Research on the Metabolism of Nitrogenous Free Radicals in Ducklings with Selenium poisoning

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1 Research o the Metabolism of Nitrogeous Free Radicals i Duckligs with Seleium poisoig Rui Wu, Cheg Xia, Hogyou Zhag College of Aimal Sciece ad Techology, Heilogjiag August First Lad Reclamatio Uiversity, Daqig, Heilogjiag ,Chia; Telephoe: or or ; Fax: , fuhewu@126.com or wwrr1967@sohu.com Abstract:The duckligs (=100) were divided ito cotrol group ad experimetal group radomly, ad seleium poisoig of duckligs was artificially caused by feedig ratio cotaiig 8 mg kg -1 Se every day. The dyamic chages of No cotet ad NOS activity i the serum ad tissues were determied by meas of the method of itric acid reductase. The results showed that the NO cotet ad NOS activity i serum ad tissues i experimetal group icreased sigificatly (P<0.05) ad they were time-depedet. It was suggested that the high level of seleium i bodies could icrease the NOS activity ad NO cotet as it destroyed the metabolism of material ad eergy as well as structure ad fuctio of tissues ad cells. These chages of NO cotet ad NOS activity might be ivolved i the metabolism of seleium poisoig. [The Joural of America Sciece. 2005;1(2):77-81]. Key words:seleium poisoig;ducklig;;tissue;nitric oxide;nitric oxide sythase Itroductio Seleium, oe kid of chemical elemet with atomic umber 34 ad atomic weight 78.96, was first foud by Swede chemist Berzelius i 1817, ad existed widely i the ature with four kids of oxidatio states such as 2,0,4 ad 6. It, oe of essetial trace elemets aimal required, owed chemical property betwee ometal ad metal ature ad existed i the cell of aimals ad plats [1]. Owig to its differet levels ad forms i soil ad plats, it ofte caused seleium deficiecy ad poisoig i farm aimals. I the seleium-deficiet areas, seleium poisoig ofte occurred because of supplemet of excessive seleium i the ratios to prevet or treat seleium deficiecy. At preset, there were may research reports o seleium poisoig of farm aimals at home ad abroad, maily o two aspects, seleium distributio ad chage of path-morphology i tissues [2,3], but there was o report o the effect of seleium poisoig o the level of itroge oxide (NO) ad Nitric Oxide Sythase (NOS) activity. Healthy ducklig were selected to study the effect of seleium poisoig o the level of NO ad NOS activity i the experimet, ad our aim was to elucidate the molecular mechaism of seleium poisoig ad to set up the basis for prevetig ad treatig cliical seleium poisoig. 1 Materials ad Methods 1.1 Materials Aimals ad ratios Oe-day-old healthy Jidig duckligs (female =50,male =50) were purchased from Zhegli duck farm i Harbi. The ratios cotaiig 0.15±0.023 mg kg -1 seleium were bought from Heilogjiag Dasheg Feed Corporatio Drugs ad istrumets Sodium seleate(na 2 SeO 3 5H 2 O), produced by Secod Reaget Factory i Sheyag; Nitroge Oxide (NO) reaget box, Nitric Oxide Sythase (NOS) reaget box ad Komasiliagla protei reaget box, produced by Jiacheg Bioegieerig Research Istitute i Najig; 7230G visible light spectrometer, made i Shaghai Aalyses Istrumet Factory; DF200A complete automatic aalysis scales, made i Chagshu Weighig Apparatus Factory i Jiagsu; Avati TM 30 low-temperature hypervelocity cetrifuge, made i Becma Corporatio, Germay. 1.2 Methods Groupig ad feedig of duckligs Oe-day-old healthy duckligs (=100) were ad libitum fed with ratio ad water, ad radomly grouped ito two (=50, respectively) after two weeks. The umber of female ad male duckligs i each group was equal. The duckligs i cotrol group (Group I) 77

2 were fed with full ratio, but the duckligs i poisoig group (Group II) were fed with full ratio cotaiig 8 mg kg -1 seleium. The duckligs i two groups were slaughtered ad collected at day 15,30,60 ad 90 (=10, respectively) Collectio ad determiatio of samples [ 4 ] 1 ml heart blood was collected ad disposed for a while i room temperature, ad serum was fially separated. Meawhile, the duckligs were slaughtered ad their heart, liver, kidey, splee, cerebrum, testis ad muscle were collected, weighed ad placed i cold salie (1:9 W/V), ad quickly grided ito 10% homogeate i ice-water by glass homogeizer, Homogeate was cetrifuged at rpm, the NO cotet ad NOS activity i serum ad tissues ad protei cotet i upper liquid of homogeate were measured, respectively. NO cotet (μmol l -1 i serum, μmol g -1 Pr i tissue)was measured with itric acid reductase; NOS activity (U mg -1 Pr) by spectrometer ad protei cotet i homogeate by Komasiliagla method Aalysis of data Data i the experimet were aalyzed by SAS software. 2 Results 2.1 Model replicatio of seleium poisoig The duckligs i Group Ⅱ had o sigificat sig i the early period of experimet, ad there was o sigificat weight differece betwee two groups at day 15 (P>0.05), but the weight of duckligs i Group Ⅰ was sigificatly higher tha that i Group Ⅱ at day 30 (P<0.05), ad after day 60 (P<0.01). The duckligs i Group II had cliical sigs: listlessess, disorderly ad deciduous hair, cryig ad brayig, white ad black maure, i later period paralysis, death owig to loss of deglutitio. There were o marked typical chages with eyes i the early period, but the chroic iflammatio occurred i the tissues, especially i liver ad kidey. I additio, there appeared hyperemia, hemorrhage ad edema i cerebrum, cerebral membrae ad alimetary tracts, ad eve extesive ulceratio or ecrosis i severe patiets. The results showed that the model replicatio of seleium poisoig i the experimet was successful because there existed 2.5 mg L -1 seleium i blood ad mg kg -1 seleium i feather. 2.2 NO cotet ad NOS activity i the serum ad tissues of duckligs with seleium poisoig The results i Table 1-2 showed that NO cotet i serum of duckligs i Group Ⅱ was higher or sigificatly higher tha that i cotrol group at day 15 (P<0.01), at day 30, 60 ad 90 (P<0.05); NO cotet i heart ad testis of duckligs i Group Ⅱ was sigificatly higher tha that i cotrol group at day 15,30,60 ad 90 (P<0.01); NO cotet i liver of duckligs i Group Ⅱ was sigificatly higher tha that i cotrol group at day 15 (P<0.05), 30,60 AND 90 (P<0.01); NO cotet i kidey of duckligs i Group Ⅱ was sigificatly higher tha that i cotrol group at day 15 ad 30 (P<0.05),60 ad 90 (P<0.01); NO cotet i splee ad cerebrum of duckligs i Group Ⅱ was sigificatly higher tha that i cotrol group at day 15 ad 30, 60 ad 90 (P<0.05); NO cotet i muscle i Group Ⅱ was sigificatly higher tha that i cotrol group at day 15, 60 ad 90 (P<0.05). The results i Table 3-4 showed that there was o sigificat NOS activity differece i serum of duckligs betwee Group Ⅱ ad cotrol group at day 15,30,60 ad 90 (P>0.05); NOS activity i heart of duckligs i Group Ⅱ was sigificatly higher tha that i cotrol group at day 30,60 ad 90 (P<0.05); NOS activity i liver splee, cerebrum, testis ad muscle of duckligs i Group Ⅱ was sigificatly higher tha that i cotrol group at day 15,30,60 ad 90 (P<0.01); NOS activity i kidey of duckligs i Group Ⅱ was higher tha that i cotrol group at day 15 ad 30 (P<0.05), at day 60 ad 90 (P<0.01). Table 1. The cotets of NO i serum ad tissue-homogeate i each group of 15,30-day old 1) Heart Cotrol group Poisoig group 15d 30d 15d 30d ± ± ± ** 56.84±6.400 * ± ± ±5.201** 58.00±6.834** 78

3 Liver ± ± ±5.238* 79.25±8.366** Kidey ± ± ±3.201* 32.56±4.158* Splee ± ± ±4.302* 44.96±5.362* Cerebrum ± ± ±8.654 * 79.56±9.015 * Testis ± ± ±5.685 ** 59.20±7.018 ** Muscle ± ± ±9.987 * 76.26± ) * mea P<0.05 o the same day,** mea P<0.01 o the same day,the same as below. Table 2. The cotets of NO i serum ad tissue-homogeate i each group of 60,90-day old Cotrol group Poisoig group 60d 90d 60d 90d ± ± ±5.000 * 49.45±5.641 * Heart ± ± ±6.982 ** 61.25±8.010 ** Liver ± ± ±9.260 ** 97.56± ** Kidey ± ± ±5.298 ** 50.21±6.325 ** Splee ± ± ±6.301 * 68.25±8.300 * Cerebrum ± ± ±9.563 * 94.58± * Testis ± ± ±9.265 ** 95.00± ** Muscle ± ± ± * 97.26± * Table 3. The activity of NOS i serum ad tissue-homogeate i each group of 15, 30-day old Heart Liver Kidey Splee Cotrol group Poisoig group 15d 30d 15d 30d ± ± ± ± ± ± ± ±0.710 * ± ± ±0.621 ** ±1.023 ** ± ± ±0.421 * 4.326±0.510 * ± ± ±0.210 ** 1.541±0.164 ** 79

4 Cerebrum ± ± ±0.021 ** 2.651±0.026 ** Testis ± ± ±0.203 ** 0.684±0.053 ** Muscle ± ± ±0.210 ** 4.650±0.521 ** Table 4. The activity of NOS i serum ad tissue-homogeate i each group of 60, 90-day old Cotrol group Poisoig group 60d 90d 60d 90d ± ± ± ±0.029 Heart ± ± ±0.710 * 7.984±0.752 * Liver ± ± ±0.900 ** ±2.013 ** Kidey ± ± ±0.510 ** 7.952±0.640 ** Splee ± ± ±0.098 ** 1.589±0.087 ** Cerebrum ± ± ±0.362 ** 3.295±0.042 ** Testis ± ± ±0.214 ** 2.950±0.032 ** Muscle ± ± ±0.654 ** 8.258±0.400 ** 3 Aalysis ad Discussio NO is oe kid of small molecule gas ad typical free radical with low solubility i water ad high solubility i lipid, which ca diffuse easily through the cell membrae.its chemical reactio is very strog as it possesses extra electro. Its biological half-life spa is 3 to 5s. NO is oe of products from which L-Arg is decompoud ito L-Cit uder the catalysis of NOS with the help of NADPH, haemochrome ad calmoduli [5,6]. NOS is the rate-limitig ezyme of the reactio, ad it ca be divided ito three types, euro NOS (NOS), edothelial NOS (enos) ad iducible NOS (inos), accordig to its source ad effect property [6]. NO i body has double-side effect. It has importat physiological effects whe NO metabolism is ormal ad its cocetratio is low (pmol -1 or fmolp -1 ), but it has importat pathological effects whe NO metabolism is disordered ad its cocetratio is high (moll -1 ). It ca damage cells ad destroy iformatio trasmissio [7,8]. The results i the experimet showed that NOS activity i serum rose slightly, but NOS activity i tissues rose markedly i poisoig group as compared with oes i cotrol group, which suggested that the high level of seleium was able to iduce ehacemet i NOS activity i serum, heart, liver, kidey, splee, cerebrum, testis ad muscle, amog which the chage i liver was the most otable, ext i cerebrum, splee, testis, muscle, kidey ad heart, i spite of isigificat differece i NOS activity i serum at day 15,30,60 ad 90,ad i heart at day 15,ad it was also higher as compared with oe i cotrol group. The results showed that excess of seleium could cause ehacemet i NOS activity i serum ad tissues, ad the icrease i NO cotet. Because NOS was the major rate-limitig factor, the chage i NO cotet could be cosistet with chage i NOS activity. It was aalyzed that the chages i NOS activity ad NO cotet resulted probably from two factors. Fist, seleium poisoig resulted i damage to DNA by active oxyge iduced by complex reactio of seleium compoud [9]. As mrna was oe importat adjustmet locatio i NOS activity, it was able to cause the chage i NOS activity ad damage to DNA, which led to gee mutatio ad cacer. It was reported that sigle chai i DNA i huma TK6 mature lymphocyte broke after it cotacted NO (5.5-22mmolL -1 ), ad the coditio became more severe with icrease of dosage ad 80

5 legtheig of time [10]. Secod, the seleium poisoig led to disorder of ormal biochemical fuctio of cell, ad amog which the calcium metabolic disorder was most importat because Ca 2+ was essetial to keep NOS activity. Whe aimal was i the coditio of low calcium, inos tightly combied with calmoduli, causig the opeig of calcium chael ad trasmissio of biological electrical curret from flavoprotei to protoheme which resulted i the productio of NOS, ad the productio of a large umber of NO. The excess of NO cotet i tissues had pathological effect o all orgas such as digestive system, cardiovascular system, ervous system ad urogeital system etc, resultig i damage to cell structure ad fuctio. The ustable measuremet of NOS activity i cotrol group resulted probably from the fault i the experimet. The chage of NOS activity i liver was most apparet, therefore, the pathological damage i liver was the severest because the liver was oe of major target orgas damaged by the seleium poisoig. Moreover, the chage of NOS activity ad NO cotet i cerebrum suggested that seleium poisoig iflueced the fuctio of cetral ervous system. It was reported that NO i cetral ervous system promoted the release of some materials, icludig the acetylcholie ad γ-amio butyrate from dopamie etc, i the superoxgitrite form, ad these trasmitters had importat ifluece o the fuctio the cetral ervous system [11]. Therefore, it was iferred that the chage of NOS activity ad NO cotet i the cerebrum resulted i fuctioal abormality of acetylcholie erve owig probably to its effect o the release of acetylcholie erve trasmitters, ad fially led to the cliical typical erve symptom. As metioed above, toxicological mechaism of seleium aouced by way of research of NOS activity ad NO cotet was complete ew research iterest. It had a importat part i the prevetio ad treatmet of the disease, ad i the ew developmet of medicie. The molecular biology research of NOS, from ow o, was ivolved i localizatio, extractio purificatio, classificatio, aalysis of gee aligmet, determiatio of NOS mrna, ad cloig of NOS gee, ad trasmissio of gee i cell by way of the carrier, whose aim was to cure some disease etc. Correspodece to: Rui Wu College of Aimal Sciece ad Techology Heilogjiag August First Lad Reclamatio Uiversity Daqig, Heilogjiag ,Chia Telephoe: or or Fax: fuhewu@126.com or wwrr1967@sohu.com Refereces: [1] Li JX, Zhag GD, Ge XL. Forecast ad Evirometal Character of Geography Chemistry i huma with Seleium Deficiecy ad surplus[m]. Geology Press, Beijig, Chia. 2000:1 3 (i Chiese). [2] Li GQ, Wag JH, Chao GR. Study o the Cliic ad Pathology of Seleium Poisoig i Milk Goats. Acta of Northwester Agricultural Uiversity. 1994;22(1): [3] Hou JW, Qi ZY, Liu BF. The Variatio of Seleium cotet i Wool, Blood ad Tissues After Poisoed by Feedig Natural Seleiferous Feed Stuffus to Goats. Acta of Veteriary ad Zootechology Sciece. 1994;25(5): [4] Yag Q, Zhuag ZJ, Che H. Study of NO ad NOS i hippocampus of Rats with Iodie Deficiecy. Chiese Joural of Edeiology. 2001;20(1):9 10. [5] Wu R, Kag SL, Guo BM. Biology Character of Free Redical ad Apoptosis, Ier Mogolia Joural of Aimal Scieces ad Productio. 2001;22(2): [6] Wu YK, Zhag LS, Zhu P. Progress of Nitric Oxide i Biological Medicie. Chiese Joural of Veteriary Sciece.2001;21(5): [7] Kowles RG, Palacipos M, Palmer RMJ. Formatio of itric oxide from L-argiie i the cetral ervous system: A trasductio mechaism for stimulatio of the soluble guaylate cyclase. Process Natural Academic sciece USA. 1989;86: [8] Nuokawa Y, Ishida N, Taaka S, Promoter aalysis of huma iducible itric oxide sythase gee associated with cardiovascular homeostasis. Biochemistry Biophysics Research Commuicatio. 1994;200(2): [9] Weier CP, Liazsoai I, Baylis SA. Iductio of calcium-depedet itric oxide sythases by sex hormoes. Process Natural Academic sciece USA. 1994;91(11): [10] Guye T, Burso D, Crespi CL, et al. DNA damage ad mutatio i huma cells exposed to itric oxide i vitro. Process Natural Academic sciece USA. 1992;89: [11] Kuriyama K, Ohkuma S. Role of itric oxide i cetral syaptic trasmitter release. Joural Pharamacol [Japa]. 1995;69:1 81