001 0 0.5 1 1.5 2 2.5 0. 5 1 1.5 2 2.5 3 3.5 4 4 .5 5 Studied groups C G1 G2 G3 C a ta la se U /g m .H b 1.5 0.8 0.88 0.815 IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Fig. (5): Rel ation of catalase activity against worke rs (Hg > 3 g/dl) and control Fig. (6): GS Ts activities in expose d groups and normal control 0 1 2 3 4 5 6 7 8 0 0.5 1 1.5 2 2. 5 Catalase activity U/gm.Hb H g C o n c.  g /d l r = - 0.85 P < 0.001 Nor mal group Worker s (Hg > 3  g/dl) 1 1.5 2 2.5 3 3.5 4 0. 5 1 1.5 2 2 .5 3 3.5 4 4 .5 5 Studied groups C G1 G2 G3 G S T U /g m .H b 1.85 2.59 2.44 2.77 IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L. 23 (1) 2010 Fig. (7): Rel ation of serum mercury for workers (Hg > 3 g/dl) and control versus GS T activity. Fig. (8): Rel ationship of MDA for workers (Hg > 3 g/dl) and normal control against GS T activity. 0 1 2 3 4 5 6 7 8 0 0.5 1 1.5 2 2.5 3 3 .5 4 GST activity U/gm.Hb H g  g/ d l r = + 0.759 P < 0.001 Nor mal group Worker s (Hg > 3  g/dl) 0 5 10 15 20 25 30 0 0.5 1 1.5 2 2 .5 3 3. 5 4 GST activity U/gm.Hb M D A n m o l/ d l r = + 0.54 P < 0.001 Nor mal group Worker s (Hg > 3  g/dl) 2010) 1( 23مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد الشد التأكسدي فيتأثیر التعرض للزئبق نظام الدفاع االنزیمي ضد األكسدة فيو وفاء فاضل الطائي ، معین اسكندر الجبوري، انعام أمین محمد جامعة بغداد، ابن الهیثم -كلیة التربیة، قسم الكیمیاء الخالصة صناعة : مثل ،نه یدخل في العدید من الصناعاتاة للتسمم بالزئبق، إذ دیعد خالل القرون السابقة وثقت حوادث وفي الزراعة یستعمل الزئبق في تعفیر الحبوب أما في الطب فیدخل في سبیكة حشوة . البطاریات، والمحاریر، والبارومیترات .األسنان د لتسمم باألنواعلهناك میكانیكیة مهمة تؤدي إلى تصدع الخلیة وتحدث نتیجة المختلفة للزئبق، إنها میكانیكیة حث الش .التأكسدي مختلفة في شركة الفرات من غیر المدخنین وممن ال مدداالعاملین ) الذكور(هذه الدراسة أجریت على مجامیع من العمال لمدخنین فهم من الذكور األصحاء وغیر المتعرضین للزئبق، وغیر ا ) (Cأما مجامیع السیطرة.یشكون من أمراض مزمنة .عند المقارنة بمجامیع العاملین هانفس ومن األعمار : ثالث مجامیع علىسنة وقد قسمت ) 61-22(أعمار العاملین تتراوح بین G1 = سنوات) 10(تعرض أقل من مدةتتضمن العاملین. G2 = سنة) 19- 10(تعرض بین مدةتتضمن العاملین. G3 = سنة) 19( تعرض أكثر من مدةتتضمن العاملین. إن النتائج التي حصلنا علیها من خالل فحص المعاییر المختلفة قادتنا إلى إضافة مجموعة أخرى تتضمن نماذج في هذه المجموعة الحظنا . التعرض مدةالنظر عن صرفمن العاملین أعطت فحوصاتها تراكیز عالیة من الزئبق ب .تة الدفاعیة التي فحصتغییرات معنویة عالیة في معاییر المنظوم المؤشر على مقدار األكسدة (في مستویات المالون داي ألدیهاید اواضح اإن نتائج هذه الدراسة أظهرت ارتفاع ي كل مجامیع العاملین و ) الفوقیة للدهون ، 9.31: الذین وجدت لهم تراكیز عالیة للزئبق في مصل الدم، التي كانتالسیما ف ز C ،G1 ،G2 ،G3مل لكل من المجامیع 100/نانومول 18.11، 14.73، 12.99، 12.78 وأخیرًا مجموعة التراكی .على التوالي G4العالیة للزئبق كانت اد في فعالیة أنزیم السوبراوكساید دایسمیوتیز في كریات الدم الحمراء، الم نجد هناك تغیر .على التوالي(G3,G2,G1,C ) لكل من) غم هیموغلوبین/وحدة 0.77،0.72،0.73،0.67(النتائج p(كما أظهرت النتائج انخفاضًا معنویًا كبیرًا لفعالیة إنزیم الكتلیز في كریات الدم الحمراء لكل مجامیع العاملین ) 0.001 > غم من الهیموغلوبین للمجامیع /وحدة 0.45، 0.815، 0.88، 0.8، 1.5: یاتياموالقیم كانت ك. مقارنة بمجموعة األصحاء C ،G1 ،G2 ،G3 ،G4 على التوالي. ترانسفریز في مجامیع العاملین مقارنة بمجامیع األصحاء –س –بینما كان هناك ارتفاع كبیر في فعالیة إنزیم الكلوتاثایون )p ، Cغم من الهیموغلوبین للمجامیع /وحدة 3.2، 2.776، 2.441، 2.589، 1.85، كما موضح في القیم )0.001> G1 ،G2 ،G3 ،G4 على التوالي.