母源性BDE-209对子代大鼠关节软骨发育的影响
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摘要
目的:以Wistar大鼠为模型,研究母源性暴露于BDE-209下,对子代大鼠关节软骨及发育的影响。
方法:出生21d的SPF级Wistar大鼠母鼠20只,随机分成实验组与对照组各10只,实验组母鼠胃灌BDE-209造成孕前孕期的暴露,对照组胃灌等量的纯花生油,其间母鼠成年自然交配,同时两组胃灌持续至母鼠分娩前。收集仔鼠,自然哺乳生长至出生后21天(断乳日),每组随机选取20只仔鼠测量其身长,体质量,处死仔鼠取右侧膝关节,常规HE染色,观察组织形态学的改变并测量关节软骨骺板软骨厚度,行关节软骨TUNEL凋亡检测软骨细胞凋亡情况,免疫组化法进行关节软骨2型胶原染色,观察其胶原表达情况。
结果:实验收获的两组仔鼠中未见明显外观畸形,仔鼠出生后发育至断乳日,实验组仔鼠平均身长17.55±1.58cm与对照组仔鼠平均身长19.60±1.09cm相比较短,差异有统计学意义(P=0.000＜0.01),平均体质量34.65±6.15g与对照组33.77±5.81g相比,无显著差异性(P=0.646＞0.05)。实验组仔鼠关节软骨的骺板软骨平均厚度331.53±31.83um,与对照组相282.02±21.10um比,具有显著差异性(p=0.000＜0.01),实验组仔鼠关节软骨的软骨细胞表层凋亡率2.05±1.10,中层凋亡率24.05±5.73均大于对照组的表层1.25±0.91,中层13.65±3.01,其中表层具有统计学差异(P=0.017＜0.05),而中层具有显著性差异性(P=0.000＜0.01),而在关节软骨的深层,实验组软骨细胞凋亡率4.25±3.39小于对照组深层7.50±4.75,差异有统计学意义(P=0.017＜0.05)。与此同时,对照组关节软骨2型胶原表层表达率0.216±0.058,中层表达率0.204±0.057均大于实验组的表层0.166±0.045,中层0.169±0.038,差异有统计学意义(P=0.004＜0.01;P=0.027＜0.05),在软骨的深层,实验组0.163±0.049与对照组0.151±0.049相比,无明显差异性(P=0.445＞0.05)。
结论:母代接触BDE-209可造成子代大鼠身材发育矮小,骺板软骨的异常增厚,关节软骨2型胶原表达和关节软骨细胞凋亡的异常,可能会导致软骨发育障碍,矿化不良,骨化不良的结果。
Objective:To evaluate the effect of BDE-209(brominated diphenyl ethers-209) on the offspring's development of the articular cartilage(AC) through the female wistar rats exposed before pregnancy.
Methods:Twenty female wistar rats were divided into two groups which include ten rats in each groups random.Peanut oil suspensions of BDE-209 was given in doses of 1500mg/kg.d by oral gavage for the experiment group,the control group was given the same capacity of pure peanut oil at the same condition.After thirty days,the female with the male rats at a proper propotion,affirm the delivery and then end of oral gravage. 21 days later,measure the body length of neonatal rats and recording.The neonatal rats were killed and knee joint were obtained,HE staining and detecting the thickness of growth plate cartilage(GPC).Chondrocyte apoptosis was detected by teminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL) method,The expression of typeⅡcollagen were stained by immunohistochemistry.
Results:No significant was observed between the two groups in appearance teratism of the neonatal rats.The average body length in experiment group(17.55±1.58cm),the average body length in control group(19.60±1.09cm) There was significant different between the two groups in the of the neonatal rats(P=0.000＜0.01) after 21 days(P＜0.05),The average body quality in experiment group(33.77±5.81g),The average body quality in control group(34.65±6.15g),There was no significant different between the two groups(P=0.646＞0.05).the thickness of growth plate cartilage in experiment group(331.53±31.83um) was larger than the control group (282.02±21.10um)(P=0.000＜0.01).In the superficial and middle zone of articular cartilage,the positive percentages of chondrocyte apoptosis stained of experiment group(superficial zone 2.05±1.10;middle zone 24.05±5.73) were higherthan the control group(superficial zone 1.25±0.91;middle zone 13.65±3.01)(superficial zone P=0.017＜0.05;middle zone P=0.000＜0.01),but ine the deep zone of articular cartilage,the positive percentages of chondrocyte apoptosis was decrease and there was different between groups(P=0.017＜0.05)(experiment group4.25±3.39; control group 7.50±4.75),at the same time,in the superficial and middle zone of articular cartilage,the The expression of typeⅡcollagen in control group(superficial zone0.216±0.058;middle zone0.204±0.057) was higher than the experiment group(superficial zone 0.166±0.045;middle zone 0.169±0.038)(superficial P=0.004＜0.01;middle P=0.027＜0.05),in the deep zone,the experiment group(0.163±0.049),the control group (0.151±0.049),ther were no different obviousily(P=0.445＞0.05).
Conclusion:The maternal BDE-209 exposure during the pregnancy lead to the body length of neonatal rats decrease and the chondrocytes in proliferate zones and hypertrophic zones of growth plate cartilage(GPC) in limbs are excessively obstacle result in osteomalacia.
方法:出生21d的SPF级Wistar大鼠母鼠20只,随机分成实验组与对照组各10只,实验组母鼠胃灌BDE-209造成孕前孕期的暴露,对照组胃灌等量的纯花生油,其间母鼠成年自然交配,同时两组胃灌持续至母鼠分娩前。收集仔鼠,自然哺乳生长至出生后21天(断乳日),每组随机选取20只仔鼠测量其身长,体质量,处死仔鼠取右侧膝关节,常规HE染色,观察组织形态学的改变并测量关节软骨骺板软骨厚度,行关节软骨TUNEL凋亡检测软骨细胞凋亡情况,免疫组化法进行关节软骨2型胶原染色,观察其胶原表达情况。
结果:实验收获的两组仔鼠中未见明显外观畸形,仔鼠出生后发育至断乳日,实验组仔鼠平均身长17.55±1.58cm与对照组仔鼠平均身长19.60±1.09cm相比较短,差异有统计学意义(P=0.000＜0.01),平均体质量34.65±6.15g与对照组33.77±5.81g相比,无显著差异性(P=0.646＞0.05)。实验组仔鼠关节软骨的骺板软骨平均厚度331.53±31.83um,与对照组相282.02±21.10um比,具有显著差异性(p=0.000＜0.01),实验组仔鼠关节软骨的软骨细胞表层凋亡率2.05±1.10,中层凋亡率24.05±5.73均大于对照组的表层1.25±0.91,中层13.65±3.01,其中表层具有统计学差异(P=0.017＜0.05),而中层具有显著性差异性(P=0.000＜0.01),而在关节软骨的深层,实验组软骨细胞凋亡率4.25±3.39小于对照组深层7.50±4.75,差异有统计学意义(P=0.017＜0.05)。与此同时,对照组关节软骨2型胶原表层表达率0.216±0.058,中层表达率0.204±0.057均大于实验组的表层0.166±0.045,中层0.169±0.038,差异有统计学意义(P=0.004＜0.01;P=0.027＜0.05),在软骨的深层,实验组0.163±0.049与对照组0.151±0.049相比,无明显差异性(P=0.445＞0.05)。
结论:母代接触BDE-209可造成子代大鼠身材发育矮小,骺板软骨的异常增厚,关节软骨2型胶原表达和关节软骨细胞凋亡的异常,可能会导致软骨发育障碍,矿化不良,骨化不良的结果。
Objective:To evaluate the effect of BDE-209(brominated diphenyl ethers-209) on the offspring's development of the articular cartilage(AC) through the female wistar rats exposed before pregnancy.
Methods:Twenty female wistar rats were divided into two groups which include ten rats in each groups random.Peanut oil suspensions of BDE-209 was given in doses of 1500mg/kg.d by oral gavage for the experiment group,the control group was given the same capacity of pure peanut oil at the same condition.After thirty days,the female with the male rats at a proper propotion,affirm the delivery and then end of oral gravage. 21 days later,measure the body length of neonatal rats and recording.The neonatal rats were killed and knee joint were obtained,HE staining and detecting the thickness of growth plate cartilage(GPC).Chondrocyte apoptosis was detected by teminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL) method,The expression of typeⅡcollagen were stained by immunohistochemistry.
Results:No significant was observed between the two groups in appearance teratism of the neonatal rats.The average body length in experiment group(17.55±1.58cm),the average body length in control group(19.60±1.09cm) There was significant different between the two groups in the of the neonatal rats(P=0.000＜0.01) after 21 days(P＜0.05),The average body quality in experiment group(33.77±5.81g),The average body quality in control group(34.65±6.15g),There was no significant different between the two groups(P=0.646＞0.05).the thickness of growth plate cartilage in experiment group(331.53±31.83um) was larger than the control group (282.02±21.10um)(P=0.000＜0.01).In the superficial and middle zone of articular cartilage,the positive percentages of chondrocyte apoptosis stained of experiment group(superficial zone 2.05±1.10;middle zone 24.05±5.73) were higherthan the control group(superficial zone 1.25±0.91;middle zone 13.65±3.01)(superficial zone P=0.017＜0.05;middle zone P=0.000＜0.01),but ine the deep zone of articular cartilage,the positive percentages of chondrocyte apoptosis was decrease and there was different between groups(P=0.017＜0.05)(experiment group4.25±3.39; control group 7.50±4.75),at the same time,in the superficial and middle zone of articular cartilage,the The expression of typeⅡcollagen in control group(superficial zone0.216±0.058;middle zone0.204±0.057) was higher than the experiment group(superficial zone 0.166±0.045;middle zone 0.169±0.038)(superficial P=0.004＜0.01;middle P=0.027＜0.05),in the deep zone,the experiment group(0.163±0.049),the control group (0.151±0.049),ther were no different obviousily(P=0.445＞0.05).
Conclusion:The maternal BDE-209 exposure during the pregnancy lead to the body length of neonatal rats decrease and the chondrocytes in proliferate zones and hypertrophic zones of growth plate cartilage(GPC) in limbs are excessively obstacle result in osteomalacia.
引文
1 De Wit CA,An overview of brominated flame retardants in the environment,Chemosphere,2002;46:583-624
2 Andersson,O,Blomkvist,G.Polybrominated aromatic pollutants found in fish in Sweden.Chemosphere 1981,10,1051-1060
3 Legler J,Brouwer A.Are brominated flame retardants endocrine disruptors?Environ,Int,2003;29;879-85
4 AlaeeM,Arias P,Sjodin A,Bergman A.An overview of commercially used brominated flame retardants and their applications,changes in their use pattern in different countries/regions over time and possible modes of release.Environ Int 2003;29:638-89
5 Meironyte Guvenius D,Bergman A,Noren K Polybrominated diphenyl ethers in Swedish human liver and adipose tissue Arch Environ Contam,Toxicol.2001 40(4):564-70
6 Wania,F.,Dugani,C.B.,Assessing the long-range transport potential of polybrominated diphenyl ethers:a comparison of four multimedia models.Environ.Toxicol.Chem.2003,22,1252-1261
7 Ciparis,S.,Hale,R.C.,Bioavailability of polybrominated diphenyl ether flame retardants in biosolids and spiked sediment to the aquatic oligochaete,Lumbriculus variegatus.Environ.Toxicol.Chem.2005,24,916-925
8 Domingo,J.L.,Bocio,A.,Falco,G.,Llobet,J.M.,Exposure to PBDEs and PCDEs associated with the consumption of edible marine species.Environ.Sci.Technol.2006,40,4394-4399
9 Hamers, Timo; Kamstra, Jorke H; Sonneveld, Edwin; Murk, Albertinka J; Kester, Monique H A; Andersson, Patrik L; Legler, Juliette; Brouwer, Abraham;In vitro profiling of the endocrine-disrupting potency of brominated flame retardants;Toxicol Sci 2006 ,92 (1): 157-73
10 Darnerud PO, Aune M, Larsson L, Hallgren S.Plasma PBDE and thyroxine levels in rats exposed to Bromkal or BDE-47. Chemosphere. 2007,67(9):S386-92
11 Henrik Viberg, Anders Fredriksson, Eva Jakobsson ,Ulrika Orn,and Per Eriksson ; Neurobehavioral Derangements in Adult Mice Receiving Decabrominated Diphenyl Ether (PBDE 209) during a Defined Period of Neonatal Brain Development; Toxicological Sciences 2003, 76, 112-120
12 Lebeuf, Michel; Couillard, Catherine M; Legare, Benoit; Trottier, Steve ; Effects of deBDE and PCB-126 on hepatic concentrations of PBDEs and methoxy-PBDEs in Atlantic tomcod.; Environ Sci Technol 2006, 40 (10): 3211-6
13 Sean C . Lema , Irvin R . Schultzb John P. Incardona, Penny Nathaniel L. Scholza Swansona; Neural defects and cardiac arrhythmia in fish larvae following embryonic exposure to 2 , 2 ', 4 ,4 '-tetra bromo diphenyl ether ( PBDE- 47 ); Aquatic Toxicology ,2007, 82: 296 -307
14 Zuurbier, Moniek; Leijs, Marike; Schoeters, Greet; Ten Tusscher, Gavin; Koppe, Janna G; Children's exposure to polybrominated diphenyl ethers. Acta Paediatr Suppl 2006, 95 (453): 65-70
15 Watanabe ,Tatsukawa R, Formation of brominated dibenzofurans from the photolysis of flame retardant deca bromobiphnyl ether in hexane solution by UV and sunlight [J]. Bull Environ Contam Toxicol,1987,39:953-59
16 陈敦金,余琳,廖秦平,屈委月;母源性BDE2209胃灌后对子鼠学习记忆能力的影响以及血清BDE2209浓度的测定,中华围产医学杂志,2006;9(6):412-415
17 周俊,陈敦金,余艳红;孕期、哺乳期暴露十溴联苯醚对子代大鼠免疫功能的影响,南方医科大学学报,2006,26(6)738-739
18 Sohail Khattak,Guiti K-Moghtader,Kristen McMartin,Maru Barrera,Debbie Kennedy,Gideon Koren;Organic solvent exposure during pregnancy and congenital malformations—a prospective controlled study;JAMA.1999;281:1106-1109
19 Hardingham TE;Proteoglycans:their structure,interactions,and molecular organization in cartilage.Biochemical Society Transactions (1981);9(6);489-97
20 Michael A,Cremer,Edward F,Rosloniec Andrew H,Kang;The cartilage collagens:are view of their structure,organization,and role in the pathogenesis of experimental arthritis in animals and in human rheumatic disease.J Mol Med,1998,76:275-288
21 CamachoIA,SinghN,HegdeVL,et al Treatment of mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to aryl hydrocarbon receptor dependent nuclear translocation of NF-kappaB and expression of Fas ligand in thymic stromal cell sandconsequent apoptosis in Tcells [J].JImmunol,2005,175(1):90-103
22RudolfSchleyerbach,VincentC,Hascall;Structureofarticularcartilage[A].edite d by KlansE.Kuettner.Workshop Conference Hoechst-Werk Albert Wiesbaden[C].Munich,1985.4-10
23 申洪.免疫组织化学染色定量方法研究(Ⅲ)[J].中国组织化学与细胞杂志,1995,4(1):89-921
24 Mazdai A,Dodder NG,Abernathy MP,Hites HA,Bigsby RM,Polybrominated diphenyl ethers in maternal and fetal blood samples,Environ Health Perspect 2003;111:1249-52
25 Darnerud PO,Aune M,Larsson L,Hallgren S.Plasma PBDE and thyroxine levels in rats exposed to Bromkal or BDE-47.Chemosphere.2007,67(9):S386-92
26 A.Sjodin,L.Hagmar,E.Klasson-Wehler,J.Bjork and A.Bergman,Influence of the consumption of fatty Baltic Sea fish on plasma.WHO,Environmental Health Criteria 162.Brominated diphenyl ethers.International Program on Chemical Safety(IPCS),Switzerland (1994)
27Weiss C,Mirow S.An Ultrastructural study of osteoarthritic changes in the articular cartilage of human knees,J Bone Joint Surg Am,1972,54:954
28 Hashimoto S,Ochs RL,Komiya S,et al.Link age of chondrocyte apoptosis and cartilage degradation in human osteoarthritis,Arthritis Rheum,1998,41:1632
29 Blanco FJ,Guitian R,Vazquez-Martul E,etal.Osteoarthritis chondrocytes die by apoptosis.Arthritis Rheum,1998,41:284
30 Kim HA,Lee YJ,Seong SC,Choe KW,Song YW;apoptotic chondrocyte death in human osteoarthritis;J Rheumatol.2000;27(2):455-62
31 H T Kimb,M Y Lo,R Pillarisetty;Chondrocyte apoptosis following intraarticular fracture in humans;Osteoarthritis and Cartilage.2002,10(9):747-749
32 Cao L,Lee V,Adams ME,Kiani C,Zhang Y,Hu W,Yang BB;beta-Integrin-collagen interaction reduces chondrocyte apoptosis;Matrix Biology;1999,18(4):343-55
33ZemmyoM,MeharraEJ,KuhnK,CreightonAchermarmL,LotzM.Accelerated,a ging-dependent development of osteoarthritis in alphal integrin-deficient mice.Arthritis Rheum 2003,48(10):2873-80
2 Andersson,O,Blomkvist,G.Polybrominated aromatic pollutants found in fish in Sweden.Chemosphere 1981,10,1051-1060
3 Legler J,Brouwer A.Are brominated flame retardants endocrine disruptors?Environ,Int,2003;29;879-85
4 AlaeeM,Arias P,Sjodin A,Bergman A.An overview of commercially used brominated flame retardants and their applications,changes in their use pattern in different countries/regions over time and possible modes of release.Environ Int 2003;29:638-89
5 Meironyte Guvenius D,Bergman A,Noren K Polybrominated diphenyl ethers in Swedish human liver and adipose tissue Arch Environ Contam,Toxicol.2001 40(4):564-70
6 Wania,F.,Dugani,C.B.,Assessing the long-range transport potential of polybrominated diphenyl ethers:a comparison of four multimedia models.Environ.Toxicol.Chem.2003,22,1252-1261
7 Ciparis,S.,Hale,R.C.,Bioavailability of polybrominated diphenyl ether flame retardants in biosolids and spiked sediment to the aquatic oligochaete,Lumbriculus variegatus.Environ.Toxicol.Chem.2005,24,916-925
8 Domingo,J.L.,Bocio,A.,Falco,G.,Llobet,J.M.,Exposure to PBDEs and PCDEs associated with the consumption of edible marine species.Environ.Sci.Technol.2006,40,4394-4399
9 Hamers, Timo; Kamstra, Jorke H; Sonneveld, Edwin; Murk, Albertinka J; Kester, Monique H A; Andersson, Patrik L; Legler, Juliette; Brouwer, Abraham;In vitro profiling of the endocrine-disrupting potency of brominated flame retardants;Toxicol Sci 2006 ,92 (1): 157-73
10 Darnerud PO, Aune M, Larsson L, Hallgren S.Plasma PBDE and thyroxine levels in rats exposed to Bromkal or BDE-47. Chemosphere. 2007,67(9):S386-92
11 Henrik Viberg, Anders Fredriksson, Eva Jakobsson ,Ulrika Orn,and Per Eriksson ; Neurobehavioral Derangements in Adult Mice Receiving Decabrominated Diphenyl Ether (PBDE 209) during a Defined Period of Neonatal Brain Development; Toxicological Sciences 2003, 76, 112-120
12 Lebeuf, Michel; Couillard, Catherine M; Legare, Benoit; Trottier, Steve ; Effects of deBDE and PCB-126 on hepatic concentrations of PBDEs and methoxy-PBDEs in Atlantic tomcod.; Environ Sci Technol 2006, 40 (10): 3211-6
13 Sean C . Lema , Irvin R . Schultzb John P. Incardona, Penny Nathaniel L. Scholza Swansona; Neural defects and cardiac arrhythmia in fish larvae following embryonic exposure to 2 , 2 ', 4 ,4 '-tetra bromo diphenyl ether ( PBDE- 47 ); Aquatic Toxicology ,2007, 82: 296 -307
14 Zuurbier, Moniek; Leijs, Marike; Schoeters, Greet; Ten Tusscher, Gavin; Koppe, Janna G; Children's exposure to polybrominated diphenyl ethers. Acta Paediatr Suppl 2006, 95 (453): 65-70
15 Watanabe ,Tatsukawa R, Formation of brominated dibenzofurans from the photolysis of flame retardant deca bromobiphnyl ether in hexane solution by UV and sunlight [J]. Bull Environ Contam Toxicol,1987,39:953-59
16 陈敦金,余琳,廖秦平,屈委月;母源性BDE2209胃灌后对子鼠学习记忆能力的影响以及血清BDE2209浓度的测定,中华围产医学杂志,2006;9(6):412-415
17 周俊,陈敦金,余艳红;孕期、哺乳期暴露十溴联苯醚对子代大鼠免疫功能的影响,南方医科大学学报,2006,26(6)738-739
18 Sohail Khattak,Guiti K-Moghtader,Kristen McMartin,Maru Barrera,Debbie Kennedy,Gideon Koren;Organic solvent exposure during pregnancy and congenital malformations—a prospective controlled study;JAMA.1999;281:1106-1109
19 Hardingham TE;Proteoglycans:their structure,interactions,and molecular organization in cartilage.Biochemical Society Transactions (1981);9(6);489-97
20 Michael A,Cremer,Edward F,Rosloniec Andrew H,Kang;The cartilage collagens:are view of their structure,organization,and role in the pathogenesis of experimental arthritis in animals and in human rheumatic disease.J Mol Med,1998,76:275-288
21 CamachoIA,SinghN,HegdeVL,et al Treatment of mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to aryl hydrocarbon receptor dependent nuclear translocation of NF-kappaB and expression of Fas ligand in thymic stromal cell sandconsequent apoptosis in Tcells [J].JImmunol,2005,175(1):90-103
22RudolfSchleyerbach,VincentC,Hascall;Structureofarticularcartilage[A].edite d by KlansE.Kuettner.Workshop Conference Hoechst-Werk Albert Wiesbaden[C].Munich,1985.4-10
23 申洪.免疫组织化学染色定量方法研究(Ⅲ)[J].中国组织化学与细胞杂志,1995,4(1):89-921
24 Mazdai A,Dodder NG,Abernathy MP,Hites HA,Bigsby RM,Polybrominated diphenyl ethers in maternal and fetal blood samples,Environ Health Perspect 2003;111:1249-52
25 Darnerud PO,Aune M,Larsson L,Hallgren S.Plasma PBDE and thyroxine levels in rats exposed to Bromkal or BDE-47.Chemosphere.2007,67(9):S386-92
26 A.Sjodin,L.Hagmar,E.Klasson-Wehler,J.Bjork and A.Bergman,Influence of the consumption of fatty Baltic Sea fish on plasma.WHO,Environmental Health Criteria 162.Brominated diphenyl ethers.International Program on Chemical Safety(IPCS),Switzerland (1994)
27Weiss C,Mirow S.An Ultrastructural study of osteoarthritic changes in the articular cartilage of human knees,J Bone Joint Surg Am,1972,54:954
28 Hashimoto S,Ochs RL,Komiya S,et al.Link age of chondrocyte apoptosis and cartilage degradation in human osteoarthritis,Arthritis Rheum,1998,41:1632
29 Blanco FJ,Guitian R,Vazquez-Martul E,etal.Osteoarthritis chondrocytes die by apoptosis.Arthritis Rheum,1998,41:284
30 Kim HA,Lee YJ,Seong SC,Choe KW,Song YW;apoptotic chondrocyte death in human osteoarthritis;J Rheumatol.2000;27(2):455-62
31 H T Kimb,M Y Lo,R Pillarisetty;Chondrocyte apoptosis following intraarticular fracture in humans;Osteoarthritis and Cartilage.2002,10(9):747-749
32 Cao L,Lee V,Adams ME,Kiani C,Zhang Y,Hu W,Yang BB;beta-Integrin-collagen interaction reduces chondrocyte apoptosis;Matrix Biology;1999,18(4):343-55
33ZemmyoM,MeharraEJ,KuhnK,CreightonAchermarmL,LotzM.Accelerated,a ging-dependent development of osteoarthritis in alphal integrin-deficient mice.Arthritis Rheum 2003,48(10):2873-80