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英文文献原文
英文文献原文

Mercury (Hg2+) effect on enzyme activities and hepatopancreas histo structures of juvenile Chinese mitten crab Eriocheir sinensis*

Abstract We studied the effects of mercury (Hg2+) on antioxidant and digestive enzyme activities in terms of LC50 value and on hepatopancreas histo structures of juvenile Chinese mitten crabs Eriocheir sinensis in 40-day exposure to various concentrations of Hg2+ (0, 0.01, 0.05, 0.10, 0.20, and 0.30 mg/L). The results show that the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) significantly increased in the concentrations of 0.01 and 0.05 mg/L, while that of enzyme decreased in 0.10, 0.20 and 0.30 mg/L treatments. Meanwhile, Hg2+ disrupted the histo structures of the hepatopancreas, causing decreases in activities of pepsin, tryptase, amylase, and cellulose, which are synthesized in the hepatopancreas. Moreover, as the Hg2+ concentration increased, the survival rate of the crabs decreased, worst at 56.57% in 0.30 mg/L. Therefore, although crabs are able to tolerate low levels of mercury pollution, high levels lead to cellular injury and tissue damage in hepatopancreas, which then loses some of its vital physiological functions such as absorption, storage, and secretion.

Keyword: Eriocheir sinensis; mercury; hepatopancreas; antioxidant enzymes; histo structure; digestive ; enzymes

1 INTRODUCTION

Mercury is a nonessential metal for aquatic animals (Bano et al., 1989). Due to anthropogenic input and environmental transport of mercury, pollution of the aquatic environment has increased significantly in the past decade (Mason et al., 1994; Botton, 2000). Waterborne mercury penetrates into aquatic animals, accumulates in different tissues, and induces adverse effects at the molecular, cellular and tissue levels (Bianchini et al., 1996; St-Amand et al., 1999; Chou et al., 2002).

The Chinese mitten crab, Eriocheir sinensis (Edwards, 1853), is one of the most economically important crab species in China, with annual production reaching 500 000 tons and valued at US$2.2 billion in 2005. Previous investigations have described acute toxicity (Bianchini et al., 1996) and morphological changes in the gills of E. sinensis exposed to mercury (Zhao et al., 2008). However, the mechanism of mercury toxicity to E. sinensis is still largely unknown.

Previous studies show that ambient mercury exposure leads to the generation of reactive oxygen species (ROS) including O2-, OH-, O2-and H2O2 (Ali et al., 2000). ROS are very unstable and highly reactive, and can induce alterations in the structure of cellular macromolecules, such as proteins, lipids, nucleic acids, etc. (Michiels et al., 1988). ROS formation can also induce cellular damage (Verlecar et al., 2008). Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are the primary enzymes responsible for scavenging ROS. Homeostatic mechanisms act to equilibrate the amount of ROS generated under stress conditions in order to protect an organism from adverse effects(Pan et al., 2006; Yan et al., 2007). In addition, the activity of these enzymes is regulated by health status and environmental stress (Winston et al., 1991). Although several studies have reported ROS formation, antioxidant enzyme activity, and scavenger responses in crustaceans (Sridevi et al., 1998; Yan et al., 2007), little is known about the relationship between ambient Hg2+ and the antioxidant defensive systems of Eriocheir sinensis.

* Supported by the National Basic Research Program of China (973 Program) (No. 2007CB407306);

Basic Scientific Research Operation Cost of State-level Public Welfare Scientific Research Institute of Chinese Research Academy of Environmental Sciences (No. 2007KYYW08)

** Corresponding author: xhwang@https://www.sodocs.net/doc/169795714.html,

The hepatopancreas in various crustacean species functions as the major digestive gland, and it is very sensitive to heavy metals and other water-born pollutants (Bhavan et al., 2000; Yang et al., 2007; Li et al., 2007). The excretion of digestive enzymes, which reflects digestive activity, is one of the most important physiological functions of the crustacean hepatopancreas, and is affected by copper (Cu2+) (Yang et al., 2005). Histo structural analysis of the hepatopancreas has been used as a practical means of assessing the physiological adaptation of Macrobrachium rosenbergii to copper (Li et al., 2007). Hg2+ is more toxic than Cu, or other metal ions, to Macrobrachium nipponensis and Eriocheir sinensis (Zhang et al., 2008; Zhao et al., 2009). Canli et al. (1995) reported that both organic, and inorganic, mercury were mainly taken up from seawater through the gills of Norway lobsters, Nephrops norvegicus. However, little is known about the histo structural changes within the hepatopancreas of E. sinensis after Hg2+ exposure. The objectives of this study were to examine survival rates, the activity of three antioxidant enzymes (superoxide dismutase, glutathione peroxidase and catalase) and five digestive enzymes (pepsin, tryptase, amylase, cellulase and lipase), and histo structural changes in the hepatopancreas of E. sinensis exposed to different ambient Hg2+ concentrations. We hope this study will provide both cytological and cytochemical evidence regarding the physiological changes in E. sinensis as a response to increasing ambient Hg2+ concentrations.

2 MATERIALS AND METHODS

2.1 Animals

Juvenile Eriocheir Sinensis, weighing 27.360 1±2.23 g and with a carapace width of 3.48±0.32 cm, were obtained from a private farm located in Ninghe, Tianjin, China, and acclimatized for 10 days in aerated fresh tap water (pH 7.5±1.0, DO 7.5±0.5 mg/L, total hardness 213.5±5.0 mg/L as CaCO3) with an inherent Hg2+ concentration of 0.003 4 μg/L. The crabs were fed a commercial diet designed for E. sinensis by Dajiang Products, Shanghai, China (32% protein, 4.0% lipid, 8.0% fiber, 5.0% ash, with a mercury content less than 0.01 μg/kg).

2.2 Experiment procedure

Mercury test solutions were prepared by dissolving 1.354 g of HgCl2 (Fluka, Germany) in 1 000 ml of distilled water to yield stock solution with a mercury concentration of 1 g/L. The different experimental concentrations were prepared by diluting the stock solution with fresh, aerated tap water. According to the results of the acute (LC50) experiment (Zhao et al., 2009), approximately 1/60, 1/12, 1/6, 1/3 and 1/2 of the 96 h LC50 corresponded to 0.01, 0.05, 0.10, 0.20, 0.30 mg/L Hg2+ were used as the test solutions for the sub-lethal experiments. The sub-lethal experiments were performed in triplicate and 10 crabs were exposed to 10 liters of water, or test solution, in a 40cm×30cm×20cm polyethylene tank. The crabs were fed with the same commercial diet as above, twice a day (09:00 and 17:00) at 8% of body weight per day. The test Hg2+ solution was renewed daily at 16:00, and excess food and feces were removed. Throughout all experiments the light regime was 14 hours of light and 10 hours of darkness, and water temperature, pH, and dissolved oxygen averaged 21±2°C, 7.5±1, 5.5±0.5 mg/L respectively.

At the end of the 40 days of sub-lethal mercury exposure, crabs at intermolt were dissected, and the hepatopancreas was divided into two parts. One part was chopped into

0.5cm×0.5cm×0.3cm specimens and fixed in Bouin’s fixative for 48 hours for histological study. The other part was stored at -70°C until biochemical analysis.

2.3 Enzyme activity assay

To assess enzyme activity, the hepatopancreas stored at -70°C was weighed, and then homogenized in five times w/v homogenate buffer solution (10 mmol/L sodium citrate/0.1 mol/L sodium chloride; pH 7.0). Part of the crude homogenate was used directly to determine the lipase activity. The remaining homogenate was centrifuged at 10 000 g at 4°C for 15 minutes. The supernatant was separated, and divided into sub-samples for each enzyme assay. These samples were stored in 1.5 ml Eppendorf tubes at -20°C until analyses, which were done within 24 hours. SOD activity was measured by the method described by McCord et al. (1969). One unit of SOD activity was defined as the amount of enzyme that inhibited the NBT photoreduction reaction by 50% per minute per milligram of total protein present in hepatopancreas (U/mg protein). GPX activity was assayed by the method of Paglia et al. (1967). One unit of GPX activity was defined as the amount of enzyme that decreased 1 μmol NADPH per minute per milligram of total protein at a temperature of 25°C and a pH of 8.0 (U/mg protein). CAT was determined by following the reduction of hydrogen peroxide at 240 nm (Beers et al., 1952). One unit of CAT activity was defined as the amount of enzyme needed to degrade 1 μmol H2O2 per minute per milligram of total protein present in hepatopancreas (U/mg protein) at 30°C and pH 8.0.

Pepsin activity was determined according to the method described by Liu et al. (1991) and revised according to Stellmach’s methods (1988). One unit of pepsin enzyme activity(U) was defined as the amount of enzyme that released 1 μg tyrosine per minute per milligram of protein. Tryptase activity was determined according to the method of Liu et al. (1991) with casein as a substrate. One enzyme activity unit (U) was defined as the number of micromoles of tyrosine released per minute per milligram of protein. Amylase and cellulase activities were determined by the method of Pan et al. (1997), using amylopectin and carboxymethylcellulose as substrates, respectively. One unit of amylase or cellulase activity was defined as the number of micromoles of glucose released per minute per milligram of protein. Lipase activity was determined by Zhu’s method (1981) using olive oil as substrate. One enzyme activity unit (U) was defined as the amount of enzyme that catalyzed the release of 1 μg of fatty acid per minute per milligram of protein.

Protein concentrations were determined according to the method of Lowry et al. (1951) using bovine serum albumin as a standard.

2.4 Histology of the digestive gland

The fixed tissues were dehydrated in ascending concentrations of alcohol, cleared in toluene, embedded in paraffin and 6 μm sections cut using a rotary microtome. Sections tissues were then stained with hematoxylin and eosin (H&E).

2.5 Statistical analysis

Each numerical measurement was expressed as mean ± standard error, and was subjected to one-way analysis of variance (ANOVA, SPSS for Windows, version 15.0) to determine significant differences between treatments. If a significant difference was identified, differences between means were compared using Duncan's multiple range tests (1955) P<0.05 was considered significant.

3 RESULTS

3.1 Survival rates

The survival rates of crabs exposed to each test solution is shown in Fig.1. The survival rates after 40 days exposure to 0.01, 0.05, 0.10, 0.20 and 0.30 mg/L Hg2+ were 86.67%, 83.33%, 73.33%, 60% and 56.67%, respectively. Though no significant differences were observed between crabs exposed to 0.01 mg/L Hg2+ and controls (P>0.05), the survival rates of crabs exposed to 0.05, 0.10, 0.20 and 0.30 mg/L Hg2+ were significantly lower than the controls (P<0.05).

Fig1

Fig.1 The effect of waterborne Hg2+on the survival rates of Eriocheir sinensis

Vertical bars represent the standard error of the mean. Bars with the same letter are not significantly different (P>0.05)

3.2 SOD, CAT and GPX activities

The SOD, CAT and GPX activities in the hepatopancreas of the crabs in the different treatments group are shown in Table 1. Ambient Hg2+ exposure significantly affected the activity of these three antioxidant enzymes. SOD, GPX and CAT activity in the hepatopancreas of crabs exposed to 0.01 and 0.05 mg/L Hg2+ were significantly higher than in the controls. However, SOD, GPX and CAT activity in the hepatopancreas of crabs exposed to 0.10, 0.20 and 0.30 mg/L Hg2+ was significantly lower than that in the controls.

3.3 Digestive enzyme activity

The effects of Hg2+ on the activity of pepsin, tryptase, amylase, cellulase and lipase in E. sinensis hepatopancreas extracts are summarized in Table 2. The activity of pepsin, tryptase, amylase and cellulase was 1.58, 3.56, 154.47, and 2.06 U/mg protein, respectively, in the control groups, and decreased with increasing ambient Hg2+ concentration. The lowest levels of pepsin, tryptase, amylase and cellulase activity (1.14, 1.14, 63.34, and 0.67 U/mg protein, respectively) were found in the crabs exposed to 0.30 mg/L Hg2+. Unlike the other four enzymes, lipase activity showed a “low-high-low” trend in the different treatment groups. The lipase activity in crabs exposed to 0.01, 0.05 and 0.10 mg/L Hg2+ was significantly higher than that in control. However, lipase activity in crabs exposed to 0.20 and 0.30 mg/L Hg2+ was significantly lower than that in control.

Table 1 Effect of Hg2+ on antioxidant enzymes activities within the hepatopancreas of Eriocheir sinensis

Table 2 Effect of Hg2+ on digestive enzyme profiles extracted from hepatopancreas of Eriocheir sinensis

3.4 Hepatopancreas histostructure

Fig.2 shows the effects of ambient Hg2+ on the histological structures within the hepatopancreas of E. sinensis. The severity of the damage increases with the mercury dosage. Although there was no discernable histological damage to the hepatopancreas of the crabs exposed to 0.01 mg/L Hg2+, increased numbers of R cells were observed (Fig.2.2). In crabs exposed to 0.05 mg/L Hg2+, the basal lamina was thickened, and rippled from the epithelium in some areas of the tubules (Fig.2.3). Increased numbers of R cells were huddled together in some areas of the tubules. Vacuolization was observed in the hepatopancreas epithelial cells of crabs exposed to 0.10 mg/L Hg2+ (Fig.2.4). Tumid epithelial cells decreased the diameter of the tubule lumen, and a melanin-like material (M) was deposited in the vacuoles of the epithelial cells. In crabs exposed to 0.20 mg/L Hg2+, degeneration of the vacuolated cells was noticed, and the tubule lumen was almost occluded by the expansion and swelling of the tubular epithelial cells (Fig.2.5). The cell boundary between the different cells was not clear, and more melanin-like

material was observed within the cells. The integrity of the tubules within hepatopancreas was completely disrupted in the crabs exposed to 0.30 mg/L Hg2+ (Fig.2.6). Necrotic tubules in the hepatopancreas were separated from the basal lamina, and tissue debris was deposited in the tubule lumen.

4 DISCUSSION

In the present study, survival rates were significantly decreased by exposure to ambient Hg2+ concentrations as low as 0.05 mg/L. This result agrees with other studies showing that mercury is more toxic than other heavy metals such as copper and zinc (St-Amand et al., 1999; Zhang et al., 2008). Rainbow et al. (1989) reported that decapod crustaceans can regulate essential metals in the body to a threshold environmental concentration. However, mercury is a non-essential metal and, as such, cannot be regulated by the crabs, so more mercury accumulates in the body and survival rates decrease. Due to poor understanding on the mechanism involved in mercury toxicity in crustaceans, it is not clear how and why mercury is the most toxic heavy metal to crustaceans. Therefore, to understand in depth the mercury toxicity to E. sinensis, we analyzed the activity of both antioxidant and digestive enzymes, and the histology of the hepatopancreas in the crabs. Mercury is a well-known pro-oxidant that induces oxidative stress via ROS formation (Zaman et al., 1994). SOD, GPX and CAT are the primary enzymes responsible for free radical scavenging, and are involved in the protective mechanisms to mitigate oxidative stress, which appeared in phagocytosis (Elumalai et al., 2007; Verlecar et al., 2008). Their activities are related to the health status of the organism, which is affected by different factors including nutrition, environment, etc. (Winston et al., 1991; Li et al., 2008). Usually, higher levels of SOD, GPX and CAT activity indicate that more free radicals are involved for scavenging (Li et al., 2008; Verlecar et al., 2008). Therefore, significantly higher SOD, GPX and CAT activities in the crabs exposed to 0.01 and 0.05 mg/L Hg2+ might indicate that the stress induced by mercury exposure results in the accumulation of free radicals in the crabs. The crabs would then suffer serious oxidative damage if these radicals were not scavenged (Winston et al., 1991). Therefore, the enhanced activity of these three antioxidant enzymes at 0.01 and 0.05 mg/L Hg2+ may enable the crabs to maintain relative good health by scavenging free radicals produced. However, the activities of SOD, GPX and CAT were significantly inhibited in crabs exposed to 0.10, 0.20 and 0.30 mg/L Hg2+. It is well-known that there are two types of SOD in cytoplasm of mitochondria within eukaryotic cell: Cu-Zn-SOD and Mn-SOD; both can convert O2- to H2O2 (McCord et al., 1969). Cu2+/Zn2+or Mn2+, as the cofactors for SOD, plays a key role in the dismutation induced by SOD (McCord et al., 1969). As a bivalent metal, the excess Hg2+ in 0.10, 0.20 and 0.30 mg/L Hg2+ treatment groups could displace the Cu2+/Zn2+ or Mn2+ from SOD, and decrease its activity (Yan et al., 2007).

The principal H2O2 scavenging enzymes are GPX and CAT. GPX catalyses the conversion of H2O2 to H2O and O2 at the expense of reduced glutathione (Vijayavel et al., 2004). It has been reported that the active center of GPX contains Ser-Cys residues (Yan et al., 2007). Mercury is a sulfhydryl reagent, and its main biological action is to bind exposed cysteine residues on proteins (Wang et al., 1996). Therefore, the decreased GPX activity in the 0.10, 0.20 and 0.30 mg/L Hg2+ groups might be due to mercury binding to the cysteine residues in active sites of GPX, changing its structure and then decreasing its activity. Most CAT is within peroxisomes and cytosol, and its function (along with SOD) is to convert H2O2 into H2O and O2. Decreased SOD activity would lead to a fall in the level of CAT through a chain reaction (Vijayavel et al., 2004). Another possible explanation is that excess Hg2+ disrupts the structural integrity of the mitochondria and other

organelles, decreasing their ability to synthesis CAT (Zaman et al., 1994). When crabs are under severe oxidative stress, ROS accumulate in their bodies and can damage tissues, impair cell membranes function, and even disrupt vital cellular functions (Rikans et al., 1997). Similar results were reported when crustaceans were exposed to cadmium, chromium and other heavy metals (Sridevi et al., 1998; Wan et al., 2004; Pan et al., 2006).

Histopathology shows that the crustacean hepatopancreas is a sensitive organ, and liable to injury by water-born pollutants (Bhavan et al., 2000). Exposure to Hg2+ for 40 days resulted in the hepatopancreas tubules of E. sinensis losing both structural integrity and cellular organization, and the extent of this damage increased with the Hg2+concentration, which was evidenced by thickening the basal lamina, deposition of melanin-like material in cells, vacuolization of cytoplasm, and disorganization of cells. Similar observations have been made in Macrobrachium rosenbergii exposed to copper (Li et al., 2007) and Chasmagnathus granulatus exposed to microcystins (Pinho et al., 2003). The thickening of the basal lamina observed in our study may represent a defensive mechanism against the toxicant (Jiravanichpaisal et al., 1994), attributable to the production of collagenous fibers and melanin, or to coagulation and walling-off by hemocytes (Bautista et al., 1994). Pinho et al. (2003) expressed that the deposition of melanin-like material in the cells was a result of an immune reaction. S?derh?ll et al. (1992) also reported that the cellular reactions used for defense by crustaceans that have only simple and primitive immune systems are frequently followed by a process of melanization. Although the precise function of melanin in the immune responses of arthropods is still not clear, it is thought that this compound may act as a ROS scavenger (Nappi et al., 1993.)

It is widely accepted that R cells are involved in the absorption of diffusible metabolites and nutrients, and in the storage of lipids and glycogen (Al-Mohanna et al., Al-Mohanna 1989). In our study, the number of R cells per tubule increased in the crabs exposed to 0.01 and 0.05 mg/L Hg2+, and this may be the result of an elevation of the lipid content. X-ray microanalyses revealed that R cells can detoxify heavy metals by accumulating them in an insoluble form within the cytoplasm, before excretion (Hopkin et al., 1979; Lyon et al., 1984). The melanin-like materials that accumulated in the crabs exposed to 0.10 and 0.20 mg/L Hg2+ might be an indication that the detoxification was carried out by the R cells.

We also measured the activity of pepsin, tryptase, amylase and cellulase in crabs, and found that they decreased with increasing Hg2+ concentration. The study of digestive enzymes may aid in understanding the digestive physiology of the crabs under adverse conditions. The digestive enzyme activity in crustaceans plays a central role in nutritional physiology, and may directly or indirectly regulate growth (Li et al., 2008). The decline of digestive enzyme activity in the crabs in this study suggests decreased digestive activity after exposure to mercury. This provides evidence that crabs cannot obtain enough nutrition to remain healthy. Also, the digestive enzymes required for extracellular digestion are synthesized in the F cells of the hepatopancreas (Papathanassiou et al., 1984; Al-Mohanna et al., 1989). When the microstructure of the F cells is damaged by ambient Hg2+, their function of excreting digestive enzymes would be inhibited. Unlike the other four digestive enzymes studies, lipase activity was induced in crabs exposed to 0.01, 0.05 and 0.10 mg/L Hg2+. This result shows that lipid metabolism is abnormally active to relieve the mercury toxicity in crabs exposed to those groups.Similar result also appeared in crabs exposed to copper (Yang et al., 2005).

In summary, the elevated levels of SOD, GPX and CAT activity shown in the crabs exposed to

0.01 and 0.05 mg/L Hg2+ meant that the crabs were protected against the oxidative stress generated by mercury. However, when the concentration of ambient Hg2+ increased to 0.10, 0.20 and 0.30 mg/L Hg2+, the declining enzyme activity meant that this protection suffered, and the structure of the hepatopancreas was disrupted, resulting in impaired physiological function and death.

对酶的活动和中国青少年中华绒螯蟹肝胰脏组织相容结构*汞(汞离子)作用

1. Typical organization of the hepatopancreas of E. sinensis (control). Normal arrangement of hepatopancreas tubules and cells, ×400 (Bar=50 μm);

2. The hepatopancreas tubules of test crabs exposed to 0.01 mg/L Hg2+,×400 (Bar=50 μm). Increased numbers of R cells are compared with controls;

3. The hepatopancreas tubules of test crabs exposed to 0.05 mg/L Hg2+, ×400 (Bar=50 μm). Basal lamina is thickened and rippled from the epithelium. The organization of the epithelial cel ls is disordered;

4. The hepatopancreas tubules of test crabs exposed to 0.10 mg/L Hg2+, ×400 (Bar=50 μm). Epithelial cells are swelled and the Tlu is narrowed. The vacuoles in some R and B cells are increased in number and the cells appear dilapidated;

5. The hepatopancreas tubules of test crabs exposed to 0.20 mg/L Hg2+, ×400 (Bar=50 μm). B cells are cracked; the Bm is thickened; the Tlu has disappeared on this longitudinal slice;

6. The hepatopancreas tubules of test crabs exposed to 0.30 mg/L Hg2+, ×400 (Bar=50 μm). Necrotic cells (Nt) in the hepatopancreas are separated from the Bm, tissue debris appears in the Tlu. The normal structure of the hepatopancreas tubule is completely disorganized;

B: B cell (Blisterlike cell); Bm: Basement membrane; F: F cell (Fibrillar cells); Mm: melanin-like material; Nt: Necrotic cells; R: R cell (Resorptive cell); Tlu: Tubule lumen

1. 中华绒螯蟹肝胰腺的正常组织(对照组)。肝胰腺肝小管和细胞排列正常,×400(标尺=50

微米);

2. 暴露于0.01 mg / L Hg2+浓度处理组的螃蟹的肝胰腺小管,×400(标尺= 50微米)。与对

照组相比R细胞数量的增加;

3. 暴露于0.05 mg / L Hg2+浓度处理组的螃蟹的肝胰腺小管,×400(标尺= 50微米)。

基底膜增厚,并波及上皮细胞,上皮细胞组织是紊乱;

4. 暴露于0.10 mg / L Hg2+浓度处理组的螃蟹的肝胰腺小管,×400(标尺= 50微米)。

上皮细胞肿胀和管腔变窄。细胞出现残破,空泡化的R细胞和B细胞数量增加;

5. 暴露于0.20 mg / L Hg2+浓度处理组的螃蟹的肝胰腺小管,×400(标尺=50微米)。B

细胞破裂,基底膜增厚,纵向切片的管腔消失;

6. 暴露于0.30 mg / L Hg2+浓度处理组的螃蟹的肝胰腺小管,×400(标尺=50微米)。肝胰

腺中的坏死细胞(NT)与基底膜分离,管腔中出现组织碎片。肝胰腺小管的结构是完全紊乱的;

B: B细胞(泡状细胞); Bm:基底膜,F:F细胞(纤维状细胞); Mm:黑色素类物质;Nt:坏死细胞; R:R细胞(吸收细胞); TLU:管腔

图1水性Hg2+对中华绒螯蟹存活率的影响

垂直线代表平均值的标准差。有相同字母的条形没有显著差异(P>0.05)

表1 Hg2+对中华绒螯蟹肝胰腺抗氧化物酶活性的影响

数据表示为平均数±标准差(n=3)。类似字母的意思是没有统计学差异(P>0.05)。

表2 Hg2 +对中华绒螯蟹肝胰腺提取的消化酶的影响

数据表示为平均数±标准差(n=3)。类似字母的意思是没有统计学差异(P>0.05)。

7.中华绒螯蟹肝胰腺的正常组织(对照组)。

肝胰腺肝小管和细胞排列正常,×400(标尺=50微

米);

8.暴露于0.01 mg / L Hg2+浓度处理组的螃蟹的

肝胰腺小管,×400(标尺= 50微米)。与对照组相

比R细胞数量的增加;

9.暴露于0.05 mg / L Hg2+浓度处理组的螃蟹

的肝胰腺小管,×400(标尺= 50微米)。基底膜增

厚,并波及上皮细胞,上皮细胞组织是紊乱;

10.暴露于0.10 mg / L Hg2+浓度处理组的螃蟹的

肝胰腺小管,×400(标尺= 50微米)。上皮细胞肿

胀和管腔变窄。细胞出现残破,空泡化的R细胞和B

细胞数量增加;

11.暴露于0.20 mg / L Hg2+浓度处理组的螃蟹的

肝胰腺小管,×400(标尺=50微米)。 B细胞破裂,

基底膜增厚,纵向切片的管腔消失;

12.暴露于0.30 mg / L Hg2+浓度处理组的螃蟹的

肝胰腺小管,×400(标尺=50微米)。肝胰腺中的

坏死细胞(NT)与基底膜分离,管腔中出现组织碎

片。肝胰腺小管的结构是完全紊乱的;

B: B细胞(泡状细胞);Bm:基底膜,F:F细胞(纤维状细胞); Mm:黑色素类物质;Nt:坏死细胞; R:R细胞(吸收细胞); TLU:管腔

图2 环境Hg2+对中华绒螯蟹幼蟹组织结构的影响

土木工程英文文献

2.1 Reinforced Concrete Plain concrete is formed from a hardened mixture of cement ,water ,fine aggregate, coarse aggregate (crushed stone or gravel),air, and often other admixtures. The plastic mix is placed and consolidated in the formwork, then cured to facilitate the acceleration of the chemical hydration reaction lf the cement/water mix, resulting in hardened concrete. The finished product has high compressive strength, and low resistance to tension, such that its tensile strength is approximately one tenth lf its compressive strength. Consequently, tensile and shear reinforcement in the tensile regions of sections has to be provided to compensate for the weak tension regions in the reinforced concrete element. It is this deviation in the composition of a reinforces concrete section from the homogeneity of standard wood or steel sections that requires a modified approach to the basic principles of structural design. The two components of the heterogeneous reinforced concrete section are to be so arranged and proportioned that optimal use is made of the materials involved. This is possible because concrete can easily be given any desired shape by placing and compacting the wet mixture of the constituent ingredients are properly proportioned, the finished product becomes strong, durable, and, in combination with the reinforcing bars, adaptable for use as main members of any structural system. The techniques necessary for placing concrete depend on the type of member to be cast: that is, whether it is a column, a bean, a wall, a slab, a foundation. a mass columns, or an extension of previously placed and hardened concrete. For beams, columns, and walls, the forms should be well oiled after cleaning them, and the reinforcement should be cleared of rust and other harmful materials. In foundations, the earth should be compacted and thoroughly moistened to about 6 in. in depth to avoid absorption of the moisture present in the wet concrete. Concrete should always be placed in horizontal layers which are compacted by means of high frequency power-driven vibrators of either the immersion or external type, as the case requires, unless it is placed by pumping. It must be kept in mind, however, that over vibration can be harmful since it could cause segregation of the aggregate and bleeding of the concrete. Hydration of the cement takes place in the presence of moisture at temperatures above 50°F. It is necessary to maintain such a condition in order that the chemical hydration reaction can take place. If drying is too rapid, surface

英文文献翻译

中等分辨率制备分离的 快速色谱技术 W. Clark Still,* Michael K a h n , and Abhijit Mitra Departm(7nt o/ Chemistry, Columbia Uniuersity,1Veu York, Neu; York 10027 ReceiLied January 26, 1978 我们希望找到一种简单的吸附色谱技术用于有机化合物的常规净化。这种技术是适于传统的有机物大规模制备分离,该技术需使用长柱色谱法。尽管这种技术得到的效果非常好,但是其需要消耗大量的时间,并且由于频带拖尾经常出现低复原率。当分离的样本剂量大于1或者2g时,这些问题显得更加突出。近年来,几种制备系统已经进行了改进,能将分离时间减少到1-3h,并允许各成分的分辨率ΔR f≥(使用薄层色谱分析进行分析)。在这些方法中,在我们的实验室中,媒介压力色谱法1和短柱色谱法2是最成功的。最近,我们发现一种可以将分离速度大幅度提升的技术,可用于反应产物的常规提纯,我们将这种技术称为急骤色谱法。虽然这种技术的分辨率只是中等(ΔR f≥),而且构建这个系统花费非常低,并且能在10-15min内分离重量在的样本。4 急骤色谱法是以空气压力驱动的混合介质压力以及短柱色谱法为基础,专门针对快速分离,介质压力以及短柱色谱已经进行了优化。优化实验是在一组标准条件5下进行的,优化实验使用苯甲醇作为样本,放在一个20mm*5in.的硅胶柱60内,使用Tracor 970紫外检测器监测圆柱的输出。分辨率通过持续时间(r)和峰宽(w,w/2)的比率进行测定的(Figure 1),结果如图2-4所示,图2-4分别放映分辨率随着硅胶颗粒大小、洗脱液流速和样本大小的变化。

关于力的外文文献翻译、中英文翻译、外文翻译

五、外文资料翻译 Stress and Strain 1.Introduction to Mechanics of Materials Mechanics of materials is a branch of applied mechanics that deals with the behavior of solid bodies subjected to various types of loading. It is a field of study that i s known by a variety of names, including “strength of materials” and “mechanics of deformable bodies”. The solid bodies considered in this book include axially-loaded bars, shafts, beams, and columns, as well as structures that are assemblies of these components. Usually the objective of our analysis will be the determination of the stresses, strains, and deformations produced by the loads; if these quantities can be found for all values of load up to the failure load, then we will have obtained a complete picture of the mechanics behavior of the body. Theoretical analyses and experimental results have equally important roles in the study of mechanics of materials . On many occasion we will make logical derivations to obtain formulas and equations for predicting mechanics behavior, but at the same time we must recognize that these formulas cannot be used in a realistic way unless certain properties of the been made in the laboratory. Also , many problems of importance in engineering cannot be handled efficiently by theoretical means, and experimental measurements become a practical necessity. The historical development of mechanics of materials is a fascinating blend of both theory and experiment, with experiments pointing the way to useful results in some instances and with theory doing so in others①. Such famous men as Leonardo da Vinci(1452-1519) and Galileo Galilei (1564-1642) made experiments to adequate to determine the strength of wires , bars , and beams , although they did not develop any adequate theo ries (by today’s standards ) to explain their test results . By contrast , the famous mathematician Leonhard Euler(1707-1783) developed the mathematical theory any of columns and calculated the critical load of a column in 1744 , long before any experimental evidence existed to show the significance of his results ②. Thus , Euler’s theoretical results remained unused for many years, although today they form the basis of column theory. The importance of combining theoretical derivations with experimentally determined properties of materials will be evident theoretical derivations with experimentally determined properties of materials will be evident as we proceed with

土木工程外文文献及翻译

本科毕业设计 外文文献及译文 文献、资料题目:Designing Against Fire Of Building 文献、资料来源:国道数据库 文献、资料发表(出版)日期:2008.3.25 院(部):土木工程学院 专业:土木工程 班级:土木辅修091 姓名:武建伟 学号:2008121008 指导教师:周学军、李相云 翻译日期: 20012.6.1

外文文献: Designing Against Fire Of Buliding John Lynch ABSTRACT: This paper considers the design of buildings for fire safety. It is found that fire and the associ- ated effects on buildings is significantly different to other forms of loading such as gravity live loads, wind and earthquakes and their respective effects on the building structure. Fire events are derived from the human activities within buildings or from the malfunction of mechanical and electrical equipment provided within buildings to achieve a serviceable environment. It is therefore possible to directly influence the rate of fire starts within buildings by changing human behaviour, improved maintenance and improved design of mechanical and electrical systems. Furthermore, should a fire develops, it is possible to directly influence the resulting fire severity by the incorporation of fire safety systems such as sprinklers and to provide measures within the building to enable safer egress from the building. The ability to influence the rate of fire starts and the resulting fire severity is unique to the consideration of fire within buildings since other loads such as wind and earthquakes are directly a function of nature. The possible approaches for designing a building for fire safety are presented using an example of a multi-storey building constructed over a railway line. The design of both the transfer structure supporting the building over the railway and the levels above the transfer structure are considered in the context of current regulatory requirements. The principles and assumptions associ- ated with various approaches are discussed. 1 INTRODUCTION Other papers presented in this series consider the design of buildings for gravity loads, wind and earthquakes.The design of buildings against such load effects is to a large extent covered by engineering based standards referenced by the building regulations. This is not the case, to nearly the same extent, in the

计算机网络-外文文献-外文翻译-英文文献-新技术的计算机网络

New technique of the computer network Abstract The 21 century is an ages of the information economy, being the computer network technique of representative techniques this ages, will be at very fast speed develop soon in continuously creatively, and will go deep into the people's work, life and study. Therefore, control this technique and then seem to be more to deliver the importance. Now I mainly introduce the new technique of a few networks in actuality live of application. keywords Internet Network System Digital Certificates Grid Storage 1. Foreword Internet turns 36, still a work in progress Thirty-six years after computer scientists at UCLA linked two bulky computers using a 15-foot gray cable, testing a new way for exchanging data over networks, what would ultimately become the Internet remains a work in progress. University researchers are experimenting with ways to increase its capacity and speed. Programmers are trying to imbue Web pages with intelligence. And work is underway to re-engineer the network to reduce Spam (junk mail) and security troubles. All the while threats loom: Critics warn that commercial, legal and political pressures could hinder the types of innovations that made the Internet what it is today. Stephen Crocker and Vinton Cerf were among the graduate students who joined UCLA professor Len Klein rock in an engineering lab on Sept. 2, 1969, as bits of meaningless test data flowed silently between the two computers. By January, three other "nodes" joined the fledgling network.

10kV小区供配电英文文献及中文翻译

在广州甚至广东的住宅小区电气设计中,一般都会涉及到小区的高低压供配电系统的设计.如10kV高压配电系统图,低压配电系统图等等图纸一大堆.然而在真正实施过程中,供电部门(尤其是供电公司指定的所谓电力设计小公司)根本将这些图纸作为一回事,按其电脑里原有的电子档图纸将数据稍作改动以及断路器按其所好换个厂家名称便美其名曰设计(可笑不?),拿出来的图纸根本无法满足电气设计的设计意图,致使严重存在以下问题:(也不知道是职业道德问题还是根本一窍不通) 1.跟原设计的电气系统货不对板,存在与低压开关柜后出线回路严重冲突,对实际施工造成严重阻碍,经常要求设计单位改动原有电气系统图才能满足它的要求(垄断的没话说). 2.对消防负荷和非消防负荷的供电(主要在高层建筑里)应严格分回路(从母线段)都不清楚,将消防负荷和非消防负荷按一个回路出线(尤其是将电梯和消防电梯,地下室的动力合在一起等等,有的甚至将楼顶消防风机和梯间照明合在一个回路,以一个表计量). 3.系统接地保护接地型式由原设计的TN-S系统竟曲解成"TN-S-C-S"系统(室内的还需要做TN-C,好玩吧?),严格的按照所谓的"三相四线制"再做重复接地来实施,导致后续施工中存在重复浪费资源以及安全隐患等等问题.. ............................(违反建筑电气设计规范等等问题实在不好意思一一例举,给那帮人留点混饭吃的面子算了) 总之吧,在通过图纸审查后的电气设计图纸在这帮人的眼里根本不知何物,经常是完工后的高低压供配电系统已是面目全非了,能有百分之五十的保留已经是谢天谢地了. 所以.我觉得:住宅建筑电气设计,让供电部门走!大不了留点位置,让他供几个必需回路的电,爱怎么折腾让他自个怎么折腾去.. Guangzhou, Guangdong, even in the electrical design of residential quarters, generally involving high-low cell power supply system design. 10kV power distribution systems, such as maps, drawings, etc. low-voltage distribution system map a lot. But in the real implementation of the process, the power sector (especially the so-called power supply design company appointed a small company) did these drawings for one thing, according to computer drawings of the original electronic file data to make a little change, and circuit breakers by their the name of another manufacturer will be sounding good design (ridiculously?), drawing out the design simply can not meet the electrical design intent, resulting in a serious following problems: (do not know or not know nothing about ethical issues) 1. With the original design of the electrical system not meeting board, the existence and low voltage switchgear circuit after qualifying serious conflicts seriously hinder the actual construction, often require changes to the original design unit plans to meet its electrical system requirements (monopoly impress ). 2. On the fire load and fire load of non-supply (mainly in high-rise building in) should be strictly sub-loop (from the bus segment) are not clear, the fire load and fire load of non-qualifying press of a circuit (especially the elevator and fire elevator, basement, etc.

3英文文献及翻译格式示例

哈尔滨工业大学毕业设计(论文) 英文原文(原文也可以直接将PDF版打印) ASSESSING CREDIT OR DETERMINING QUANTITY? THE EVOLVING ROLE OF RATING AGENCIES Lynnette D. Purda* This version: April 21, 2011 Abstract Over the past ten years, credit rating agencies have come under intense criticism from both practitioners and academics, first for their failure to identify problems resulting in bankruptcies at Enron and Worldcom and second for providing overly optimistic ratings for structured finance products. While many investors question the value of rating agencies in light of these criticisms, they have proven remarkably resilient. This paper provides a brief background on how rating agencies secured competitive advantages in evaluating credit quality. It then reviews the empirical evidence on the information content of ratings given these advantages. I argue that the information content of ratings stems from two intertwined sources: 1) information related to credit quality and 2) information related to the firm’s ability to access debt. Based on this evidence, I suggest that the dominant role for ratings today is as a benchmark for financial contracting. In this way, ratings remain influential in establishing the supply and demand of debt securities. 译文 评级机构的发展的作用评估信用还是决定数量? 本文:2011.4.21 摘要 在过去的十年,信用评级机构一直处于来自实践者和学者的激烈的批评中,首先他们未能发现问题,导致安然和世通破产;其次对结构性金融产品提供过于乐观的评级。虽然许多投资者因为这些批评对评级机构的价值提出了质疑,但他们仍然被证明是相当有活力的。这篇文章首先在评估机构如何在信用评级质量中获得竞争优势提供一个简单地背景介绍,然后考虑到这些优势回顾了一些信息内容方面的评级经验证据。个人认为信息内容的评级来自两种交织在一起(错综复杂)的来源:1)和信贷质量相关的信息;2)和公司获取债务资本能力相关的信息。以此为据,我建议当前评级的主导作用是作为基准的金融收缩。以这种方式,在建立债券的供应和需求方面评级仍然是有效的。 - -1

土木工程外文文献翻译

专业资料 学院: 专业:土木工程 姓名: 学号: 外文出处:Structural Systems to resist (用外文写) Lateral loads 附件:1.外文资料翻译译文;2.外文原文。

附件1:外文资料翻译译文 抗侧向荷载的结构体系 常用的结构体系 若已测出荷载量达数千万磅重,那么在高层建筑设计中就没有多少可以进行极其复杂的构思余地了。确实,较好的高层建筑普遍具有构思简单、表现明晰的特点。 这并不是说没有进行宏观构思的余地。实际上,正是因为有了这种宏观的构思,新奇的高层建筑体系才得以发展,可能更重要的是:几年以前才出现的一些新概念在今天的技术中已经变得平常了。 如果忽略一些与建筑材料密切相关的概念不谈,高层建筑里最为常用的结构体系便可分为如下几类: 1.抗弯矩框架。 2.支撑框架,包括偏心支撑框架。 3.剪力墙,包括钢板剪力墙。 4.筒中框架。 5.筒中筒结构。 6.核心交互结构。 7. 框格体系或束筒体系。 特别是由于最近趋向于更复杂的建筑形式,同时也需要增加刚度以抵抗几力和地震力,大多数高层建筑都具有由框架、支撑构架、剪力墙和相关体系相结合而构成的体系。而且,就较高的建筑物而言,大多数都是由交互式构件组成三维陈列。 将这些构件结合起来的方法正是高层建筑设计方法的本质。其结合方式需要在考虑环境、功能和费用后再发展,以便提供促使建筑发展达到新高度的有效结构。这并

不是说富于想象力的结构设计就能够创造出伟大建筑。正相反,有许多例优美的建筑仅得到结构工程师适当的支持就被创造出来了,然而,如果没有天赋甚厚的建筑师的创造力的指导,那么,得以发展的就只能是好的结构,并非是伟大的建筑。无论如何,要想创造出高层建筑真正非凡的设计,两者都需要最好的。 虽然在文献中通常可以见到有关这七种体系的全面性讨论,但是在这里还值得进一步讨论。设计方法的本质贯穿于整个讨论。设计方法的本质贯穿于整个讨论中。 抗弯矩框架 抗弯矩框架也许是低,中高度的建筑中常用的体系,它具有线性水平构件和垂直构件在接头处基本刚接之特点。这种框架用作独立的体系,或者和其他体系结合起来使用,以便提供所需要水平荷载抵抗力。对于较高的高层建筑,可能会发现该本系不宜作为独立体系,这是因为在侧向力的作用下难以调动足够的刚度。 我们可以利用STRESS,STRUDL 或者其他大量合适的计算机程序进行结构分析。所谓的门架法分析或悬臂法分析在当今的技术中无一席之地,由于柱梁节点固有柔性,并且由于初步设计应该力求突出体系的弱点,所以在初析中使用框架的中心距尺寸设计是司空惯的。当然,在设计的后期阶段,实际地评价结点的变形很有必要。 支撑框架 支撑框架实际上刚度比抗弯矩框架强,在高层建筑中也得到更广泛的应用。这种体系以其结点处铰接或则接的线性水平构件、垂直构件和斜撑构件而具特色,它通常与其他体系共同用于较高的建筑,并且作为一种独立的体系用在低、中高度的建筑中。

变电站_外文翻译_外文文献_英文文献_变电站的综合概述

英文翻译 A comprehensive overview of substations Along with the economic development and the modern industry developments of quick rising, the design of the power supply system become more and more completely and system. Because the quickly increase electricity of factories, it also increases seriously to the dependable index of the economic condition, power supply in quantity. Therefore they need the higher and more perfect request to the power supply. Whether Design reasonable, not only affect directly the base investment and circulate the expenses with have the metal depletion in colour metal, but also will reflect the dependable in power supply and the safe in many facts. In a word, it is close with the economic performance and the safety of the people. The substation is an importance part of the electric power system, it is consisted of the electric appliances equipments and the Transmission and the Distribution. It obtains the electric power from the electric power system, through its function of transformation and assign, transport and safety. Then transport the power to every place with safe, dependable, and economical. As an important part of power’s transport and control, the transformer substation must change the mode of the traditional design and control, then can adapt to the modern electric power system, the development of modern industry and the of trend of the society life. Electric power industry is one of the foundations of national industry and national economic development to industry, it is a coal, oil, natural gas, hydropower, nuclear power, wind power and other energy conversion into electrical energy of the secondary energy industry, it for the other departments of the national economy fast and stable development of the provision of adequate power, and its level of development is a reflection of the country's economic development an important indicator of the level. As the power in the industry and the importance of the national economy, electricity transmission and distribution of electric energy used in these areas is an indispensable component.。Therefore, power transmission and distribution is critical. Substation is to enable superior power plant power plants or power after adjustments to the lower load of books is an important part of power transmission. Operation of its functions, the capacity of a direct impact on the size of the lower load power, thereby affecting the industrial production and power consumption.Substation system if a link failure, the system will protect the part of action. May result in power outages and so on, to the production and living a great disadvantage. Therefore, the substation in the electric power system for the protection of electricity reliability,

英文论文及中文翻译

International Journal of Minerals, Metallurgy and Materials Volume 17, Number 4, August 2010, Page 500 DOI: 10.1007/s12613-010-0348-y Corresponding author: Zhuan Li E-mail: li_zhuan@https://www.sodocs.net/doc/169795714.html, ? University of Science and Technology Beijing and Springer-Verlag Berlin Heidelberg 2010 Preparation and properties of C/C-SiC brake composites fabricated by warm compacted-in situ reaction Zhuan Li, Peng Xiao, and Xiang Xiong State Key Laboratory of Powder Metallurgy, Central South University, Changsha 410083, China (Received: 12 August 2009; revised: 28 August 2009; accepted: 2 September 2009) Abstract: Carbon fibre reinforced carbon and silicon carbide dual matrix composites (C/C-SiC) were fabricated by the warm compacted-in situ reaction. The microstructure, mechanical properties, tribological properties, and wear mechanism of C/C-SiC composites at different brake speeds were investigated. The results indicate that the composites are composed of 58wt% C, 37wt% SiC, and 5wt% Si. The density and open porosity are 2.0 g·cm–3 and 10%, respectively. The C/C-SiC brake composites exhibit good mechanical properties. The flexural strength can reach up to 160 MPa, and the impact strength can reach 2.5 kJ·m–2. The C/C-SiC brake composites show excellent tribological performances. The friction coefficient is between 0.57 and 0.67 at the brake speeds from 8 to 24 m·s?1. The brake is stable, and the wear rate is less than 2.02×10?6 cm3·J?1. These results show that the C/C-SiC brake composites are the promising candidates for advanced brake and clutch systems. Keywords: C/C-SiC; ceramic matrix composites; tribological properties; microstructure [This work was financially supported by the National High-Tech Research and Development Program of China (No.2006AA03Z560) and the Graduate Degree Thesis Innovation Foundation of Central South University (No.2008yb019).] 温压-原位反应法制备C / C-SiC刹车复合材料的工艺和性能 李专,肖鹏,熊翔 粉末冶金国家重点实验室,中南大学,湖南长沙410083,中国(收稿日期:2009年8月12日修订:2009年8月28日;接受日期:2009年9月2日) 摘要:采用温压?原位反应法制备炭纤维增强炭和碳化硅双基体(C/C-SiC)复合材

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