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 ; enzymes1 INTRODUCTIONMercury 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@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 METHODS2.1 AnimalsJuvenile 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 procedureMercury 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 into0.5cm×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 assayTo 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 glandThe 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 analysisEach 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 RESULTS3.1 Survival ratesThe 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).Fig1Fig.1 The effect of waterborne Hg2+on the survival rates of Eriocheir sinensisVertical 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 activitiesThe 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 activityThe 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 sinensisTable 2 Effect of Hg2+ on digestive enzyme profiles extracted from hepatopancreas of Eriocheir sinensis3.4 Hepatopancreas histostructureFig.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-likematerial 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 DISCUSSIONIn 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 otherorganelles, 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. Söderhäll 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 to0.01 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 lumen1. 中华绒螯蟹肝胰腺的正常组织（对照组）。