Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 68 The Role of Oxidative Stress In Lead Poisoning Bashier Al-Ubaidy*, Dawsar K. Al-Khashali** , Nawfal A. Numan** Receive d 20-9-2002 Accepte d 24-4-2005 ABSTRACT To inves tiga te the re la tionship b etwee n increas ed lipid peroxid atio n, a nd the lowe ring o f bo th plas ma total p ro te ins and albumin in lea d-expos ed wo rkers , a nd the effe ctiv eness of a ntio xida nts (vit. C and E) in mod ulating oxid ativ e stress in tho se wo rkers. Thirty male and females workers e mploye d in the Iraq i storage ba ttery (age range 2 0-40 ye ars) were pa rticipating in this stud y. Additio nally, 11 he althy subjec ts were se rv ed as hea lthy controls , with the sa me age ra nge comp ared to workers group, to a void the effects o f age variatio ns on the studied pa ra meters. Blo od le ad lev els, e rythro cytes and p la sma MDA, e rythro cyte s and p la sma GS H, total protein and albumin lev els in hea lthy controls a nd lea d-e xp ose d worke rs p re - and po st-treatme nts with antioxid ant were mea sure d. Co mpa riso n w ith he althy control gro up s re vea l 36 0% inc re as e in bloo d le ad le ve ls , 15 0% increa se in erythroc yte MDA, 1 17% inc rea se in p la sma MDA, 28 % d ecrea se in e rythro cyte GS H, 5 6% de creas e in plasma GSH, 13% de creas e in to ta l pla sma p rote in a nd 2 3% dec re as e in a lbumin lev els in le ad -expo sed w orkers. Tre atment with a co mbination of antioxid ant vita mins (100 0 mg/da y vit. C and 200 mg/d ay vit. E) for one mo nth produced s ignifica nt red uction 12 % in lea d le vels, 54 % in erythroc yte MDA, 53 % in p la sma MDA; s ignific ant incre as e 41% in e rythro cyte GSH, 1 20% in plas ma GSH and 11% in plasma albumin lev els in comp aris on with pre-trea tme nt le vels . In co nclusion, there is a be neficial e ffec t o f antioxid ants o n the oxid ativ e stress parame ters that no t only related to the ir a bility to remo ve lea d from ta rget cells, b ut also as so ciated with a ntioxida nt pote ntia l for b olstering thiol a ntio xidant c apa city, a nd this makes the se v itamins a goo d candida te for therap eutic intervention in lea d poiso ning. الخالصة  ل مـن ن الكـم الهاـئ ـم رغم ال ة٬ وـب ن السـابق ر السـني عـب ة كثف رة م ها بصو ست را كل التي تمت د ن المشا سمم بالرصاص م ر الت عتب ي واهر السـمية لهـذه ـظ ر مـن ال ر الكثـي ى تفسـي عـل وفة غير قادرة معر ة التسمم ال كي ميكاني ن ع٬ فا موضو هذا ال ة حول راكم مت ومات ال عل الم ات ال .المـادة رـي دى النظ ـح واســطة إ ي المحـدث ب كسـد هــاد التأ رح أن اإلج مي للرصـاص٬ تقـت أثير الســ ة الـت ي تبحـث فــي كيفـي متداولــة والـت مم هذا التس ن عراض الناتجة ع شوء العديد من األ هم في ن ور م ون له د ن يك مل أ لذا تمت دراسة المالونداي ألديهايد .الرصاص٬ من المحت )MDA(وتا ن٬ والكل ة الدهو زناخ ر ل مؤش ر عتب ن ٬ الذي ي م )GSH(ثايو د ـت عية٬ ـق ألكسدة الطبي ع ا موان ومين٬ اللذان يعتبران من واأللب ٬ كسدة ع األ وان عطاء م راق٬ قبل وبعد إ ريات في الع طا ة لصناعة الب ن في المنشأة العام ملي عا وال ن للرصاص عرضي مال المت ها لدى الع فحص ربيك سكو مض األ حا من ول ) Vit. C(والتي تتض وفير وك مـا ) Vit. E(واأللفات والبالز حمـراء كريات الدم ال ة ختبار مدى قابلي كما تم ا ه علي ألكسدة موانع ا ر من ثم قياس مدى تأثي روجين و هيد كسيد ال محدث ببيرو هاد التأكسدي ال مة اإلج مقاو ائج الدراسـة .على هرت نـت أظ وى ست عرضين لل MDAوجود زيادة في م مال الم الزما لدى الع راء والب ريات الدم الحم من ك ظ في في كل ملحو خفاض مع ان رصاص٬ ومين واأللب ن ت الكلوتاثايو هيدروجين . مستويا سيد ال وك حدث ببير ي الم سد جهاد التأك ما لإل والبالز كريات ة ال سي سا وحظ زيادة في ح ك ل كذل ه عرضين ل مستوى الرصاص في الدم لدى العمال الم ي رتفاع ف ع ا رافقة م ي تم استخدامها أدت إلى . مت كسدة الت ع األ وان ي إن م سـن ـف تح ي ر الت معايي ميع ال ت ج جهـاد تم ر اإل را ماية للجسم ضد أضـ ر الح ة في توفي من إمكانية تأثيرها من الناحية السريري ز مما يعز دراستها عرض للرصاص ي الناتج عن الت كسد .التأ * Dep ar tme nt o f To xicology, Al-Rasheed Militar y Hospital ** De partment of Pharmac ology and Toxicology, Colleg e of Phar mac y, University of Baghdad Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 69 INTRODUCTION Lea d, s imilar to ma ny hea vy metals, is a co mplex toxin, exe rting numerous pa thophys io lo gic effe cts in many orga n systems (1). At the molecula r le vels, lea d interacts with biomolecule s and functio ns in different ways , like binding to numerous structural a nd enzymatic proteins (2), interfe re nce with me ta bo lic pa thways of mito cho ndria (3), a nd exhibiting mutage nic and ca rc inoge nic effects in mammalia n ce lls (4). Oxida tive s tres s which re fe rs to a ce llular situation cha ra cterized by elev atio n o f the stud y state co nce ntra tions of rea ctiv e oxyge n sp ecies (ROS ), a nd this could b e a p os sible co ntributo r to the p athogenes is o f lea d pois oning(5). S ome in-vitro and in-v iv o studie s showed a n elev ated p rod uc tion o f ROS d ue to le ad trea tme nt (6,7,8), a nd increas ed lipid pe roxid ation a ss ociate d with alte re d antioxid ant de fe nse sys tems (9). The e ffec ts of lea d on the o xida tive stres s pa ra meters like glutathione (GSH), and malondialdehyd e (MDA), sugges ts ROS a s a pos sible c ontrib utor to ce ll damage due to lea d expos ure (10). The increa se in lip id p eroxid atio n during le ad p oiso ning were found to b e ac comp anie d by alteratio ns in the antioxid ant de fe nse system, inc luding dec re as ed GSH le ve ls in a ll b od y comp artments (11). This s tudy wa s co nducted to inv es tiga te the exte nt to which ROS-re la ted pro ce ss es are invo lve d in le ad po is oning and the p os sibility of therap eutic inte rv ention with antioxid ant vita mins in this ca se. -: METHOD andSUBJACT This study was ca rrie d out on wo rkers emp lo yed in the Iraqi storage battery plant, and se le ctio n was made o n the ba sis that they must be directly involve d with lead e xpo sure , and ha ve bee n employed for at lea st 8 months, be fo re the inv es tiga tion were carrie d out. Thirty, ma le a nd fema le s vo luntee rs (age ra nge 20-40 years) from the workers of the b attery plant, partic ip ate in this s tudy. Eleve n hea lthy subjects s erved as hea lthy c ontrols, with the sa me age ra nge comp ared to worke rs gro up , to av oid the effects of age v aria tions on the stud ie d pa rameters. The a verage working time (hrs/da y) for e ac h worke r is 6 hrs , with a p erio d of e xpo sure to le ad ranging fro m 8 months to 2 8 yea rs . Individual s ymp to m s urvey wa s pe rformed b y clinical and p hysica l ev aluation o f the wo rkers invo lve d in the stud y, c oncerning the prese nc e of le ad-as soc ia ted signs and symptoms for the purpo se o f prop er s elec tion. Bloo d sample s (10 ml) were drawn by v ein puncture from ea ch subject p rior to starting trea tme nt with a ntio xida nts (as ba se line sample). After tha t, a ll s ubjec ts rece ive a comb ination of a ntioxida nt v ita mins (a sc orb ic acid 1 000 mg/da y a nd α-toc opherol 200 mg/day) orally for a p erio d of 4 wee ks , then sec ond blood s amp le was d ra wn fo r ev alua tion of the e ffec t of trea tment on the studied parame ters . Blo od samples were plac ed into hepa rinized tubes a nd re frige rated until sepa ration of e rythrocytes and ana lysis. Bloo d le ad lev els were mea sure d by grap hite Fra nc e a tomic a bs orptio n s pe ctro pho to meter acc ording to the me thod of Pars on et al (12). Erythro cyte s a nd p la sma malondialdehyde (MDA) le vel as ind ic ator o f lip id p eroxida tion were a sse ss ed utilizing thioba rb ituric ac id ass ay me thod o f Stoc k and Do rma nd y (13), and the susc eptibility of p la sma a nd e rythro cyte s to in-v itro hyd rogen p ero xide -ind uce d oxida tive stre ss wa s mea sured a cc ord ing to the method of Gilb ert e t al (14), and the re sults we re exp re ss ed as nmole (MDA)/gm Hb, bas ed on the mo la r e xtinctio n c oeffic ie nt of MDA (1 .5 6x1 05 M-1 cm-1). Gluta thione leve ls we re determine d in e rythro cyte s and p la sma acc ording to the me thod of Go din e t al (15). Plasma albumin le vels we re determined utilizing a rea dy-ma de kit fo r this purp ose (Ra ndo x co mpany, England) a cc ord ing to the metho d of Douma s e t al (16). To ta l plasma protein was me asure d ac cording to the Biuret metho d (17). Erythroc yte he moglob in conce ntra tion wa s meas ured using Drapkin’s re age nt metho d (18). Statistic al a nalys is o f the data was d one using Stude nt’s t-test, and P -v alues of les s than 0.05 were c ons id ered s ignifica nt. Tab le (1) showe d a s ignifica nt e le vation in le ad le vels in blood of e xpo se d worke rs (3 60%) c ompa re d to c ontrols, pro duced 1 2% dec re as e in lea d le vels , which is a s ignifica nt value c omp ared with pre -trea tme nt le vels. The res ults of the s tudy ind ic ates tha t the b ase line e rythro cyte s and plas ma MDA le ve ls we re eleva te d b y 1 50% and 11 7% res pec tive ly comp ared to that o f co ntro ls (Ta ble 1). MDA le vels in bo th co mpartment dec re as e a fter trea tme nt with a c ombina tion o f a ntioxida nt vita mins (1 00 0 mg/day a sc orbic acid and 200 mg/day (α-toco phe ro l) for o ne month, which was s ignifica nt, co mpa re d to the pretrea tme nt value s (Table 1 ). The res ponse o f e rythro cytes and plas ma to in-v itro hydroge n p eroxide cha llenge showe d tha t, MDA prod uction in both co mpartments of the e xp ose d wo rker’s blood we re s ignifica ntly highe r, c omp ared to that o f c ontrols. Tre atment with antioxida nts as indicate d be fo re , signific antly increas e the Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 70 re sistance o f erythroc ytes a nd plas ma of le ad- expos ed subjec ts to the hyd rogen pe ro xide- induced lip id peroxid atio n, refle cted by a significant de creas e in the MDA p ro duc tio n after antioxid ant trea tment, c omp are d to pre- trea tment le ve ls (Figure s I a nd II). Table (1): Bloo d le ad le ve ls, e rythro cytes and p la sma MDA le ve ls in le a d-expose d wo rke rs pre - and pos t-trea tme nts with v it. C and vit. E Eac h value re pre s e nts me an ± SE * Signific antly diffe re nt from co ntrol (P<0.05 ). ¥ Significa ntly diffe re nt with re s pe c t to pre - tre a tme nt (P<0 .0 5). Fig .1 Ery throcyte MDA of Co ntrol Subje cts a nd lead – e xpose d Worke rs Be for and Afte r Tre atme nt with Vita mins (C&E)in re s ponse to in vitro Cha lle nge with Vario us H2O 2 Co nce ntra tion Fig.2 Plas ma MDA o f Control Subjec ts and le ad – ex pose d Worke rs Be for and Afte r Treatme nt with Vita mins (C&E)in re spo nse to in vitro Challe nge with Va rious H2O 2 Conce ntra tion In lea d-exp os ed worke rs , there was 28% and 56% d ep le tion in e rythro cytes a nd pla sma glutathione (GSH) lev els res pe ctiv ely, obs erved be fo re antioxid ant trea tme nt, and comp ared to co ntro ls (Table 2). After trea tme nt with a ntio xidants for one mo nth, there were a significa nt incre as e in GSH leve ls in b oth c ompa rtments (4 1% and 12 0% re spe ctively) comp ared to pretre atment leve ls (Ta ble 1). Tota l plas ma protein and a lb umin, the general antioxid ants in the bo dy, we re fo und to be affe cted due to le ad expo sure, and their leve ls in lea d workers were s ignifica ntly dec re as ed (13% a nd 23 % re spe ctiv ely) comp ared to c ontrols. Antioxid ant trea tme nt produced a significant elev atio n o n p la sma albumin only a fte r o ne-month dura tion of trea tme nt (Ta ble 3). P arameter s Co ntrol n =11 L ea d expo sure wo rk ers Before treatme nt n=30 After treatment n=30 Blood lead μ g/dl 10 ± 0 .64 46 .4 ±2 .07* 41.03 ± 1 .96* ¥ Erythrocyte MDA nmole/gm Hb 7.7 ± 0.29 19 .81 ± 20* 9 .07 ± 0 .58* ¥ Plasm a MDA nmole/L 0.97 ± 0.08 2.11 ±0 .08* 1 .00 ± 0 .06 ¥ 0 40 80 120 160 0 1 2.5 5 7.5 H2O2 (m M) E ry th ro cy te M D A ( n m o le /g . H b) control before treatment after treatment 0 4 8 12 16 0 1 2.5 5 7.5 H2O2 (mM) P la sm a M D A ( u m ol e/ L ) control before treatment after treatment H2O 2 (mM) H2O 2( mM) Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 71 Ta ble (2): Ery throcytes a nd plas ma glutathione (GSH) le ve ls in le ad-e xpose d worke rs pre - a nd po st-tre atme nt with vit. C and v it. E. Eac h value re pre s e nts me an ± SE * Signific antly diffe re nt from co ntrol (P<0.05 ). ¥ Significa ntly diffe re nt with re s pe c t to pre - tre a tme nt (P<0 .0 5). Table (3): Plas ma to ta l prote in and albumin leve ls in le ad-ex pose d workers pre - a nd pos t-trea tme nt wit h vit. C and v it. E. Eac h value re pre se nts me an ± S E * Signific antly diffe re nt from co ntrol (P<0.05 ). ¥ Significa ntly diffe re nt with re s pe c t to pre - tre a tme nt (P<0 .0 5). DISCUSSION Free radica ls a ctiv ity ha s be en implic ated in the p athogenes is of a va riety o f huma n dise ase s and the analysis o f our data s howe d that, o xidative stres s wa s quite clea r in lea d expos ed wo rkers (Ta ble 1, Figure I a nd II), a s no tice d by increa se d erythrocytes and plas ma MDA le vels, which is in agre ement with other stud ie s (19, 20). The me chanisms b y whic h le ad ca use s it’s de le te rious e ffe cts has yet to b e elucida ted, ho weve r, part of le ad’s effect may be d ue to the ac cumulatio n of d elta -a minolev ulinic a cid dehydrata se (ALAD), an e nzyme in the heme synthes is pathw ay, which c atalyzes the condensa tion o f two mo le cule s o f -ALA to porphobilinoge n(21) .At a pH range of 7.0-8 .0 -ALA enolizes, and the re sulted eno l unde rgo autoo xida tion re sulting in the fo rma tion of sup eroxide a nd hydro xyl ra dica ls . ALA has also be en s hown to undergo es iro n-ca ta lyze d oxida tion with ROS ge ne ration, and to ind uce Ca+2 relea se fro m mitoc ho ndria thro ugh oxida tive d ama ge to inne r me mbrane (22). The effec t of antioxid ant v itamins (a sco rb ic ac id a nd to cop herol) on lipid peroxid atio n parame te r, MDA, as shown in ta ble (1) a nd figure I a nd II, s ugges ted that they d id p rod uc e a de creas e in the ba sal MDA le vels and the susc eptibility of b oth, erythrocyte s and plas ma to the oxid ativ e stress induc ed in-v itro b y H2 O2. The a ntioxida nt trea tme nt le ad to increas e in e rythro cyte s and plasma GSH le vels (Tab le 2), whic h may be due to a d irec t sc ave nging a ctiv ities of the generated ROS, a nd de crea sing utilizatio n and dama ge of GSH, or indirect through the imp ro veme nt o f the oxida nt/antioxida nt balance in the c ells a fte r tre atment (23). In no rma l co nditio ns , as we ll as during oxida tive stre ss (lead e xp osure ) a d aily d os e of asc orbic ac id a nd -to cop he rol, a pp ear to be protect the oxido -red uctive s ta te o f red blood cells, by modula ting the e xtent o f lipid peroxid atio n, a s well as the a ctiv ities of the antioxida nt enzyme s (24). In this s tudy, daily s up pleme ntatio n with a comb ination of asc orbic ac id a nd - to cop he rol, re sulted in a s ignifica nt dec re as e in le ad leve ls in the blood a fter one month (Tab le 1), and this may provide a n e conomic and convenient me thod of reducing blood lead le vels, po ss ib ly by d ecrea sing inte stinal abs orptio n of lea d (25), or it may inc re as e the re nal excre tio n of this me ta l. Alb umin is kno wn to act a s a n e ffec tive antioxida nt, due to its a bility to b ind the catalytic co ppe r io ns (26), fre e fa tty ac id , and hyp ochlorus ac id (HOCl), and a ls o showe d a signific ant ca pab ility to des tro y H2O2 in the prese nc e of red uce d glutathio ne (27). The pres ent study c le arly de mons tra te d the re la tionship betwee n inc re as ed lipid peroxid atio n, a nd the lowe ring o f bo th p la sma to ta l proteins and a lb umin in le ad -e xpo sed workers (Tab le 3 ), which ma y be attribute d to the s tructural mod ifica tion, which may lead eve ntua lly to imp air the a ntio xidant prop erties of albumin, and eve n may a ct to ind uce oxida tive stress , thro ugh it’s action as a prooxid ant in pres enc e of ca talytic ions (28). Parame te rs Contro l n =11 Le ad e xposure worke rs Be fo re tre a t - me nt n=30 Afte r tre at - me nt n=3 0 Erythro cyte GS H μmo le /gm H b 11 .92 ± 0.36 8.62 ± 0.4* 12.1 7 ± 0 .37* ¥ Pla sma GS H μ mo le /L 0 .88 ± 0.15 0.39 ± 0 .05* 0.86 ± 0 .12 ¥ Parame te rs Contro l n =11 Le ad e xposure wo rke rs Be fo re tre at- me nt Afte r tre at- me nt Total plas ma protein gm/dl 7.51 ± 0.11 6.56 ± 0.13* 6.6 ± 0.12* Plas ma albumin gm/dl 5.06 ± 0.1 3.88 ± 0.05* 4.29 ± 0.11¥ * Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 72 Antioxida nts trea tment re sulted in s ignific ant elev atio n in a lb umin le vels (Tab le 3), whic h may b e due to the ir direc t s cav enging a ctiv ity, or p ro te ctio n of a lb umin agains t ROS-induce d da mage . 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