2009 4 4 516-523 Asian Journal of Ecotoxicology Vol 4, 2009 No 4, 516-523 a GST 1 2 1 * 3 1 1 1 361005 2 116023 3 361021 a B a P B a P Sparus macrocephalus -S- GST 7 - EROD GST B a P 3 - - a 3 -OH B a P 3 B a P - B a P 05 10 20 50μg L -1 GST 2d 12h 1d 2d GST B a P R 12h =0966 p<005 R 1d = 0953 p<005 R 2d =0824 p<005 B a P 05 10 20 50μg L -1 7d 3 -OH B a P B a P - R=0943 p<005 GST EROD 3 -OH B a P R GST =0740 p <005 R EROD =0839 p <005 20μg L -1 B a P EROD GST 3 -OH B a P EROD GST B a P 3 GST EROD B a P -S- 7- - 3- a 1673-5897 2009 4-516 - 08 X1715 A Effects of Benzo a pyrene Exposure on Hepatic GST Activity in Black Porgy Sparus macrocephalus and Variation Relationships with Hepatic Metabolic Enzymes and Biliary Metabolites MU Jing-li 1 2 WANG Xin-hong 1 * LIN Jian-qing 3 LIN Xiao-long 1 HONG Hua -sheng 1 1 State Key Laboratory of Marine Environmental Science, Environmental Science Research Center, Xiamen University, Xiamen 361005 2 National Marine Environmental Monitoring Center, Dalian 116023 3 College of Bioengineering, Jimei University, Xiamen 361021 Received 13 December 2008 accepted 4 January 2009 Abstract Effects of benzo a pyrene B a P exposure on the activity of glutathione S -transferase GST in black porgy Sparus macrocephalus liver was studied The dose and time response of two enzymes 7 -ethoxyresorufin O -deethylase EROD and GST involved in B a P metabolism and the amounts of B a P metabolites excreted into the bile were investigated Results showed that the time -response trend on of induction of hepatic GST of black porgy was bell -shaped with a plateau at B a P exposure for 2 days A significant dose -related increase of the GST activity in liver was observed at the exposure time of 12h, 1d and 2d R 12h =0966, p<005; R 1d =09529, p<005; R 2d =0824, p<005 Compared with our former study, the induction of hepatic EROD, GST activities and biliary 3 -OH B a P of black porgy were significantly induced by B a P 05, 10, 20 and 50μg L -1 after exposure for 7 days and a significant dose -response relationship between 3 -OH B a P and B a P was observed R=0943, p<005 After exposed to 20μg L -1 B a P, similar variation trends of hepatic EROD, GST activities and biliary 3-OH B a P had been observed in black porgy, but those variations were not consistency with the time change completely Those results demonstrated that the variation between hepatic metabolic enzymes and biliary metabolites were very complex The exposure concentrations and exposure time were the most important factors which influence the variations of metabolic enzymes and metabolites Keywords metabolites glutathione S -transferase 7 -ethoxyresorufin O -deethylase metabolic mechanism bile 3 -OH B a P Sparus macrocephalus 2008-12 - 13 2009-01 - 04 863 No 2007AA09Z126 No 40106012 40476048 20777060 No 200805090 No 2008613 No 200712 1979 * Corresponding author E-mail: xhwang@xmueducn
4 a GST 517 1 Introduction a Benzo a pyrene B a P EROD Polycyclic aromatic PAHs 2007 hydrocarbons PAHs Sebastiscus marmoralus B a P Conney, 1982 Nacci et al, 2002 3 B a P B a P EROD B a P B a P B a P P4501A CYP1A B a P Sparus macrocephalus B a P Little et al, 1984 Melikian et al, 1989 Flowers et al, 1996 Gao et al, 2005 7-7 - ethoxyresorufin O -deethylase EROD CYP1A1-2002b B a P EROD EROD B a P 3 - - CYP4501A1 B a P a 3 -OH B a P 3 - Goks 覬 r and F 覬 lin, 1992 Buhler and Wang -Buhler, 1998 Whyte et al, 2000 -S - Glutathione S - transferase GST Glutathione B a P GSH B a P Habig 2 Materials and methods et al, 1974 Hasspieler et al, 1994 Van Schanke et al, 2001 B a P 21 HPLC B a P Ruddock et al, 2002 Hosnedl et al, 2003 Zhu et al, 2008 EROD GST B a P >96% Sigma -Aldrich PAHs 4 05 10 20 50μg L -1 Gagnon and Holdway, 2002 1 1 50 2L 2h 6h 12h 1d 2d 4d Gravato and Santos, 2002 Gagnon and Holdway, 2002 2007 Klummp 2002b EROD GST 14d 1- - 1 -OH Py PAHs 7d Gorbi Regoli 2004 EROD Fuentes -Rios 2005 05 50μg L -1 56d EROD GST EROD GST 3 2003 Klumpp et al, 2002a GST GST PAHs B a P 912±13 cm 1120±15 g 115cm 63cm 50cm 7d 14d 6
518 4 22 3 Results and analysis 30±15 mg 15mL ph=76 31 B a P GST 2min -4 B a P GST 10000r min -1 20min EROD 1 GST GST GST Habig 1974 - GST 14d 01mL CDNB 175mL 2,4-37 1min GST 025mL 37 5min GSH GST 37 1min 1cm GSH B a P 340nm A1 37 3min A2 GST = A2 -A1 21875/Δt GST Bradford Stegeman and Lech, 1991 Hasspieler et al, 1994 Bradford, 1976 EROD B a P GST 2007 2009 23 ± SPSS Pearson 2d B a P B a P 4d GST GSH B a P GST 2004 B a P 5μg L -1 110 50μg L -1 B a P GST ANOVA Tukey
4 a GST 519 5μg L -1 B a P 1d GST 32 EROD GST 3 -OH B a P 7d B a P EROD B B a P 3 -OH B a P a P - GST EROD GST 3-OH B a P 3 B a P 12h 1d 2d 2 B a P R 12h =0966 p <005 R 1d =0953 p <005 R 2d =0824 p<005 B a P 321 EROD GST 3 -OH B a P 3 GST B a P B a P - GST Hepatic GST activity/ nmol min -1 mg -1 140 120 100 80 60 40 20 12h 茛 1d 2d 茛 茛 茛 茛 0 1 2 3 4 5 6 B a P B a P concentration/ μg L -1 茛 2007 2009 B a P CYP450 CYP1A B a P a -7,8 - -9,10 - BPDE B a P 3 -OH B a P 9 - - a 9 -OH B a P GSH Little et al, 1984 Willett et al, 2000 EROD CYP1A B a P 2 GST B a P GST a R 12h=0966, p<005; R 1d=0952, p<005; R 2d=0824, p<005 a GSH Fig2 Variation of GST activity in liver of black porgy Habig et al, 1974 Kirby et al, 1999 exposed to B a P R 12h=0966, p<005; R 1d=0952, p<005; R 2d=0824, p<005 3 -OH B a P B a P - GST 7d 3 -OH B a P Van der - 0943 p<005 Oost 2003 43 39 3 EROD GST 33% 48% GST GST B a P GST 7d EROD GST 3 -OH B a P 0839 p<005 GST 0 740 p < 0 05 4 B a P
520 4 B a P EROD PAHs Van Schanke Gorbi Regoli 2004 2001 Limanda limanda B a P Anguilla anguilla PAHs 7d B a P B a P EROD EROD GST B a P PAHs 3 B a P B a P EROD GST EROD B a P PAHs EROD GST EROD Wang 2008 PAHs B a P B a P B a P - 3 -OH B a P B a P B a P CYP1A B a P B a P PAHs 3-OH B a P 3-OH B a P concentration/ ng μl -1 05 04 03 02 01 EROD Hepatic EROD activity/ pmol min -1 mg -1 800 600 400 200 3-OH B a P EROD GST 00 0 0 0 05 1 2 5 B a P B a P concentration/ μg L -1 3 B a P 7d EROD GST 3-OH B a P B a P - Fig3 Dose-effect relationships between B a P exposure concentrations and EROD, GST activities and 3-OH B a P concentration after exposure for 7 days 100 80 60 40 20 GST Hepatic GST activity/ nmol min -1 mg -1 Log EROD 30 28 26 24 22 EROD GST 20 165-25 -20-15 -10-05 00 days units of 3-OH B a P, GST, EROD are ng μl -1, pmol min -1 mg -1, nmol min -1 mg-1, respectively Log 3-OH B a P 4 B a P 7d EROD GST 3-OH B a P 3-OH B a P GST EROD ng μl -1 pmol min -1 mg-1 nmol min -1 mg-1 Fig4 The correlativity between hepatic EROD, GST activities and 3-OH B a P concentration after B a P exposure for 7 190 185 180 175 170 Log GST 322 EROD GST 3 -OH B a P 3-20μg L -1 B a P EROD GST 3-OH B a P 3 5 EROD GST 3 -OH B a P 2d GST EROD 3 -OH B a P 7d 3 - B a P CYP1A B a P B a P GST B a P 3 -OH B a P GSH
4 a GST 521 B a P B a P B a P B a P GSH GSH Habig et al, 1974 Conney, 1982 Whyte et al, 2000 B a P 2d B a P PAHs GST 3-OH B a P 5 EROD B a P GST - B a P B a P B a P Willett 2000 Ictalurus Punctatus B a P Dicentrarchus labrax 012μmol L -1 B a P B a P 3d GST EROD GST B a P B a P EROD GST GST B a P 4h 8h 16h 2007 3 Gravato Santos 05μg L -1 B a P 8 GST Gravato Santos 2003 2003 4h EROD 3-OH B a P 3-OH B a P concentration/ ng μl -1 04 03 02 01 00 3-OH B a P a EROD GST 2h 6h 12h 1d 2d 4d 7d 14d 21d Time 1000 800 600 400 200 0 EROD Hepatic EROD activity/ pmol min -1 mg -1 120 100 80 60 40 20 GST Hepatic GST activity/ nmol min -1 mg -1 5 Fig5 3-OH B a P 3-OH B a P concentration/ ng μl -1 04 03 02 01 3-OH B a P EROD GST b 00 2h 6h 12h 1d 2d 4d 7d 14d 21d 0 20 Time 20μg L -1 B a P EROD GST 3-OH B a P a: 20μg L -1 b: Changes of hepatic EROD, GST activities and 3-OH B a P concentration with time under the exposure of 20μg L -1 B a P a: 20μg L -1 B a P; b: acetone control 1000 800 600 400 200 EROD Hepatic EROD activity/ pmol min -1 mg -1 120 100 80 60 40 GST Hepatic GST activity/ nmol min -1 mg -1
522 4 8h GST 12 : 4875-4917 B a P 16h B a P B a P dehydrogenase J 20μg L -1 B a P EROD GST 3 -OH B a P 3 Gravato 7d 20μg L -1 EROD GST 3 -OH B a P Pollution Bulletin, 44 3 : 230-237 20μg L -1 B a P Y, Smith E C A, Lebwohl M, Wei H 2005 Benzo a P4501A J Free Radical Biology and Medicine, 39 9 : 1177-1183 aquatic toxicology and environmental monitoring J B a P EROD GST 3 -OH B a P B a P Reseach, 58 2-5 : 511-515 3 B a P EROD GST B a P PAHs Ecotoxicology and Environmental Safety, 55 3 : 352-358 PAHs EROD Hasspieler B M, Behar J V, Digiulio R T 1994 Glutathione - 1969 dependent defense in channel catfish Ictalurus punctatus and brown bullhead Ameriurus nebulosus J Ecotoxicology and Environmental Safety, 28 1 : 82-90 References Bradford M M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein -dye binding J Analytical Biochemistry, 72: 248-254 Buhler D R, Wang -Buhler J L 1998 Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring J Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology, 121 1-3 : 107-137 Conney A H 1982 Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: G H A Clowes Memorial Lecture J Cancer Research, 42 Flowers L, Bleczinski W, Burczynski M E, Harvey R G, Penning T M 1996 Dispositon and biological activity of benzo a pyrene - 7,8 -dione A genotoxic metabolite generated by dihydrodiol Biochemistry, 35 42 : 13664-13672 Fuentes -Rios D, Orrego R, Rudolph A, Mendoza G, Gavilá J F, Barra R 2005 EROD activity and biliary fluorescence in Schroederichthys chilensis Guichenot 1848 : Biomarkers of PAH exposure in coastal environments of the South Pacific Ocean J Chemosphere, 61 2 : 192-199 Gagnon M M, Holdway D A 2002 EROD activity, serum SDH and PAH biliary metabolites in sand flathead Platycephalus bassensis collected in Port Phillip Bay, Australia J Marine Gao D, Luo Y, Guevara D, Wang Y, Rui M, Goldwyn B, Lu pyrene and its metabolites combined with ultraviolet A synergistically induce 8 -hydroxy -2 -deoxyguanosine via reactive oxygen species Goks 覬 r A, F 覬 lin L 1992 The cytochrome P -450 system in fish, Aquatic Toxicology, 22 4 : 287-311 Gorbi S, Regoli F 2004 Induction of cytochrome P4501A and biliary PAH metabolites in European eel Anguilla anguilla Seasonal, dose -and time -response J Marine Environmental Gravato C, Santos M A 2002 Liver phase and phase enzymatic induction and genotoxic responses of beta naphthoflavone water -exposed sea bass J Ecotoxicology and Environmental Safety, 52 1 : 62-68 Gravato C, Santos M A 2003 Genotoxicity biomarkers association with B a P biotransformation in Dicentrarchus labrax L J Habig W H, Pabst M J, Jakoby W B 1974 Glutathione S - Transferases The first enzymetic step in mercapturic acid formation J Biological chemical, 249 22 : 7130-7139 Hosnedl T, Kocourek V, Tomaniová M, Volka K 2003 1 - Hydroxypyrene as a biomarker for fish exposure to polycyclic aromatic hydrocarbons J Bulletin of Environmental Contained and Toxicological, 71 3 : 465-472 Kirby M F, Matthiessen P, Neall P, Tylor T, Allchin C R, Kelly C A, Maxwell D L, Thain J E 1999 Hepatic EROD activity in flounder Platichthys flesus as an indicator of contaminant exposure in English estuaries J Marine Pollution Bulletin, 38 8 : 676-686 Klumpp D W, Hong H S, Humphrey C, Wang X H, Codi S 2002a Toxic contaminants and their biological effects in coastal waters of Xiamen, China I Organic pollutants in mussel and
4 a GST 523 fish tissues J Marine Pollution Bulletin, 44 8 : 752-760 Klumpp D W, Humphrey C, Hong H S, Feng T 2002b Toxic contaminants and their biological effects in coastal waters of Xiamen, China Biomarkers and embryo malformation rates as indicators of pollution stress in fish J Marine Pollution Bulletin, 44 8 : 761-769 Little P J, James M O, Pritchard J B, Bend J R 1984 Benzo a pyrene metabolism in hepatic microsomes from feral and 3 - methycholanthrene-treated southern flounder, Paralichthys lethostigma J Journal of Environmental Pathology, Toxicology and Oncology, 5 4-5 : 309-320 Melikian A A, Bagheri K, Hecht S S, Hoffmann D 1989 Metabolism of benzo a pyrene and 7 beta, 8 alpha dihydroxy -9 alpha,10 alpha -epoxy -7,8,9,10 -tetrahydrobenzo a pyrene in lung and liver of newborn mice J Chemico - Biological Interactions, 69 2-3 : 245-257 Mu J L, Wang X H, Lin J Q 2007 Dose- and time -dependent formation of 3 -hydroxy -benzo a pyrene in the bile of black porgy Sparus macrocephalus exposed to benzo a pyrene J Environmental Chemical, 26 4 : 487-490 in Chinese Mu J L, Wang X H, Wang S H, Lin J Q, Wang K J, Hong H S 2009 Study on the kinetics of hepatic EROD activities in black porgy Sparusmacro cephalus exposed to benzo a pyrene J Marine Science, 33 1 : 68-71 in Chinese Nacci D E, Kohan M, Pelletier M, George E 2002 Effects of benzo a pyrene exposure on a fish population resistant to the toxic effects of dioxin -like compounds J Aquatic Toxicology, 57 4 : 203-215 Ruddock P J, Bird D J, McCalley D V 2002 Bile metabolites of polycyclic aromatic hydrocarbons in three species of fish from the Severn Estuary J Ecotoxicological Environmental Safety, 51 2 : 97-105 Stegeman J J, Lech J J 1991 Cytochrome P -450 monooxygenase systems in aquatic species: carcinogen metabolism and biomarkers for carcinogen and pollutant exposure J Environmental Health Perspectives, 90: 101-109 Van der Oost R, Beyer J, Vermuelen N P E 2003 Fish bioaccumulation and biomarkers in environmental risk assessment: a review J Environmental Toxicology and Pharmacology, 13: 57-149 Van Schanke A, Holtz F, Van der Meer J P, Boon J P, Ariese F, Stroomberg G, Van den Berg M, Everaarts J M 2001 Dose - and time-dependent formation of biliary benzo a pyrene metabolites in the marine flatfish dab Limanda limanda J Environmental Toxicology and Chemistry, 20 8 : 1641-1647 Wang C G, Zheng Y X, Zheng W Y, Yu Q 2004 The effect of exposure of benzo a pyrene, pyrene and their mixture on hepatic glutathione -S -transferase activity in Mugil so -iuy J Marine Science, 28 3 : 40-43 in Chinese Wang Y, Zhang R H, Zuo Z H, Chen Y X, Wang C G 2008 Relation of hepatic EROD activity and cytochrome P4501A level in Sebastiscus marmoratus exposed to benzo a pyrene J Journal of Environmental Sciences, 20: 101-104 Whyte J J, Jung R E, Schmitt C J, Tillitt D E 2000 Ethoxyresorufin -O -deethylase EROD activity in fish as a biomarker of chemical exposure J Critical Reviews in Toxicology, 30 4 : 347-570 Willett K L, Gardinali P R, Lienesch L A, Di Giulio R T 2000 Comparative metabolism and excretion of benzo a pyrene in 2 species of ictalurid catfish J Toxicological Sciences, 58 1 : 68-76 Zheng R H, Zhao Y, Chen Y X, Zuo Z H, Wang C G 2007 Effects of tributyl tin on the metabolism of benzo a pyrene in Sebastiscus marmoratus J Acta Oceanologica Sinica, 29 4 : 165-169 in Chinese Zhu S, Li L, Thornton C, Carvalho P, Avery B A, Willett K L 2008 Simultaneous determination of benzo a pyrene and eight of its metabolites in Fundulus heteroclitus bile using ultra - performance liquid chromatography with mass spectrometry J Journal of Chromatography B, 863 1 : 141-149 Zhu X S, Meng F P, Yang Z X 2003 Comparative study on sensitivities of brain acetylcholinesterase from five marine fishes to two organophosphorus pesticides J Shanghai Environmental Science, 27 3 : 567-571 in Chinese,, 2007 Sparus macrocephalus a 3 - - - J, 26 4 : 487-490,,,,, 2009 a EROD J, 33 1 : 68-71,,, 2004 a J, 28 3 : 40-43,,,, 2007 Sebastiscus marmoratus a J, 29 4 : 165-169,, 2003 5 AChE 2 J, 27 3 : 567-571