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1.
Grass carp(Ctenopharyngodon idella) is one of the most important species in China.Decabromodiphenyl ethane(DBDPE) is a brominated flame retardant that has been used widely in industry, and has been observed to accumulate in the tissues of fish from South China. Evidence has shown that DBDPE is toxic to aquatic animals, but the molecular response has been unclear. MicroRNAs(miRNAs) are small noncoding and negative regulatory RNAs that are 20–24 nucleotides in length, which are involved in a wide range of biological processes. We took advantage of deep-sequencing techniques to accurately and comprehensively profile the kidney mi RNA expression of grass carp after 8 weeks of oral exposure to DBDPE. After mapping sequencing data to the genome and Expressed Sequence Tags(ESTs) of grass carp, we identified 493 miRNAs in the sequenced grass carp samples, which included 51 new miRNAs. The results indicated that 5 miRNAs were significantly down-regulated and 36 miRNAs were significantly up-regulated(FDR 0.001,1.5-fold change) after DBDPE exposure. Real-time quantitative PCR(RT-q PCR) was performed on 4 miRNAs from the two samples, and the sequencing and RT-q PCR data were consistent. This study provides the first comprehensive identification of grass carp miRNAs, and the first expression analysis of grass carp miRNAs following DBDPE exposure.The results indicated that miRNAs have potential for use as biomarkers.  相似文献   

2.
Twelve perfluoroalkyl substances (PFASs) and nine organochlorine pesticides (OCPs) were quantified in surface sediments from the Huaihe River, China, along which there are intensive industrial and agricultural activities. Concentrations of PFASs ranged from 0.06 to 0.46 ng/g dry weight (dw), and concentrations of OCPs ranged from 1.48 to 32.65 ng/g dw. Compared with other areas in China, concentrations of PFASs were lesser than the national mean value, while concentrations of OCPs were moderate. Concentrations of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) ranged from n.d. (not detected) to 0.03 and n.d. to 0.10 ng/g dw, respectively. Among the three groups of OCPs, mean concentrations of hexachlorocyclohexane and its isomers (HCHs), dichlorodiphenyltrichloroethane and its metabolites (DDTs) and hexachlorobenzene (HCB) were 5.62 ± 4.35, 2.43 ± 3.12 and 1.55 ± 4.17 ng/g dw, respectively. Concentrations of HCHs and DDTs decreased from upstream to downstream along the mainstream of the Huaihe River. When compared to sediment quality guidelines (SQGs), concentrations of HCHs, DDTs and HCB would pose adverse biological effects. In general, contamination by PFASs in the upstream of the Huaihe River was more severe than that in the downstream, which was mainly caused by interception from dams, locks and industrial emissions. And OCPs from tributaries, especially the Yinghe River and Wohe River, were higher than those from Huaihe mainstream, and primarily came from historical inputs.  相似文献   

3.
Persistent organochlorine compounds were analyzed by means of GC-ECD in surface sediment samples from two selected rivers in Tianjin, Haihe River and Dagu Drainage River. A total of 16 surface sediment sites were selected along the both rivers. The frequency of detection of T-HCH and T-DDT in sediment samples both was up to 100%, which illustrated that the contamination of HCH and DDT was widespread in Haihe and Dagu Drainage Rivers. Results indicated that the concentrations of various pesticides in sediments from Haihe River were in the range of 3.30-75.96 ng/g dw for T-HCH and 1.57-211.57 ng/g dw for T-DDT. Compared with Haihe River, Dagu Drainage River was contaminated by HCHs and DDTs along the all locations and the values of T-HCH and T-DDT residues in sediments ranged from 2.30 to 124.61 ng/g dw and from 11.28 to 237.30 ng/g dw, respectively, The possible pollution sources were analyzed through monitoring results of organochlorine pesticides(OCPs) residues in sediments from the two rivers. The investigation also indicated that HCH was still used as pesticide in Tianjin partial area.  相似文献   

4.
Gaobeidian Lake, located in Beijing, China, serves as a recipient lake for e uents from a large municipal sewage treatment plant (MSTP). In order to evaluate the e ects of discharging MSTP e uent on the mercury contamination of the local aquatic ecosystem, sediment cores, water, plankton, fish, and turtle samples were collected from Gaobeidian Lake for mercury speciation analysis. High concentrations of total mercury (T-Hg) were detected in sediment cores (5.24–17.0 g/g dry weight (dw), average: 10.1 g/g). The ratio of methylmercury (MeHg) to T-Hg was less than 0.3% in sediments and ranged from 35% to 76% in biota samples. The highest level of T-Hg and MeHg were found in aquatic bryophyte and crucian carp (3673 and 437 ng/g dw, respectively). The relative contents of MeHg were significantly correlated with trophic levels (R2 = 0.5506, p < 0.001), which confirmed that MeHg can be bio-transferred and biomagnified via food chain in this aquatic ecosystem.  相似文献   

5.
Chlorination disinfection has been widely used in reclaimed water treatment plants to ensure water quality. In order to assess the downstream quality risk of a running reclaimed water disinfection process, a set of dynamic equations was developed to simulate reactions in the disinfection process concerning variables of bacteria, chemical oxygen demand (COD), ammonia and monochloramine. The model was calibrated by the observations obtained from a pilot disinfection process which was designed to simulate the actual process in a reclaimed water treatment plant. A Monte Carlo algorithm was applied to calculate the predictive effluent quality distributions that were used in the established hierarchical assessment system for the downstream quality risk, and the key factors affecting the downstream quality risk were defined using the Regional Sensitivity Analysis method. The results showed that the seasonal upstream quality variation caused considerable downstream quality risk; the effluent ammonia was significantly influenced by its upstream concentration; the upstream COD was a key factor determining the process effluent risk of bacterial, COD and residual disinfectant indexes; and lower COD and ammonia concentrations in the infiuent would mean better downstream quality.  相似文献   

6.
Common silver barb,Puntius gonionotus,exposed to the nominal concentration of 0.06 mg/L Cd for 60 d,were assessed for histopathological alterations(gills,liver and kidney),metal accumulation,and metallothionein(MT)mRNA expression.Fish exhibited pathological symptoms such as hypertrophy and hyperplasia of primary and secondary gill lamellae,vacuolization in hepatocytes,and prominent tubular and glomerular damage in the kidney.In addition,kidney accumulated the highest content of cadmium,more than gills and liver.Expression of MT mRNA was increased in both liver and kidney of treated fish.Hepatic MT levels remained high after fish were removed to Cd-free water.In contrast,MT expression in kidney was peaked after 28 d of treatment and drastically dropped when fish were removed to Cd-free water.The high concentrations of Cd in hepatic tissues indicated an accumulation site or permanent damage on this tissue.  相似文献   

7.
Long-term changes of fish biotic integrity in the Sanmenxia Wetland,North China,since the 1950 s were assessed using the fish index of biological integrity(FIBI). The water and sediment quality was evaluated by the water quality index(WQI) and sediment pollution index(SPI). The results showed that FIBI continuously decreased from 46 to 20 during the past 5 decades,which indicated that the fish community state shifted from fair to very poor conditions,and damming by itself did not affect the fish biotic integrity. At the same time,WQI fell from 83 to 44.1,and SPI increased from 0.99 to 2.14 since the 1960 s,resulting from fast regional socio-economic development and insufficient wastewater treatment. Correlation analysis suggested that water quality significantly affected biotic integrity(r = 0.867,p 〈 0.05) through direct effects on the fish community. As a representative example of many wetlands in North China,our study clearly demonstrated that the biological integrity was degraded,induced both by water quality deterioration and sediment pollution,further driven by the contradiction between rapid development of regional economy and lagging development of sewage treatment facilities,which were thought to be the main factor responsible for the degradation of biological integrity.  相似文献   

8.
An increasing number of industrial, agricultural, and commercial chemicals in the aquatic environment leads to various deleterious effects on organisms, which is becoming an increasingly serious problem in China. In this study, the comet assay was conducted to investigate the genotoxicity to human body caused by organic concentrates in the drinking water sources of Nanjing City from Yangtze River of China, and health and ecology risk due to expose to these organic pollutants were evaluated with the multimedia environmental assessment system (MEAS). For all the water samples, they were collected from four different locations in the drinking water sourcr samples, es of Nanjing City. The results of the comet assay showed that all the organic concentrates from the water samples could induce different levels DNA damages on human peripheral blood lymphocytes, and a statistically significant difference (p〈0.01) was observed compared with the solvent control, which demonstrated the genotoxicity was in existence. According to the ambient severity (AS) of individual compound, we had sorted out the main organic pollutants in the drinking water source of the four waterworks, and the results showed that there was some potential hazard to human body for all the source water, namely the total ambient severity (TAS) of health for each water source was more than 1. However, the TAS of ecology for each water source was less than 1, which indicated that it was safe to ecology. The results of this investigation demonstrate the application of the comet assay and the MEAS in aquatic environmental monitoring studies, and the comet assay found to be fast, sensitive, and suitable for genotoxicity monitoring programs of drinking water source.  相似文献   

9.
Gaobeidian Lake, located in Beijing, China, serves as a recipient lake for efluents from a large municipal sewage treatment plant (MSTP). In order to evaluate the effects of discharging MSTP efluent on the mercury contamination of the local aquatic ecosystem, sediment cores, water, plankton, fish, and turtle samples were collected from Gaobeidian Lake for mercury speciation analysis. High concentrations of total mercury (T-Hg) were detected in sediment cores (5.24–17.0 μg/g dry weight (dw), average: 10.1 μg/g). The ratio of methylmercury (MeHg) to T-Hg was less than 0.3% in sediments and ranged from 35% to 76% in biota samples. The highest level of T-Hg and MeHg were found in aquatic bryophyte and crucian carp (3673 and 437 ng/g dw, respectively). The relative contents of MeHg were significantly correlated with trophic levels (R2 = 0.5506, p 0.001), which confirmed that MeHg can be bio-transferred and biomagnified via food chain in this aquatic ecosystem.  相似文献   

10.
Glutathione(GSH) and GSH-related enzymes play a great role in protecting organisms from oxidative damage. The GSH level and GSH-related enzymes activities were investigated as well as the growth yield and malonyldialdehyde(MDA) content in the Antarctic ice microalga Chlamydomonas sp. ICE-L exposure to the different cadmium concentration in this paper. The results showed that the higher concentration Cd inhibited the growth of ICE-L significantly and Cd would induce formation of MDA. At the same time, it is clear that GSH level, glutathione peroxidases(GPx) activity and glutathione S-transferases(GST), activity were higher in ICE-L exposed to Cd than the control. But GR activity dropped notably when ICE-L were cultured in the medium containing Cd. Increase of GSH level, GPx and GST activities acclimate to oxidative stress induced by Cd and protect Antarctic ice microalga Chlamydomonas sp. ICE-L from toxicity caused by Cd exposure. These parameters may be used to assess the biological impact of Cd in the Antarctic pole region environment.  相似文献   

11.
刘慧  王晓蓉  王为木  沈骅 《环境科学》2005,26(1):173-176
选择幼龄鲫鱼为材料 ,研究锌(Zn2+)及其配合物(Zn-EDTA)低浓度长期暴露(40d)对鲫鱼(Carassius auratus)肝脏锌富集和抗氧化防御系统的影响 .结果表明 ,鲫鱼肝脏中锌的积累量随锌暴露浓度的升高而增加,且Zn2+处理明显高于Zn EDTA处理 .2种形态锌对鲫鱼肝脏超氧化物歧化酶 (SOD)、过氧化氢酶 (CAT)和谷胱甘肽过氧化物酶 (GPx)均表现出抑制作用 ,其中Zn2+暴露浓度与酶活性之间存在良好的剂量效应关系;Zn2+在低浓度时对谷胱甘肽转硫酶(GST)有诱导作用 ,高浓度时为抑制作用 .Zn EDTA对GST活性始终产生抑制作用 .CAT与GPx对锌暴露比GST和SOD更为敏感 ,适合作为水环境锌污染的早期监测指标 .  相似文献   

12.
北部湾文蛤对石油烃和多氯联苯的氧化应激响应   总被引:1,自引:0,他引:1  
为探讨海域现场双壳类动物对有机污染物积累的抗氧化响应,在北部湾潮间带11个采样点采集文蛤(Meretrix meretrix)样品,测定其软组织中w(TPHs)(TPHs为石油烃)、w(PCBs)(PCBs为多氯联苯)以及鳃、内脏中GSH(还原型谷胱甘肽),GSSG(氧化型谷胱甘肽)、SOD(超氧化物歧化酶)、CAT(过氧化氢酶)、GPx(谷胱甘肽过氧化物酶)、GST(谷胱甘肽转硫酶)、TBARS(硫代巴比妥酸反应物)7种氧化应激物的水平,分析其空间分布特征,并进行有机污染物含量与氧化应激物响应值之间的相关性分析. 结果表明:文蛤软组织中w(TPHs)和w(PCBs)分别为78.22~300.71μg/g和4.23~26.68ng/g,最大值均出现在S10采样点(防城港西湾);内脏中CAT活性较高,其他氧化应激物均在鳃中有较高水平. 与对照采样点S1(湛江流沙湾)相比,S2、S3、S6、S10、S11等5个采样点文蛤组织内SOD、CAT、GST、GPx、GSH水平较低;大多数采样点文蛤鳃中w(GSSG)较低,而S3、S4、S6、S7、S9、S11等6个采样点文蛤内脏中w(GSSG)较高;大多数采样点文蛤组织中TBARS含量较低,表明其抗氧化防御机能尚未丧失. TPHs显著抑制文蛤鳃中的w(GSSG) (R=-0.64,P<0.05),PCBs显著抑制文蛤鳃中的GPx(R=-0.72,P<0.05)和GST(R=-0.72,P<0.05)的活性,表明w(GSSG)及GPx和GST的活性可作为指示北部湾有机污染的生物标志物.   相似文献   

13.
The aim of the present study was to investigate the effect of vitamin E on pro/anti-oxidant status in the liver, brain and heart of Newcastle disease virus (NDV) infected chickens. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and the levels of reduced glutathione and malonaldehyde were estimated in selected tissues of uninfected, NDV-infected and NDV + vit. E-treated chickens. A significant increase in MDA levels in brain and liver (p < 0.05) was observed in NDV-infected chickens when compared to controls. The activities of SOD, CAT, GPx, GR, GST and levels of GSH were significantly (p < 0.05) decreased in brain and liver of NDV-infected chickens over controls. On the other hand, a significant decreased MDA levels and enhanced antioxidant enzyme activity levels were observed in NDV + vit. E-treated animals compared to NDV-infected chickens. Histopathological studies revealed that liver of NDV infected chicken shows focal coagulation and infiltration of hepatocytes, whereas neuronal necrosis and degeneration of Purkinje cells were observed in brain and moderate infiltration of inflammatory cells was observed in heart. However such histological alterations were not observed in NDV + vit. E-treated animals. The results of the present study, thus demonstrated that antioxidant defense mechanism is impaired after the induction of NDV, suggesting its critical role in cellular injury in brain and liver. Further, the results also suggest that vitamin E treatment will ameliorate the antioxidant status in the infected animals. The findings could be beneficial to understand the role of oxidative stress in the pathogenesis of NDV and therapeutic interventions of antioxidants.  相似文献   

14.
阴离子表面活性剂SDS和SDBS对紫贻贝生化指标的影响研究   总被引:6,自引:0,他引:6  
刘波  俞志明  宋秀贤  周斌 《环境科学》2007,28(1):165-169
以青岛胶州湾现场调查数据为依据,选择阴离子表面活性剂十二烷基硫酸钠(SDS)和十二烷基苯磺酸钠(SDBS)作为污染物,以近海底栖生物紫贻贝为受试生物,研究了长期暴露后紫贻贝生化指标(SOD,CAT,GSH,GPx,GST,iNOS,AKP)的变化.结果表明,经过72 d不同浓度暴露后,SDBS实验组紫贻贝体内的SOD、CAT和iNOS活性均有显著下降(除CAT 0.1 mg/L组外),GSH、GST和GPx在3.0 mg/L SDS和SDBS组较各自对照组均有显著升高.SDBS对紫贻贝生化指标影响的显著性水平大于SDS. 统计分析显示,SDBS暴露组下GST与GPx呈显著正相关关系,iNOS与SOD也表现出一定正相关,但GSH与CAT、GSH与SOD呈现显著负相关关系.此外,结果发现后闭壳肌中iNOS可能是一个具有应用前景的阴离子表面活性剂暴露生物标志物.  相似文献   

15.
李亚宁  周启星  胡献刚  罗义 《环境科学》2008,29(7):2012-2017
在实验室模拟条件下分别采用动、静态生物慢性实验的方法,研究了不同浓度的四溴双酚-A(Tetrabromobisphenol A, TBBPA)对颤蚓(Monopylephorus limosus)体内抗氧化酶系统及相Ⅱ结合酶系统活性的影响以及颤蚓暴露于单一浓度的TBBPA后,其体内抗氧化酶系统的活性随时间的响应.结果表明,暴露 8d 后,颤蚓体内SOD的活性发生显著变化,变化趋势明显分为升高、降低、再升高3阶段,在0.05ms/L时SOD的活性被最大诱导(p<0.01),达到对照组的7.8倍,各浓度组SOD的活性均显著高于对照组,由对照组的1.5倍到对照组的7.8倍;同时, CAT的活性变化则呈升高、降低、再升高、再降低4阶段,0.5mg/L时其活性达到最大值(p<0.01),除0.005mg/L和0.25mg/L浓度组CAT活性受抑制外,其余浓度组CAT的活性均高于对照组,由对照组的1.1倍到1.9倍,染毒浓度为0.25ms/L时, GST的活性达到最大诱导(p<0.01),其活性先缓慢上升后下降,并且各处理组GST的活性均显著高于对照组(p<0.05).在单一浓度TBBPA污染暴露的10d时间内,颤蚓体内SOD的活性变化趋势形成了一个变形的"M" 形曲线,在第3d时其活性达到最大诱导,而CAT活性的变化则表现为一个不规则的"N"形曲线,在第5d,时其活性达到最大诱导,并且SOD的活性比CAT的活性受时间的影响小,相对来说更稳定.可见, SOD与GST的活性变化似乎更能反映出TBBPA对颤蚓的污染效应及其毒性作用,而SOD的活性变化似乎更为灵敏,但是二者能否作为指示TBBPA污染的生物标志物尚需进一步研究.  相似文献   

16.
经急性毒性实验,得到萘对成体斑马鱼96 h的半数致死浓度(LC50)为11.8 mg·L-1.在此基础上,设置5个浓度梯度:0、 1/6 LC50、 1/4 LC50、 1/3 LC50、 1/2 LC50,研究了在不同的暴露时间下(0.5、 1、 2、 4、 、 14d),萘对斑马鱼抗氧化防御系统的影响.结果表明,还原型谷胱甘肽(glutathione, GSH)、谷胱甘肽过氧化物酶(glutathione peroxidase, GPx)和谷胱甘肽硫转移酶(glutathione S-transferase, GST)对萘非常敏感,在暴露0.5 d时就受到抑制或诱导.0.5 d后GPx活性整体上被诱导,只在第14 d时高浓度组(1/3 LC50和1/2 LC50浓度组)GPx活性被抑制;GST和GSH则总体上是低于对照的.超氧化物岐化酶(superoxide dismutase, SOD)活性在2d后呈现先诱导后抑制;过氧化氢酶(catalase, CAT)活性在1 d后整体上受抑制.萘对斑马鱼内脏团抗氧化防御系统能产生影响,其变化可作为生物标志物,来评价暴露于多环芳烃有机污染的鱼类的生物学效应.  相似文献   

17.
The effects of phenanthrene (Phe) on hepatosomatic index (HSI) and hepatic enzymatic activities in hybrid tilapia (Oreochromis niloticus O. aureus ) were investigated via the static freshwater exposure at dosage of 50, 100, and 400 g/L for 4–14 d. Compared with the control group, HSI was significantly decreased (P < 0.05) at 400 g/L at day 14. Increased enzymatic activities (P < 0.05) were observed for catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) at either 100 or 400 g/L at day 8 and 14, as well as for CAT at 50 g/L at day 14, except for GPx at 400 g/L at day 8. Ethoxyresorufin O-deethylase (EROD) activity was significantly increased (P < 0.05) at all dosage at day 4 as well as at 50 g/L at day 8, but significantly decreased at either 100 or 400 g/L at day 14 (P < 0.05). Glutathione-S-transferase (GST) activity was not a ected. The results suggest that CAT, GPx, SOD and EROD, as well as HSI in tilapia may be used as the biomarkers or indexes for evaluating or monitoring the pollution of polycyclic aromatic hydrocarbons (PAHs) such as Phe.  相似文献   

18.
选择0#柴油和平湖原油乳化液对缢蛏(Sinonovacula constricta)进行氧化胁迫实验,选取典型的抗氧化酶-超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷光甘肽硫转移酶(GST)及过氧化物酶(POD)用于衡量油类污染物对生物体造成的氧化压力大小.此外,结合综合生物标志物响应(Integrated Biomarker Responses,IBR)指标,对2种石油污染物对缢蛏的毒性响应进行定量化评价.结果表明,不同浓度的0#柴油和平湖原油对缢蛏消化腺中的4种酶表现出不同程度的诱导效应,各试验组在暴露前期均表现出诱导或抑制,但对4种酶的影响存在时间顺序性,SOD、CAT和GST的酶活性表现为升高-降低的过程,POD表现为降低-升高的过程,活性达到峰值的时间SOD和CAT要早于GST和POD.结合计算出的IBR数值来看,高浓度0#柴油能够引起最为显著的生物效应变化,显示该石油污染物高毒性的特征,0#柴油生物毒性大于平湖原油生物毒性.  相似文献   

19.
研究了脊尾白虾(Exopalaemon carinicauda)在对苯并[a]芘(benzo[a]pyrene,BaP)的富集(15 d)、释放(15 d)过程中肝脏的生物标志物(EROD、GST、SOD、CAT、GPx、MDA)的响应,结果显示:在富集阶段,第1 d低浓度实验组(0.05 g/L)和高浓度实验组(0.45 g/L)六种生物标志物即均显著受到诱导(P0.05),诱导程度与暴露浓度成正相关,而后不同种类生物标志物呈现差异性的变化趋势,EROD继续上升并在第10 d后达到稳定状态,GST继续上升并在第10 d后受到抑制下降,SOD、CAT、GPx均受到抑制逐渐下降,且均在15 d时低于对照组水平,MDA呈现持续上升的趋势,直接反映了肝脏的氧化损伤程度。在释放阶段,低浓度实验组各生物标志物的恢复速度较高浓度实验组要快,且均能恢复到接近对照组水平(P0.05),高浓度实验组各生物标志物则均未能恢复到对照组水平(P0.05)。实验结果表明,脊尾白虾肝脏具有一定恢复能力,各生物标志物对BaP暴露的敏感性具有一定差异,BaP暴露的浓度及时间是影响各生物标志物响应变化的主要因素。  相似文献   

20.
Glycogen is a vital energy substrate for anaerobic organisms, and the size of glycogen stores can be a limiting factor for anoxia tolerance of animals. To this end, glycogen stores in 12 different tissues of the crucian carp (Carassius carassius L.), an anoxia-tolerant fish species, were examined. Glycogen content of different tissues was 2–10 times higher in winter (0.68–18.20% of tissue wet weight) than in summer (0.12–4.23%). In scale, bone and brain glycogen stores were strongly dependent on body mass (range between 0.6 and 785 g), small fish having significantly more glycogen than large fish (p < 0.05). In fin and skin, size dependence was evident in winter, but not in summer, while in other tissues (ventricle, atrium, intestine, liver, muscle, and spleen), no size dependence was found. The liver was much bigger in small than large fish (p < 0.001), and there was a prominent enlargement of the liver in winter irrespective of fish size. As a consequence, the whole body glycogen reserves, measured as a sum of glycogen from different tissues, varied from 6.1% of the body mass in the 1-g fish to 2.0% in the 800-g fish. Since anaerobic metabolic rate scales down with body size, the whole body glycogen reserves could provide energy for approximately 79 and 88 days of anoxia in small and large fish, respectively. There was, however, a drastic difference in tissue distribution of glycogen between large and small fish: in the small fish, the liver was the major glycogen store (68% of the stores), while in the large fish, the white myotomal muscle was the principal deposit of glycogen (57%). Since muscle glycogen is considered to be unavailable for blood glucose regulation, its usefulness in anoxia tolerance of the large crucian carp might be limited, although not excluded. Therefore, mobilization of muscle glycogen under anoxia needs to be rigorously tested.  相似文献   

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