TAP CHi KHOA HOC VA CONG NGHE Tap 47, s6 6, 2009 Tr. 59-67 ANH HU'QNG CUA BUTACHLOR LEN HE THONG CHONG OXY HOA CUA cA TRAM CO {Ctenopharyngodon idellus) PHAN VAN TRi, TRAN THANH PHONG, DO QUY HAI 1. DAT VAN DE Str dung rong rai cac thuoc bao ve thuc vat (BVTV) 6' tren dong ruong dugc xem la mot trong nhirng yeu cau ca ban nham dam bao viec on dinh nang suat ctia cay trong. Ben canh cac mat tich cue, viec su' dung khong kiem soat cac hoa chat trir sau va diet co trong cac loai thuoc bao ve thuc vat gay ra nhirng tac hai cho moi sinh va anh huang den quan the dong vat a nuac. Dae biet, cac loai ca da chiu anh huang true tiep cua thuoc BVTV trong qua trinh san xuat. Cac hoa chat nay va cac chat chuyen hoa tii' chting gay doc doi vai ca the sinh vat c6 lien quan den stress oxy hoa (Ozcan et al. 2004). Cac thuoc BVTV co the gay ra stress oxy hoa dan den su tao thanh cac goc tir do va su bien doi trong cac chat chong oxy hoa hoac he thong enzim bat cac goc oxy tu do. Sir peroxide hoa lipid co ca che phan ttr lien quan vai dgc tinh dugc cam trng boi thuoc BVTV (Ozcan et al. 2004). Nhieu cong trinh nghien ctru da de cap den anh huong ctia cac hoa chat trir sau va diet co len sir thay doi hoat do enzim chong oxy hoa va cac chi so hoa sinh a ca (Crestani et al. 2007; Diana et al. 2006; Ozcan et al. 2004; Xionghai et al. 2007). Hoat tinh ctia cac enzim chong oxy hoa, trang thai glutation, mirc do ctia sir peroxide hoa lipid, su cam irng ctia cac isozyme, thuang dugc str dung nhu la cac marker sinh hoc de danh gia mirc do doc tinh ctia thuoc BVTV. Ket qua nghien ciru ctia chimg toi nham gop phan danh gia tac dong ciia butachlor len cac enzyme chong oxy hoa va cac chi so hoa sinh a cac mo, ca quan khac nhau ctia ca tram co. 2. NGUYEN LIEU VA PHtTONG PHAP NGHIEN CUtJ 2.1. Nguyen lieu nghien ciru Ca Tram co {Ctenopharyngodon idellus) da truong thanh, trong lugng ca khoang 800 - 1200 g, mua tai cac ho nuoi gia dinh va tai cac chg a Thira Thien Hue. Butachlor la chat diet co, thuoc nhom chat acetamide, dugc phep str dung tai Viet Nam. Cong thtrc hoa hgc la: C17H26CINO2 (N-Butoximetyl-2-clo-2,6-dietylacetanilide). Butachlor la thuoc doc bang III, LD50 qua mieng 3.300 mg/kg, LD50 qua da: 4080 mg/kg. Butachlor la thuoc trir CO ngi hap, co tac dung chon Igc, trii' co long vuc, duoi phung, chae, lac 2.2. Phu-o'ng phap nghien ciJu 2.2.1. Chudn bi mdu Truoc tien ca dugc nuoi on djnh trong nuoc khoang 24 gia. Tilp do xii' li voi butachlor a nong do 3,6 ppm va 1,8 ppm trong 24 gia. Ca sau khi xu li dugc tach cac mo va ca quan nhu gan, mang, ca trang, ca do, tim va lach dl sti' dung cho nghien cuu. 59 Mau (1 g) dugc nghiin va dong nhat bang each cho them 9 ml dung dich NaCI 0,62%o. Lay 0,5 ml djch miu dl do chi so peroxide hoa lipid. Phan con lai dugc li tam a 4°C, 17000 vong/phtit trong 10 phut, sau do diing djch trong de xac djnh boat do enzim superoxide dismutase (SOD), catalase (C-ase) va peroxidase (P-ase). MIU (1 g) dugc nghiin vai 9 ml trichloacetic acid (TCA) 5%, li tam a 5000 vong/phiit trong 5 phtit, lay phan dich de xac dinh ham lugng glutathion dang khti' (GSH). 2.2.2. Xdc dinh hogt do enzyme SOD Hoat do SOD dugc xac djnh dua vao u'c che su tao thanh adrenochrom, la san pham ctia adrenalin qua qua trinh tu oxy boa a pH = 10,2 va so mau a buac song 480 nm (Matkovics et al. 1977). 2.2.3. Xdc dinh hogt do enzyme C-ase •, • : ;; Hoat do C-ase dugc xac djnh theo phuong phap chuan do bang KMn04 0,1 N, dua vao lugng H2O2 bj phan hiiy boi enzim tao thanh H2O va O2 (Do Quy Hai 1986). 2.2.4. Xdc dinh hogt do enzyme P-ase Hoat do enzyme P-ase dugc xac djnh bang each do kha nang xtic tac ctia enzyme len toe do ciia phan irng chuyen boa nhua guaiacol thanh tetraguaiacol vai sir tham gia ctia H2O2 va so mau 6' buoc song 470 nm (Do Quy Hai 1986). 2.2.5. Xdc dmh hdm lucmg GSH Ham lugng GSH dugc xac djnh bang thuoc thtr Ellinan (Jozef, Raymond 1968), dua tren ca SO' cac nhom chira SH khong co ban chat protein phan irng voi 5,5-dithiobis-2 nitrobenzoic acid (DTNB) va so mau a buac song 412 nm. . , 2.2.6. Chi so peroxide hda lipid (LP) Chi so LP duoc xac djnh dua vao san pham trung gian malondialdehyde (MDA) dugc tao ra do su peroxide hoa lipid. Trong moi truong acid MDA tao thanh phuc mau hong ben vai ^id 2-thiobarbituric (TBA) va so mau a buoc song 532 nm (Placer et al. 1966). 2.3. Xir li so lieu H' Moi thi nghiem dugc lap lai 3 lan. Ket qua thi nghiem dugc xti' li thong ke dl tinh gia,tri trung binh va phan tich LSD voi p < 0,05 b5ng chuong trinh SAS. , -, •V 3. KET QUA VA THAO LUAN 3.1. Anh huong cua butachlor len cac enzyme chong oxy hoa Ket qua su thay doi boat do enzim chong oxy.hoa duai tac dung ctia butachlor a n6ng do 3,6 ppm va 1,8 ppm sau 24 gia xu li dugc trinh bay a cac hinh 1, 2 va 3. 60 3.2. Anh hu'bng ciia butachlor len SOD Sau 24 gia xtr li vai butachlor, hoat do SOD a mang, tim va lach deu giam. C» gan, ca trang va ca do hoat do nay tang a ning do thap ciia butachlor va giam a nong do cao. Nhu vay, a nong do cao (3,6 ppm), boat do SOD a cac mo deu giam so voi doi chu'ng (hinh 1). Theo nghien ciru ciia Xionghai va dong tac gia (2007), khi xti' li chat diet co alachlor len ca chep {Carassiiis auraliis) trong 60 ngay thi hoat tinh SOD a gan tang dan ciing vai nong do alachlor. Hoat tinh SOD tang nhe a nong do thap ciia alachlor (4-16 pg/l). Khi nong do alachlor tang, boat tinh SOD dirge tich luy co y nghTa va dat cue dai (tang 150% so voi doi chirng) a nong do 250 pg/l. Trong nghien cu'u ciia chiing toi, khi xti' li vai butachlor nong do 1,8 ppm, hoat do SOD tang a gan (20,6%), ca trang (129%)) va co' do (17,36%) so voi doi chiing. Hoat tinh SOD dugc cam u'ng co y nghTa a gan, ca trang va ca do, dan den sir tang cac goc superoxide 6' nong do butachlor 1,8 ppm. SOD xiic tac chuyen goc superoxide thanh HiOi va O2 va la enzim dau tien xii' ii goc oxy (Kappus 1985). Su ire che hoat tinh SOD a nong do 6 nhiem cao phii hgp vdi cong bo ciia Zhang va dong tac gia (2004). 16 . 14 c B 12 o p 10 Q o (f) c: <gj o- 4 TO- O X Mang Ca tring I Butachlor 3.6 ppm D Butachlor 1.8 ppm [H 06i chtrng Jl Gan IVlanq Catranq Co do Tim Lach Hinh I. Hoat do rieng cua enzyme SOD a cac mo ca tram co (LSDo,o5= 0,15) 3.3. Anh hu'd'ng cua butachlor len P-ase Xir li voi butachlor a ca hai nong do lam giam boat do P-ase a gan, tim va lach. Trong khi do, a mang, ca do va ca trang boat do P-ase tang so voi doi chirng voi butachlor nong do 1,8 ppm va giam khi xu' li a ning do 3,6 ppm (hinh 2). Nghien ciru ctia Do Quy Hai va Cap Xuan Tii (2000), cho thay duoi tac dpng cua thuoc trir sau monitor boat do peroxidase a mo gan, tim va than ciia chuot nhSt trJng {Mus musculus) deu giam so voi doi chting. 61 B o I CL CD E 3^ cu (/) 03 40 35 30 25 20 15 'm B Butachlor 3.6 ppm C Butachlor 1.8 ppm ^ Doi chtrng •^ 10 <o- •a o I u Gan Mang Ca trang t Co (36 Tim Lach Hinh 2. Hoat do rieng ciia enzim peroxidase a cac mo ca tram co (LSDQ.OS -0,14) 3.4. Anh hu'ong ciia butachlor len C-ase 0.7 c B "o i_ CL E OJ (/) ro 6 cn c <a) <o- •a "ro- o I 06 0.5 0,4 0.3 02 01 I LJ li • Butachlor 3.6 ppm D Butachlor 1.8 ppm • Doi chu'ng ry Gan Mang Ca tring Cc do Tim Lach Hinh 3. Hoat do rieng enzim catalase a cac mo ca tram co (LSDmis - 0.02) Tuang tu \6'\ P-ase, boat do C-ase a gan. tim va lach deu giam a ca hai nong do xti' li. vai nong dp butachlor cao hoat do C-ase giam manh. 6 mang va ca trang, boat do C-ase tang a nong 62 do thap va giam a nong do cao ciia butachlor. Trong khi do, hoat dp C-ase a ca do tang so voi doi chirng a ca 2 nong dp xtr li va dat gia trj Ion nhat a nong dp butachlor 1.8 ppm (hinh 3). Hoat tinh C-ase tang cao de phan u'ng lai su tieu thu oxy tang do tiem nang san xuat H2O2 Ion (Ritola et al. 2002). C-ase la mpt enzim cam ting bao ve he thong sinh hpc chong lai cac loai oxy phan trng (Romeo et al. 2000). Phan irng khac nhau ctia catalase dugc nhan thay a ca sau khi xtr li bang cac tac nhan moi truang ngoai. Hoat tinh ctia enzim nay cung tang len a gan, mang, tim, co' ciia ca C. carpio va Iclaluriis punclalus sau khi xii' li vai dichlorvos (Hai et al. 1997"). C-ase CO mat chii yeu a peroxisome, chju trach nhiem lam giam H2O2 tao thanh tir sir chuyen hoa cac acid beo ehuoi dai trong peroxisome (Winston et al. 1991). Hoat ti'nh ctia catalase nhay cam vai cac tac nhan gay 6 nhiem a gan ctia ca nuoc ngpt (Uner et al. 2001). Trong nghien ciru ciia chiing toi hoat tinh ciia C-ase a gan, tim va lach bj tre che a ca hai nong dp butachlor. Dieu nay co ie do su tich luy H2O2 lam hong C-ase. Theo ket qua nghien ciru ctia Crestani va dong tac gia (2007), su giam boat dp ciia enzim catalase da the hien a gan ca tre khi xti' li vai clomazone (0,5 hoac 1,0 mg/l) sau 12, 24 va 96 gia. Ngoai ra, sir phan img lai stress oxy hoa va tiem nang chong oxy boa ciia ca thay doi theo loai, moi truong song va dieu kien nuoi duong. Stress oxy hoa co the loai bo boat tinh ctia enzim chong oxy hoa, dan den thiet hai oxy va lam mat co' che den bii (Zhang et al. 2004). 0 cac mo nghien ciru, su tang hoac giam lioat dp enzim chong oxy hoa phu thuoc vao tirng loai mo va tiiy vao nong dp butachlor xir li. Sir giam hoat dp ciia enzim co the do tac nhan xu li da lam hiiy hoai enzim hoac lam enzim mat hoat tinh (Do Quy Hai, Cap Xuan Tii 2000). 3.5. Anh hu'ong ciia butachlor len mot so chi so hoa sinh Ket qua sir thay doi cac chi so hoa sinh duoi tac dung ciia hoat chat butachlor a iiai nong dp 3,60 ppm va 1,80 ppm sau 24 gia xir li dupc trinh bay a cac hinh 4 va 5. 3.6. Anh hu'ong ciia butachlor Ien ham luong GSH p 2.5 • Butachlor 3.6 ppm n Butachlor 1.8 ppm S Doi chtrng I CO 1.5 0.5 B Gan Mang Ca trang Cado Tim Lach Hmh 4. Ham lugng GSH a cac mo ca trim co (LSDo,o5=0,03) 63 Ham lugng GSH a nong dp 3,6 ppm deu tang so voi doi chirng a tat ca cac mo, ngoai trii' mang. Khi xtr li voi butachlor 3,6 ppm, ham lupng GSH dat gia trj Ion nhat a mo tim la 2,65 pM/g mau tuoi (tang 194,4%), tilp theo la mo gan dat 2,56 pM/g mau tuai (tang 82,85%o) (hinh 4). GSH la mpt hgp chat sulfliydryl khoi lugng phan ttr thap chti yeu co trong bao tuang, no hoat dgng nhu la mgt chat phan ung bao ve te bao chong lai nhieu tac nhan gay 6 nhiem. Vi vay, GSH la hang rao bao ve dau tien chong stress o.xy hoa. Theo nghien cu'u Xionghai va dong tac gia (2007), khi te bao tiep xiic vai tac nhan 6 nhiem nhu alachlor, thi tac nhan nay se bj loai bo do tiep xtic true tiep vai GSH hoac thong qua cac glutathione S-transferase (GST), lam giam nong dp GSH (Zhang et al. 2004). Ket qua nghien ctru ctia chting toi phii hgp voi nghien cuu ciia Xionghai va dong tac gia (2007). Trong qua trinh stress oxy hoa, ham lugng GSH co the tang nhu la mgt co' che thich nghi bang each tang tong hgp GSH (Diana et al. 2006). Tuy nhien, khi stress oxy hoa manh me, co the lam giam ham lugng GSH, ket qua lam hong ca che thich nghi (Zhang et al. 2004). Sir giam GSH lam giam kha nang bat goc tu do ctia te bao (Eiia et al. 2003) va co the lam tang mue dp nguy hiem ciia stress oxy boa do ket qua giam kha nang bao ve ctia te bao (Diana et al. 2006). Hai va dong tac gia (1997) nhan thay ham lugng GSH a gan va co' ctia ca chep da giam sau 24 gia xir li vai 1 va 5 mg/l dichlorvos (organophosphate) va gay hong su oxy boa. 3.7. Anh huong ciia butachlor len gia trj LP Chi so LP tang len a tat ca cac mo, ngoai trir mo gan a ca hai nong do (hinh 5). Gia trj peroxide hoa lipid cao nhat la 165,30 nM MDA/g mau ttro'i (tang 140,37%) a mo lach va dat 76,14 nM MDA/g mau tuai (tang 322,77%) a mo tim, khi xti' li vai butachlor 3,6 ppm (hinh 5). Dieu nay chirng to butachlor tac dpng manh len mang te bao nhung chua pha hiiy cau triic mang. ,-^ Z3 1<(U E cn ^ Q 2 S c 180 160 - 140 120 -• 100 80 , D. 60 O 40 20 • Butachlor 3.6 ppm n Butachlor 1.8 ppm mi D6i chtrng fhih Gan Mang Catrang Ca do Tim Hinh 5. Chi s6 LP a cac mo ca trim co (LSDo,o5= 3,41) Lach 64 Theo nghien ciru cua Diana va ddng tac gia (2006), anh huang ctia thuoc trii' sau co nguon goc lan hiru ca la methyl parathion 2 mg/l da cam irng lam tang gia trj peroxide hoa lipid a ca trang va mang ciia ca Bryon cephalus sau 96 gia xu' li. Trong khi a gan, gia trj peroxide hoa lipid thay doi khong co y nghTa. Theo Ozcan va dong tac gia (2004), thuoc trir sau co the tao ra stress oxy hoa dan den su hinh thanh cac goc tu do va gay ra su peroxide boa lipid, phan anh boat tinh bao ve ctia enzim chong oxy boa. Khi danh gia anh huang ctia cac thuoc BVTV khac nhau len su peroxide hoa lipid va hoat tinh ctia enzim chong oxy hoa a cac mo khac nhau ctia ca cho thay mac dau co mpt so khac biet trong phan ting giua cac loai ca, nhung phan trng thich nghi da dugc quan sat va ca che chung ctia phan irng thich nghi la su cam ung cua enzim chong oxy boa. Phan irng chong oxy hoa cung da dugc nhan thay la khac nhau trong so cac mo. Ket qua tuang tu cung da dugc cong bo 6' cac loai ca khac (Radi, Matkovics 1988; Winston, Di Giulio 1991). Butachlor tac dpng manh len he enzim chong oxy hoa a nong dp thap (1,8 ppm) lam cho boat dp cac enzim deu giam a tat ca cac mo, trong khi gia trj ctia chi so LP tang len. 0 nong dp cao (3,6 ppm) cua butachlor, co su tang hoac giam hoat do ciia cac enzim chong oxy hoa trong qua trinh chong chju vai su tac dgng ctia thuoc. Dieu nay cho thay butachlor a nong dp 3,6 ppm co tac dung dpc len tat ca cac mo, rieng a mo tim va lach nong dp 1,8 ppm cung da bat dau gay dpc. 4. KET LUAN Enzim SOD, P-ase, C-ase a cac mo deu nhay cam, thay doi boat dp chong lai tac dpng dpc hai cua butachlor a ca hai nong dp 3,6 ppm va 1,8 ppm. Butachlor a nong dp 3,6 ppm va 1,8 ppm co tac dpng xau len mang te bao thong qua sir gia tang gia trj peroxide boa lipid. Ca hai nong dp butachlor 3,6 ppm va 1,8 ppm co tac dung gay dpc ro rang 6' mo tim va lach. TAI LIEU THAM KHAO 1. 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Q. - Effects of chronic exposure of 2,4-dichlorophenol on the antioxidant system in liver of freshwater fish Carassius auratus. Chemosphere 55 (2) (2004) 167-174. 66 SUMMARY EFFECTS OF BUTACHLOR ON ANTIOXIDANT SYSTEM OF GRASS CARP {Ctenopharyngodon idellus) Pesticides has been widely used in agriculture all over the world. They cause contamination of natural aquatic environment, that can affect the organism, particularly fish. In this paper, the biochemical perturbations of grass carp {Ctenopharyngodon idellus) exposed to butachlor at different concentrations (1.8 ppm and 3.6 ppm) over 24 hours were investigated. After exposure, fish liver, gill, white muscle, red muscle, heart and pancreas were excised and used for experiments. The activity of superoxide dismutase (SOD), catalase (CAT), the content of glutathione (GSH), and lipid peroxidation were determined. Our result showed that SOD, P-ase and C-ase were all sensitive and changed their antioxidant activities against pesticide toxicity. These enzymes could be induced by a slight oxidative stress due to compensatory response; however, a severe oxidative stress suppressed the activities of these enzymes due to oxidative damage and the loss of compensatory mechanisms. Butachlor may enhance the lipid peroxidation value by direct interaction with the cell membrane. These changes reflected that the antioxidant systems of the tested fishes were affected. The present work demonstrated that butachlor induced oxidative stress in Ctenopharyngodon idellus. It is evident that the use of butachlor in agriculture and aquaculture must be carefully evaluated. Our results suggested that the parameters analyzed could be good biomarkers of exposure to oxidative stress caused by butachlor. Keyvi'ords. Antioxidant enzyme, butachlor, Ctenopharyngodon idellus, lipid peroxidation, oxidative stress. Dia chi: Nhgn hdi ngdy 9 thdng 4 ndm 2009 Truong dai hpc Khoa hpc, Dai hpc Hue. 67 . 3.1. Anh huong cua butachlor len cac enzyme chong oxy hoa Ket qua su thay doi boat do enzim chong oxy. hoa duai tac dung ctia butachlor a n6ng do. stress oxy hoa (Ozcan et al. 2004). Cac thuoc BVTV co the gay ra stress oxy hoa dan den su tao thanh cac goc tir do va su bien doi trong cac chat chong oxy