ORIGINAL PAPERS. The Effect of Oral Chromium Administration on Antioxidant Barrier in Rats

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1 ORIGINAL PAPERS Adv Clin Exp Med 2004, 13, 3, ISSN X MAŁGORZATA KIEŁCZYKOWSKA, KAZIMIERZ PASTERNAK, IRENA MUSIK, JOLANTA WROŃSKA The Effect of Oral Chromium Administration on Antioxidant Barrier in Rats Wpływ doustnej intoksykacji chromem na układ antyoksydacyjny szczurów Department of General Chemistry, Medical University of Lublin, Poland Abstract Background. ROS (reactive oxygen species) generated in organism as a consequence of prooxidative processes disturb its functions and can cause many severe diseases. The system of antioxidant substances was created to pre vent ROS toxic influence. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) belong to the most im portant antioxidant enzymes and are used as markers of oxidative stress. Chromium, metal widely used in industry exerts allergic, carcinogenic and hepatoxic influence. Chromium exposure can also cause pulmonary and renal di sturbs. It was showed that this metal effects prooxidative processes. On the other hand chromium is used as a con stituent of drugs. Objectives. Described facts and dependences made us investigate the influence of chromium oral administration on GPx and SOD levels in serum and tissues of rats. Material and Methods. Our study was carried out on aged two months, male Wistar rats ( g). The ani mals were divided into two groups 20 animals each: tested group (I Cr 500 ppm) received a water solution of Cr(NO 3 ) 3 9H 2 O in the form of drinking water, at the dose of 500 mg Cr dm 3 and control group (II K) rece ived redestilled water. A half of animals of each group was killed after three weeks and the rest after six weeks. Daily consumption of provided fluids was about 25 cm 3. Blood from the heart as well as the tissues of liver, kid ney, femoral muscle, brain, spleen and heart muscle were collected. Serum was separated. 10% (w/v) tissue homo genates were prepared in 0.1 mol dm 3 Tris HCl buffer, ph = 7.4. In serum and supernatants GPx and SOD acti vities were determined using RANSEL and RANSOD kits produced by RANDOX. Comparisons between control and tested groups as well as between I Cr 500 ppm groups after 3 weeks and Cr groups after 6 weeks were con sidered significant with p < In serum enzyme activities were calculated in U/ml whereas in tissues GPx in U/g of protein and SOD in U/mg of protein. Results. Chromium administration influenced mainly GPx level vs. control. In heart muscle, femoral muscle, bra in and liver it changed during all the experiment (in heart muscle after 3 weeks ± 29.8 vs. control ± ± 22.8, after 6 weeks ± 59.7 vs. control ± 40.5; in femoral muscle after 3 weeks 23.6 ± 3.9 vs. control 53.0 ± 12.6, after 6 weeks ± 15.5 vs. control 28.6 ± 6.0; in brain after 3 weeks 47.1 ± 12.1 vs. control 85.7 ± ± 20.2, after 6 weeks 50.0 ± 9,7 vs. control 30.8 ± 9.7; in liver after 3 weeks ± 45.6 vs. control ± 31.6, after 6 weeks ± 51.0 vs. control ± 158.5). In spleen it was altered only after 6 weeks (167.2 ± 20.4 vs. control ± 37.0) whereas in serum and kidney changes were observed only after 3 weeks (serum 6.6 ± 0.8 vs. control 8.2 ± 1.0; kidney ± 51.0 vs. control ± 40.5). SOD level vs. control was changed in serum after 6 weeks (13.6 ± 1.9 vs. control 23.4 ± 3.5), in heart muscle during all the experiment (after 3 weeks 12.8 ± 2.0 vs. control 2.7 ± 0.5, after 6 weeks 9.2 ± 1.5 vs. control 6.4 ± 0.8), in liver after 3 weeks (20.9 ± 4.4 vs. control 10.4 ± ± 3.2), in kidney after 6 weeks 16.0 ± 3.4 vs. control 9.3 ± 1.4). In Cr administered group GPx activity enhanced very significantly in serum (6.6 ± 0.8 after 3 weeks vs ± 1.5 after 6 weeks), heart muscle (300.1 ± 29.8 after 3 weeks vs ± 59.7 after 6 weeks) and femoral muscle (23.6 ± 3.9 after 3 weeks vs ± 15.5 after 6 we eks). SOD was decreased in serum, heart muscle and but the changes were not so marked (in serum 43.1 ± 8.8 after 3 weeks vs ± 1.9 after 6 weeks; in heart muscle 12.8 ± 2.0 after 3 weeks vs. 9.2 ± 1.5 after 6 weeks, in brain 19.8 ± 4.6 after 3 weeks vs. 1.4 ± 0.5 after 6 weeks). Conclusion. The obtained results let us suggest that although Cr administration in diet influence both GPx and SOD activity, GPx seems to be a better marker of oxidative stress caused by chromium exposure (Adv Clin Exp Med 2004, 13, 3, ). Key words: chromium, glutathione peroxidase, superoxide dismutase, rats.

2 396 M. KIEŁCZYKOWSKA et al. Streszczenie Wprowadzenie. RFT reaktywne formy tlenu, powstające w organizmie podczas zachodzenia procesów prooksy dacyjnych, zaburzają jego funkcjonowanie i mogą być przyczyną wielu groźnych chorób. W celu ochrony przed toksycznym wpływem RFT organizm wykształcił zespół substancji antyoksydacyjnych. Peroksydaza glutationowa (GPx) i dysmutaza ponadtlenkowa (SOD) należą do najważniejszych enzymów antyoksydacyjnych i znajdują za stosowanie jako markery stresu oksydacyjnego. Chrom, metal powszechnie stosowany w przemyśle, wywiera karcinogenny i hepatotoksyczny wpływ i może powodować alergie. Narażenie na ten pierwiastek może być przy czyną zaburzeń czynności płuc i nerek. Wykazano, że chrom wpływa na przebieg procesów oksydacyjnych. Z dru giej strony pierwiastek ten jest używany jako składnik leków. Cel pracy. Przedstawione fakty i zależności skłoniły autorów do podjęcia badań nad wpływem doustnego poda wania chromu na aktywność GPx i SOD w surowicy i tkankach szczurów. Materiał i metody. Badania zostały przeprowadzone na dwumiesięcznych szczurach, samcach rasy Wistar ( g). Zwierzęta podzielono na dwie grupy po 20 zwierząt każda: badaną (I Cr 500 ppm), która otrzymy wała do picia roztwór Cr(NO 3 ) 3 9H 2 O w dawce 500 mg Cr dm 3 i kontrolną (II K), która otrzymywała do pi cia wodę redestylowaną. Połowę zwierząt po trzech tygodniach, a pozostałe po sześciu usypiano przez podanie ke taminy, a następnie pobierano do badań krew, wątrobę, nerkę, mózg, mięsień uda, śledzionę i mięsień sercowy. Dzienne pobranie dostarczanych płynów wynosiło około 25 cm 3. Z krwi oddzielono surowicę. 10% (m/v) homo genaty tkankowe przygotowano w 0,1 molowym buforze Tris HCl o ph = 7,4. W surowicy i nadsączach tkanko wych oznaczono aktywności peroksydazy glutationowej i dysmutazy ponadtlenkowej stosując zestawy RANSEL i RANSOD firmy RANDOX. Różnice między grupą kontrolną i badaną oraz grupą I Cr 500 ppm po trzech i po sześciu tygodniach uznano za istotne statystycznie przy p < 0,05. Aktywności enzymów w surowicy podano w U/ml, podczas gdy w tkankach: GPx w U/g białka, SOD w U/mg białka. Wyniki. Podawanie chromu wpływało głównie na aktywność GPx w stosunku do kontroli. W mięśniu sercowym, mięśniu uda, mózgu i wątrobie zmieniała się podczas całego eksperymentu (w mięśniu sercowym po 3 tyg. 300,1 ± 29,8 vs. K 145,2 ± 22,8, po 6 tyg. 550,6 ± 59,7 vs. K 374,5 ± 40,5; w mięśniu uda po 3 tyg. 23,6 ± 3,9 vs. K 53,0 ± 12,6, po 6 tyg. 114,3 ± 15,5 vs. K 28,6 ± 6,0; w mózgu po 3 tyg. 47,1 ± 12,1 vs. K 85,7 ± 20,2, po 6 tyg. 50,0 ± 9,7 vs. K 30,8 ± 9,7; w wątrobie po 3 tyg. 375,4 ± 45,6 vs. K 284,6 ± 31,6, po 6 tyg. 422,9 ± 51,0 vs. K 820,0 ± 158,5). W śledzionie zmieniała się jedynie po 6 tyg. (167,2 ± 20,4 vs. K 358,3 ± 37,0), podczas gdy w surowicy i nerce zmiany obserwowano jedynie po 3 tyg. (surowica 6,6 ± 0,8 vs. K 8,2 ± 1,0; nerka 418,9 ± ± 51,0 vs. K 239,2 ± 40,5). Aktywność SOD vs. K zmieniała się w surowicy po 6 tyg. (13,6 ± 1,9 vs. K 23,4 ± ± 3,5), w mięśniu sercowym podczas całego eksperymentu (po 3 tyg. 12,8 ± 2,0 vs. K 2,7 ± 0,5, po 6 tyg. 9,2 ± ± 1,5 vs. K 6,4 ± 0,8), w wątrobie po 3 tyg. (20,9 ± 4,4 vs. K 10,4 ± 3,2), w nerce po 6 tyg. (16,0 ± 3,4 vs. K 9,3 ± 1,4). W grupie otrzymującej chrom aktywność GPx znacząco podwyższała się w surowicy (6,6 ± 0,8 po 3 tyg. vs. 11,2 ± 1,5 po 6 tyg.), mięśniu sercowym (300,1 ± 29,8 po 3 tyg. vs. 550,6 ± 59,7 po 6 tyg.) i w mięśniu uda (23,6 ± 3,9 po 3 tyg. vs. 114,3 ± 15,5 po 6 tyg.). Aktywność SOD zmniejszała się w surowicy, mięśniu serco wym i mózgu, ale te zmiany nie były tak znaczące (w surowicy 43,1 ± 8,8 po 3 tyg. vs. 13,6 ± 1,9 po 6 tyg.; w mię śniu sercowym (12,8 ± 2,0 po 3 tyg. vs. 9,2 ± 1,5 po 6 tyg.: w mózgu 19,8 ± 4,6 po 3 tyg. vs. 1,4 ± 0,5 po 6 tyg.). Wnioski. Otrzymane wyniki pozwalają przypuszczać, że chociaż podawanie chromu w pożywieniu wywiera wpływ zarówno na aktywność GPx, jak i SOD, peroksydaza glutationowa wydaje się lepszym markerem stresu oksydacyjnego spowodowanego narażeniem na ten pierwiastek (Adv Clin Exp Med 2004, 13, 3, ). Słowa kluczowe: chrom, peroksydaza glutationowa, dysmutaza ponadtlenkowa, szczury. Peroxidative processes occurring in organism can cause many severe diseases [1, 2]. The organi sms prevent these deleterious mechanisms using the antioxidant barrier system of enzymatic and non enzymatic substances which can neutralize to xic radicals and molecules. Glutathione peroxida se (GPx) EC and superoxide dismutase (SOD) EC belong to the most important antioxidant enzymes [2]. Their correct action plays the great role in organism functions. Chromium belongs to metals used in industry. It can exert allergic, carcinogenic and hepatoxic influence, cause pulmonary disorders and renal di sturbances [1, 3 6]. Chromium affects prooxidative processes [7], and influences antioxidant enzymes activity [4]. The organisms are exposured mainly to Cr(III) and Cr(VI). Some authors consider Cr(VI) to be the main toxic form of this metal [8]. However it was showed that the biological reduc tants can reduce Cr(III) to Cr(II) which can take part in reactive oxygen species (ROS) generation [3, 5]. Cr(III) was also found out to cause ROS ge neration [3]. What s more Cr(VI) can also be redu ced in cells to Cr(III) and this reaction contribute to prooxidative processes [5]. Chromium takes part in enzymatic processes, causes DNA damages [5], influences insulin activity, lipid and protein metabolism [9, 10]. Intraperitoneal K 2 Cr 2 O 7 admi nistration to rats causes chromium to deposit in skin [11]. The chrome plating factory workers were found out to have significantly increased Cr level in blood and urine in comparison to non exposed people [1]. On the other hand this metal was used as a constituent of the preparation admi nistered to workers exposed to lead to improve an tioxidant barrier [12].

3 The Chromium Effect on Antioxidant Barrier in Rats 397 Above mentioned facts made us undertake the investigations on the influence of Cr(III) oral into xication on GPx and SOD activity in serum and tissues of rats. Material and Methods Our study was carried out on aged two months, male Wistar rats ( g). The animals were di vided into two groups (twenty animals each): te sted group I Cr 500 ppm received a water solu tion of Cr(NO 3 ) 3 9H 2 O in the form of drinking wa ter, at the dose of 500 mg Cr dm 3, control group II K received redestilled water. The animals were received food and water ad libitum. Daily con sumption of provided fluids was about 25 cm 3. A half of animals of each group was killed after three weeks and the rest after six weeks. Each time the rats were sacrificed under ketamine narcosis and blood from the heart as well as the tissues of liver, kidney, femoral muscle, brain, spleen and heart mu scle were collected. Serum was separated. 10% (w/v) tissue homogenates were prepared in 0.1 mol dm 3 Tris HCl buffer, ph = 7.4. Supernatants were obtained by centrifugation at 5000 g for 30 min. In serum and supernatants GPx and SOD activi ties were determined using RANSEL and RANSOD kits produced by RANDOX. Protein was measured using method of Bradford [13]. The assays were car ried out with the help of SPECORD M40 (Zeiss, Je na) spectrophotometer. Comparisons between con trol and tested groups as well as between Cr groups after 3 weeks and Cr groups after 6 weeks were ma de using the Cochran Cox or t Student tests. Values were considered significant with p < Results The changes of enzymes activities in the con trol group could be the result of aging of rats. It has not influenced the results because each time the obtained values were compared to the respec tive control ones. In serum, GPx and SOD activities vs. control were decreased GPx after 3 weeks and SOD after 6 weeks. In Cr group GPx increased during the intoxication while SOD decreased (Tab. 1). In spleen GPx nad SOD levels vs. control re mained unchanged except GPx after 6 weeks. The enzymes activities were also stable in Cr group (Tab. 2). In heart muscle both enzymes levels vs. con trol were enhanced during all the intoxication. Just as, in the case of serum the chromium administra tion caused the GPx increase and SOD decrease in Cr group (Tab. 3). In femoral muscle and brain only GPx activity vs. control changed decreased after 3 weeks and increased after 6 weeks. In Cr group GPx in femoral muscle was enhanced and SOD in brain depressed as a consequence of intoxication. In liver both enzymes activities vs. control were increased after 3 weeks. After 6 weeks GPx level was depressed and SOD unchanged. In kidney GPx level vs. control was increased after 3 weeks and unchanged after 6 weeks unlike SOD which was not altered after 3 weeks and enhanced after 6 weeks. Chromium oral administration affected mainly GPx level vs. control. SOD vs. control was not in fluenced in spleen, femoral muscle and brain, in serum and kidney changed not before 6 weeks whereas in liver was restored after 6 weeks. Discussion Susa et al. studied the effect of K 2 Cr 2 O 7 on rat hepatocytes. The GPx and SOD activities were de pressed significantly as a consequence of treatment [5]. In our experiment GPx level in liver vs. control decreased as a result of longer, 6 weeks intoxica tion. Chromium intragastral intoxication caused SOD activation in male rat s hepatocytes [4]. We observed increased SOD level vs. control in liver at the first part of the experiment. Table 1. GPx and SOD activity in serum of rats receiving Cr in drinking water Tabela 1. Aktywność GPx i SOD w surowicy krwi szczurów otrzymujących Cr w wodzie pitnej Group GPx (U/ml) SOD (U/ml) (Grupa) after 3 weeks after 6 weeks after 3 weeks after 6 weeks (po 3 tyg.) (po 6 tyg.) (po 3 tyg.) (po 6 tyg.) I Cr 500 ppm 6.6 ± 0.8 * 11.2 ± 1.5 a 43.1 ± ± 1.9 ** b II K 8.2 ± ± ± ± 3.5 Statistical significance vs. control: * p < 0.05, ** p < Statistical significance between Cr group after 3 weeks and Cr group after 6 weeks: a p < 0.001, b p < Istotność statystyczna względem grupy kontrolnej: * p < 0,05, ** p < 0,001. Istotność statystyczna między grupą otrzymującą Cr po 3 i 6 tygodniach: a p < 0,001, b p < 0,01.

4 398 M. KIEŁCZYKOWSKA et al. Table 2. GPx activity (U/g of protein) in tissues of rats receiving Cr in drinking water Tabela 2. Aktywność GPx (U/g białka) w tkankach szczurów otrzymujących Cr w wodzie pitnej Tissue I Cr 500 ppm I K (Narząd) after 3 weeks after 6 weeks after 3 weeks after 6 weeks (po 3 tyg.) (po 6 tyg.) (po 3 tyg.) (po 6 tyg.) Spleen ± ± 20.4*** ± ± 37.0 (Śledziona) Heart muscle ± 29.8*** ± 59.7** a ± ± 40.5 (Mięsień sercowy) Femoral muscle 23.6 ± 3.9* ± 15.5*** a 53.0 ± ± 6.0 (Mięsień uda) Brain 47.1 ± 12.1* 50.0 ± 9.7* 85.7 ± ± 9.7 (Mózg) Liver ± 45.6* ± 51.0** ± ± (Wątroba) Kidney ± 51.0*** ± ± ± 72.1 (Nerka) Statistical significance vs. control: * p < 0.05, ** p < 0.01, *** p < Statistical significance between Cr group after 3 weeks and Cr group after 6 weeks: a p < Istotność statystyczna względem grupy kontrolnej: * p < 0,05, ** p < 0,001, *** p < 0,001. Istotność statystyczna między grupą otrzymującą Cr po 3 i 6 tygodniach: p < Table 3. SOD activity (U/mg of protein) in tissues of rats receiving Cr in drinking water Tabela 3. Aktywność SOD (U/mg białka) w tkankach szczurów otrzymujących Cr w wodzie pitnej Tissue I Cr 500 ppm I K (Narząd) after 3 weeks after 6 weeks after 3 weeks after 6 weeks (po 3 tyg.) (po 6 tyg.) (po 3 tyg.) (po 6 tyg.) Spleen 3.5 ± ± ± ± 0.5 (Śledziona) Heart muscle 12.8 ± 2.0*** 9.2 ± 1.5* A 2.7 ± ± 0.8 (Mięsień sercowy) Femoral muscle 4.5 ± ± ± ± 1.0 (Mięsień uda) Brain 19.8 ± ± 0.5 a 23.4 ± ± 0.5 (Mózg) Liver 20.9 ± 4.4** 15.7 ± ± ± 4.6 (Wątroba) Kidney 13.4 ± ± 3.4* 17.9 ± ± 1.4 (Nerka) Statistical significance vs. control: * p < 0.05, ** p < 0.01, *** p < Statistical significance between Cr group after 3 weeks and Cr group after 6 weeks: a p < Istotność statystyczna względem grupy kontrolnej: * p < 0,05, ** p < 0,01, *** p < 0,001. Istotność statystyczna między grupą otrzymującą Cr po 3 i 6 tygodniach: p < 0,01. Anderson et al. undertook the studies on zinc and chromium supplementation effect on oxidative stress in patients with type 2 diabetes. RBC Cu Zn SOD and Se GPx were not changed [14]. In occupationally exposed chrome plating fac tory workers antioxidant enzymes (SOD, GPx, CAT) in blood were not changed [1]. In our stud ies GPx vs. control in serum was not altered in the second part of experiment (6 weeks) whereas SOD vs. control remained stable in the first part (3 weeks.) Gromadzińska et al. carried out the investiga tions on the erythrocyte and plasma GPx activity of tannery workers. They were divided into two groups, in the dependence on the degree of Cr exposure. Erythrocyte and plasma GPx was signi ficantly increased vs. control, non exposed group in the case of workers exposured to higher Cr con

5 The Chromium Effect on Antioxidant Barrier in Rats 399 centration in air. There was also statistically signi ficant difference between low and high Cr groups in the case of plasma GPx its level enhanced when Cr content in air increased [6]. In our studies GPx activity in serum vs. control at first decreased and as a consequence of longer, 6 weeks intoxica tion enhanced, but not significantly whereas long, occupational exposure lead to its increase. We al so observed that in Cr administered group GPx le vel increased during the experiment. References [1] Huang YL, Chen CY, Sheu JY, Chuang IC, Pan JH, Lin TH: Lipid peroxidation in workers exposed to hexa valent chromium. J Toxicol Environ Health A. 1999, 56, [2] Matés JM, Pérez Gómez C, Blanca M: Chemical and biological activity of free radical scavengers in allergic diseases. Clin Chim Acta 2000, 296, [3] Bagchi D, Hassoun EA, Bagchi M, Stohs SJ: Chromium induced excretion of urinary lipid metabolites, DNA damage, nitric oxide production, and generation of reactive oxygen species in Sprague Dawley rats. Comp Bio chem Physiol 1995, 110C, 2, [4] Karimov KI, Inoiatova FK, Inoiatov FS: Toxic effects of various water pollutants on structural and functional parameters of hepatocytes. Vopr Med Khim 2002, 48, 2, [5] Susa N, Ueno S, Furukawa Y, Ueda J, Sugiyama M: Potent Protective Effect of Melatonin on Chromium(VI) Induced DNA Single Strand Breaks, Cytotoxicity, and Lipid Peroxidation in Primary Cultures of Rats Hepatocy tes. Toxicol Appl Pharmacol 1997, 144, [6] Gromadzińska J, Wasowicz W, Skłodowska M, Bulikowski W, Rydzyński K: The Influence of Atmospheric Chromiun on Selenium Content and Glutatione Peroxidase Activity in Blood of Tannery Workers. Environ Health Perspect 1996, 104, 12, [7] Stohs SJ, Bagchi D, Hassoun E, Bagchi M: Oxidative mechanisms in the toxicity of chromium and cadmium ions. J Environ Pathol Toxicol Oncol 2000, 19, 3, [8] Domingo JL: Metal induced developmental toxicity in mammals: a review. J Toxicol Environ Health 1994, 42, 2, [9] Speich M, Pineau A, Ballereau F: Minerals, trace elements and related biological variables in athletes and du ring physical activity. Clin Chim Acta 2001, 312, [10] Floriańczyk B: Pierwiastki śladowe w cukrzycy. Post Med Klin Dośw 1996, 5, 4, [11] Bulikowski W: Zapobiegawcze działanie chlorku magnezu u garbarzy narażonych na chrom. Biul Magnezol 1996, 1, 2, 8, [12] Machartova V, Racek J, Kohout J, Senft V, Trefil L: Effect of antioxidant therapy on indicators of free radical activity in workers at risk of lead exposure. Vnitr Lek 2000, 46, 8, [13] Bradford MM: A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein Dye Binding. Anal Biochem, 1976, 72, [14] Anderson RA, Roussel AM, Zouari N, Mahjoub S, Matheau JM, Kerkeni A: Potential antioxidant effects of zinc and chromium supplementation in people with type 2 diabetes mellitus. J Am Coll Nutr 2001, 20, 3, Address for correspondence: Małgorzata Kiełczykowska Department of General Chemistry, Medical University of Lublin Staszica Lublin Poland Received: Revised: Accepted: Praca wpłynęła do Redakcji: r. Po recenzji: r. Zaakceptowano do druku: r.

6 IV Konferencja Sekcji Endokrynologii Molekularnej Polskiego Towarzystwa Endokrynologicznego Termin 2 3 października 2004 r. Miejsce obrad Sala Konferencyjna Muzeum Archeologicznego w Poznaniu Pałac Górków Poznań ul. Wodna 27 Program naukowy Wykłady plenarne prac oryginalnych z zakresu endokrynologii molekularnej (postępy w badaniach molekularno genetycznych endokrynopatii choroby tarczycy, przysadki, nadnerczy, przytarczyc, zaburzenia różnicowania i dojrzewania płci, otyłość, nadciśnienie tętnicze, cukrzyca, osteoporoza, zaburzenia gospodarki wapniowo fosforanowej, nowotwory gruczołów endokrynnych); diagnostyka molekularna; terapia genowa z rekombinowanymi hormonami. Organizator przewodnicząca Komitetu Organizacyjnego dr hab. med. Katarzyna Łącka tel. (61) kom fax: (61) e mail: K_Lacka@wp.pl; i_gradecka_kubik@op.pl