Proteasome and antioxidant responses in Cottus gobio during a combined exposure to heat stress and cadmium

Temperature and trace metals are common environmental stressors, and their importance is increasing due to global climate change and anthropogenic pollution. Oxidative damage and antioxidant properties have been studied in liver and gills of the European bullhead (Cottus gobio) subjected to cadmium (CdCl(2) at nominal concentrations of 0.01 and 1mg/L) for 4 days at either 15°C or 21°C. First, exposure to 1mg Cd/L induced a high mortality rate (67%) in fish held at 21°C. Regarding the antioxidant enzymes, exposure to 0.01 mg Cd/L significantly increased the activity of superoxide dismutase (SOD) and decreased the activity of glutathione reductase (GR) in liver, independently of heat stress. In gills, exposure to 21°C resulted in a significantly increased activity of glutathione peroxidase (GPx), whereas the activity of glutathione S-transferase (GST) was significantly reduced as compared to fish exposed to 15°C. Furthermore, regardless of Cd stress, exposure to elevated temperature resulted in a significant decrease of lipid peroxidation (LPO) level in liver and in a significant increase in the activity of chymotrypsin-like 20S proteasome in both studied tissues of C. gobio. Overall, the present results indicated that elevated temperature and cadmium exposure independently influenced the antioxidant defense system in bullhead with clear tissue-specific and stress-specific antioxidant responses. Further, elevated temperature affected the hepatic lipid peroxidation and the activity of 20S proteasome in both tissues.     

Proteomic Analysis of Cd-Responsive Proteins in Solanum torvum

Proteomic changes induced by Cd have been described in plants in different scenarios. However, there has been no proteomic study on Cd toxicity, including any low Cd-accumulating species. Here, we investigate the response of a low Cd-accumulating species, Solanum torvum, to Cd toxicity at the root proteomic level using two-dimensional gel electrophoresis (2-DGE). The root 2-DGE map consisted of at least 927 reproducible protein spots, of which 45 were classified as differentially expressed proteins based on three replicated separations. MALDI-TOF MS analysis identified 19 of these spots, and MALDI-TOF/TOF MS analysis identified 8 of the spots. The eight proteins identified were two S-adenosylmethionine (SAM) synthetases, actin, an ATP synthase subunit, two tubulin proteins, alcohol dehydrogenase (ADH), and 14-3-3 protein 4. These proteins are involved in phytohormone synthesis, defense responses, energy metabolism, and cytoskeleton construction. Thus, our proteomic analysis revealed that Cd stress promotes an increase in the abundance of proteins involved in antioxidant defenses and anti-stress protection.     

Proteomics analysis of liver samples from puffer fish Takifugu rubripes exposed to excessive fluoride: An insight into molecular response to fluorosis

Comparative proteomics was performed to identify proteins in the liver of Takifugu rubripes in response to excessive fluoride exposure. Sixteen fish were randomly divided into a control group and an experimental group. The control group was raised in soft water alone (F^? = 0.4 mg/L), and the experimental group was raised in the same water with sodium fluoride at a high concentration of 35 mg/L. After 3 days, proteins were extracted from the fish livers and then subjected to two‐dimensional polyacrylamide gel electrophoresis analysis. The matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) was applied to identify the proteins that were differentially expressed from the two groups of fish. Among an average of 816 and 918 proteins detected in the control and treated groups, respectively, 16 proteins were upregulated and 35 were downregulated (P < 0.01) in the fluoride‐treated group as compared with those in the control group. Twenty‐four highly differentially expressed proteins were further analyzed by MALDI‐TOF/TOF‐MS, and eight were identified by Mascot. These eight proteins include disulfide isomerase ER‐60, 4SNc‐Tudor domain protein, SMC3 protein, Cyclin D1, and mitogen‐activated protein kinase 10, as well as three unknown proteins. Consistent with their previously known functions, these identified proteins seem to be involved in apoptosis and other functions associated with fluorosis. These results will greatly contribute to our understanding of the effects of fluoride exposure on the physiological and biochemical functions of Takifugu and the toxicological mechanism of fluoride causing fluorosis in both fish and human. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:21–28, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20308     

The effect of environmental salinity on the proteome of the sea bass (Dicentrarchus labrax L.)

The European sea bass, Dicentrarchus labrax L., tolerates a range of salinities from freshwater to hyper-saline. To study differences in protein expression, fish were reared in both freshwater and seawater. After 3-month acclimation, gill and intestine epithelia were collected and the soluble protein extracted. In all, 362 spots were differentially expressed in the gills and intestines of fishes reared in seawater compared to those from freshwater. Fifty differential protein spots were excised from a colloidal Coomassie-stained gel. Nine separate protein spots were identified unambiguously by mass spectrometry and database searching. Among the six proteins over-expressed in gill cells in seawater, five were cytoskeleton proteins and one was the aromatase cytochrome P450. In gill cells under freshwater conditions, the two over-expressed proteins identified were the prolactin receptor and the major histocompatibility complex class II β -antigen. In intestinal cells under freshwater conditions, the Iroquois homeobox protein Ziro5 was upregulated over ninefold. The expression of these proteins, their possible direct or indirect roles in the adaptation of D. labrax to salinity, and their correspondences with a previous study are discussed.     

Effect of cadmium exposure on the globin protein expression in 4th instar larvae of Chironomus riparius Mg. (Diptera: Chironomidae): An ecotoxicoproteomics approach

In order to identify Chironomus hemoglobin (Hb) as a biomarker of ecotoxicity monitoring; herein, the effects of cadmium chloride (Cd) on Hb parameters were investigated in the 4th instar larvae of Chironomus riparius. The expressions of globin mRNA and hemolymph protein, using ecotoxicoproteomic approach, were investigated. Conventional ecotoxicity tests were also conducted to validate the ecotoxicological relevance of the response of Chironomus Hb as a biomarker. The proteomic analysis indicated that exposure to Cd lead alteration in the expression of hemolymph protein, with the total expressions of 12 hemolymph protein spots decreasing in response to treatment, with that of two increasing in response to Cd exposure. In addition, all of the spots differentially expressed in response to Cd treatment were identified as globin proteins. The decreased total Hb content observed in the hemolymph of larvae exposed to Cd suggested that the decreased expression of selected globin proteins in response to Cd exposure impacted on Hb synthesis. The overall results suggested that Hb could be a target molecule for exposure to Cd in C. riparius, with a proteomic approach appearing to be an ideal tool for the discovery of biomarkers in ecotoxicological research.     

Effect of battery-manufacturing effluent on endogenous antioxidant in freshwater fish

Abstract

This study monitors and assesses the effect of battery-manufacturing effluent containing metals Pb, Zn and Cd on endogenous antioxidants. Malonaldehyde (MDA), reduced glutathione sulfyhydryl (GSH) and catalase (CAT) which are known biomarkers of effluent were exposed to 0%, 25%, 50%, 75% and 90% amendments for 74h on the gills, liver and kidneys of C. punctata. There was more metal Zn accumulation in the gills and GSH contents increased significantly in the gills (P<0.01), liver accumulation of Pb was found to be more (P<0.05), whereas lowest accumulation of Pb was found in kidneys and the highest accumulation of Cd (P<0.05). Over all amendments of the effluents, MDA contents were increased in the gills, liver and kidneys (P<0.01). GSH levels were decreased among the liver and kidneys compared to the gills (P<0.01) at 90% amendment. Effluent exposure caused a significant decrease in the activities of CAT in the gills, liver and kidneys (P<0.01, 0.05 and 0.05) of fish. Increased MDA activity was indicative of the formation of free radicals in the fish with exposure to amendments of battery manufacturing effluent, while increased levels of GSH pointed to the occurrence of a scavenging mechanism of free radicals.     

Sub-lethal effect of synthetic pyrethroid pesticide on metabolic enzymes and protein profile of non-target Zebra fish,Danio rerio

Extensive application of pesticide in agricultural field affects the enzymatic activity of non-target animals, including fishes. In this study, the impact of sublethal concentration of fenvalerate on marker enzymes of freshwater Zebra fish was evaluated. Pesticide-induced stress can specifically affect non target fishes, through elevated level of reactive oxygen species which is responsible for biochemical, cell metabolism and physiological activities. The oxidative stress mediated by fenvalerate at sub lethal concentrations after 28 days of exposure of Zebra fish. Following 28 days of exposure of pesticide, catalase, superoxide dismutase, aspartate amino transferases, alanine amino transferase, alkaline phosphatase and acid phosphatase were assessed. Results revealed reduction of superoxide dismutase activity after 28 days of exposure in sub lethal concentration of fenvalerate in liver and gills. In liver, catalase activity was found to be less in fenvalerate exposed fish than control fish. In liver, increase of 75.75% aspartate amino transferase and 38% increase in alanine amino transferase in gills. SGPT activity was relatively higher than SGOT suggests more contribution of phyruvalate than oxaloacetate formation. Fenvalerate induced changes in acid phosphatase and alkaline phosphatase activity in the liver and gills of Zebra fish after four weeks of exposure. Fenvalerate induced expression of various stress proteins in gill, liver, followed by muscle. Some proteins lost its intensity due to fenvalerate toxicity. Result revealed that enzyme assays and SDS-PAGE analysis for protein subunits determination is relevant tool to monitor stress in freshwater ecosystem. The findings suggest that in monitoring fenvalerate toxicity programme, enzyme activities can be potent diagnostic tool for fenvalerate induced toxicity.     

Effects of Cadmium and High Temperature on Some Parameters of Calcium Metabolism in the Killifish (Aphanius fasciatus)

This study aims to investigate the influence of high temperature on cadmium (Cd) toxicity in Aphanius fasciatus (Pisces: Cyprinodontidae). For this reason, Cd, mineral, and organic content in the vertebral column as well as the histological structure of gills and bone were compared in fishes exposed for 30 days to Cd (2 mg/L CdCl₂) and/or high temperature (26 °C). Cd exposure caused a negative correlation between Cd and Ca concentrations (r?=?0.98, p?<?0.05), as well as a significant decrease in inorganic components (p?<?0.05) and ash weight/dry weight ratio (p?<?0.05) in the vertebral column. These changes were accompanied by an increased frequency of histological alterations in gills and bone. Concomitant treatment with Cd and high temperature increases Cd accumulation and Ca depletion in the skeletal tissue and increases the frequency and the severity of histological alterations. These results confirm that temperature increases Cd toxicity and needs to be taken into account for the accurate prediction and assessment of Cd-induced spinal deformities in fish.     

Proteomics reveals new insights into the role of light in cadmium response in Arabidopsis cell suspension cultures

Light plays an important role in plant growth, development, and response to environmental stresses. To investigate the effects of light on the plant responses to cadmium (Cd) stress, we performed a comparative physiological and proteomic analysis of light‐ and dark‐grown Arabidopsis cells after exposure to Cd. Treatment with different concentrations of Cd resulted in stress‐related phenotypes such as cell growth inhibition and decline of cell viability. Notably, light‐grown cells were more sensitive to heavy metal toxicity than dark‐grown cells, and the basis for this appears to be the elevated Cd accumulation, which is twice as much under light than dark growth conditions. Protein profiles analyzed by 2D DIGE revealed a total of 162 protein spots significantly changing in abundance in response to Cd under at least one of these two growing conditions. One hundred and ten of these differentially expressed protein spots were positively identified by MS/MS and they are involved in multiple cellular responses and metabolic pathways. Sulfur metabolism‐related proteins increased in relative abundance both in light‐ and dark‐grown cells after exposure to Cd. Proteins involved in carbohydrate metabolism, redox homeostasis, and anti‐oxidative processes were decreased both in light‐ and dark‐grown cells, with the decrease being lower in the latter case. Remarkably, proteins associated with cell wall biosynthesis, protein folding, and degradation showed a light‐dependent response to Cd stress, with the expression level increased in darkness but suppressed in light. The possible biological importance of these changes is discussed.     

Protein intensity changes in the hemoglobin and plasma electrophoretic patterns of Oreochromis niloticus in response to single and combined Zn and Cd exposure

The purpose of this study was to evaluate the effects of metals on the electrophoretic patterns of hemoglobin and blood plasma proteins of Oreochromis niloticus. Fish were exposed to 0.5 and 5.0 mg/L Zn, 0.1 and 1.0 mg/L Cd, and 0.5 mg/L Zn + 0.1 mg/L Cd, and 5.0 mg/L Zn + 1.0 mg/L Cd mixtures for 7 and 28 days. In all concentrations tested, electrophoretic pattern of hemoglobin and plasma proteins by cellulose acetate electrophoresis consist of three and eight bands, respectively. The three bands for hemoglobin are one cathodic (Hb1) and two anodic (Hb2 and Hb3) bands. The protein intensity in hemoglobins of fish following Zn, Cd, and Zn + Cd exposures decreased in Hb1, whereas it increased in Hb3. The eight bands for plasma proteins are 60, 78, 87, and 94 kDA high molecular weight proteins (HMP) for four bands and 120, 132, 176, and 273 kDA very high molecular weight proteins (VHMP) for four bands. The level of 60, 78, and 94 kDA HMP and 120, 132, and 176 kDA VHMP increased in response to single and combined Zn and Cd exposure. Also, there was increasing level of the metals in the whole blood with increasing concentrations of metals in the exposure medium and with increasing duration of exposure.     

基于转录组测序的Cd胁迫下芹菜细胞自噬相关基因的筛选

细胞自噬是植物逆境应答过程中最常见的保护机制之一。动物中,自噬相关基因抵御镉(Cd)毒害的功能研究较清楚,但植物却知之甚少。文中以芹菜品种‘皇后’为试材,采用外源Cd(终浓度为0、2、4、8mg/L)添加营养液水培处理,利用转录组测序(RNA-seq)技术筛选细胞自噬相关差异基因并进行q RT-PCR验证。结果表明Cd胁迫对芹菜植株产生了明显的毒害作用,并与浓度间产生了量效关系。在筛选的8个差异表达的自噬相关基因中,ATG8a、ATG8f、ATG13、AMPK-1、AMPK-2基因随Cd浓度升高表达上调,ATG12、VPS30和VPS34则先上调后下降,说明自噬相关基因可能通过表达上调增加了自噬小体结构以抵御Cd毒性作用;而高浓度Cd(8mg/L)可能超出芹菜的耐受范围,导致多个自噬基因又出现表达下调趋势。以上结果有助于后期自噬相关基因的功能研究,为进一步探讨芹菜对Cd胁迫的耐性机制提供参考依据。     

Fish Cholinesterases as Biomarkers of Sublethal Effects of Organophosphorus and Carbamates in Tissues of Labeo Rohita

Organophosphates and carbamates are major agrochemicals that strongly affect different neuroenzymes and the growth of various fish species. Here, we study the effect of sublethal concentrations of profenofos and carbofuran on the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) and the associated health risk in fish. Labeo rohita fingerlings were exposed to three sublethal concentrations of profenofos and carbofuran. The minimum cholinesterase activities in the brain, gills, muscle, kidney, liver, and blood were after exposure to profenofos (0.06 mg/L). The minimum AChE and BuChE activities in the brain, gills, muscle, kidney, liver, and blood were after exposure to carbofuran (0.28 and 0.198 mg/L). Exposure to both types of pesticides affected the functions of these organs, including metabolism and neurotransmission, to various extents at different exposure concentrations. These findings suggest that they are required to be properly monitored in the environment, to reduce their toxic effects on nontarget organisms     

镉对尖紫蛤抗氧化酶活性及脂质过氧化的影响

为阐明镉(Cd2+)对尖紫蛤消化盲囊和鳃抗氧化酶的毒性影响程度,研究了不同浓度的Cd2+(0.005、0.05、0.5 mg/L)在不同暴露时间(24h、72h、120h)对尖紫蛤鳃和消化盲囊中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)的活性以及丙二醛(MDA)含量的影响。结果表明,在Cd2+浓度为0.005 mg/L时,在整个实验期间Cd2+对消化盲囊和鳃内的SOD、CAT、GSH-PX活性并无显著影响。在Cd2+浓度为0.05 mg/L和0.5 mg/L时,SOD、CAT、GSH-PX在鳃和消化盲囊中的活性都呈现出明显的时间剂量依赖关系。在0.05 mg/L暴露时,鳃和消化盲囊中的SOD、CAT和GSH-PX的活性随时间逐渐增强,在72h时达到最大值,但在120h时略有降低。在0.5 mg/L暴露时,消化盲囊中SOD、CAT及鳃中CAT活性在24h时上升达到最大值,但鳃中SOD直到72h才达到最大值,并均在120h下降到最低,其中消化盲囊中SOD和CAT活性在120h低于对照组,这可能与消化盲囊对Cd2+的敏感性高于鳃有关。在0.5 mg/L暴露的鳃中,GSH-PX在24h、72h活性并不上升,在120h甚至低于正常水平,0.5 mg/L暴露的消化盲囊中,24h时迅速增高,然后逐渐下降到正常值。这可能与Cd2+结合了GSH-PX的活性中心,降低了GSH-PX的活性有关。与3种酶活性随着时间延长和剂量的增加,酶活性会降低的变化趋势不同,鳃和消化盲囊中的MDA的含量随时间延长和剂量的增加而增加,并不出现下降,这表明尖紫蛤鳃和消化盲囊中的MDA含量可以灵敏的反映机体内的氧化损伤程度,但不能敏感的反映水体中Cd2+的污染情况。     

Response to nickel in the proteome of the metal accumulator plant Brassica juncea

To gain a comprehensive understanding of the molecular mechanism of heavy metal accumulation in Brassica juncea, comparative proteomic approaches were used to analysis protein profiles in leaf tissues of 6-week-old B. juncea after exposure to 100 µM Ni. Proteomic analysis revealed that 61 protein spots showed 1.5-fold change in protein abundance after Ni exposure as compared to that of corresponding spots in control. Out of the 61 differentially expressed protein spots, 37 protein spots were ambiguously identified by matrix assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS). The majority of these identified proteins were found to be involved in sulphur metabolism, protection against oxidative stress, clearly indicated that heavy metal sequestration and antioxidant system were activated by Ni treatment. The induced expression of photosynthesis and ATP generation-related proteins were also observed in plants exposed to metals, suggesting the tolerance and accumulation is an energy-demanding process. The identification of these proteins in response to Ni can lead a deep understanding of heavy metal accumulation and tolerance in B. juncea.     

A note on copper bioaccumulation in Mozambique tilapia,Oreochromis mossambicus (Osteichthyes: Cichlidae)

Copper (Cu) is one of the most commonly reported metal pollutants in African water bodies, but there are few studies on African freshwater fish species of copper accumulation and copper toxicity. Adult O. mossambicus were exposed to 0mg l^?1 (control) and 0.75mg l^?1 Cu for 96h and 0 (control), 0.11, 0.29 and 0.47mg l^?1 copper for 64 days. Samples of liver and gills were collected after 96h, and after 1, 32 and 64 days, respectively. There were significant differences in the mean Cu accumulation values in the liver and gills between the control and the Cu-exposed fish after the 96-h exposure. In fish exposed to 0.11 and 0.29mg l^?1 Cu for 64 days there was an increase in copper level in the tissues. In fish exposed to 0.47mg l^?1 Cu the concentration in the gill and liver tissue did not increase between Day 1 and Day 32. At this time, Cu accumulation in the liver was higher than for fish exposed to 0.11 and 0.29mg l^?1 Cu for 64 days. Exposure to approximately 0.47mg l^?1 Cu for more than 32 days induced mortality.     

Differential expression of proteins induced by lead in the Dwarf Sunflower Helianthus annuus

The Dwarf Sunflower (Helianthus annuus) is a hyperaccumulator of toxic metals including cadmium (Cd), mercury (Hg), nickel (Ni), and lead (Pb). In order to identify stress response to Pb, plants were exposed to a mixture of 30 mg/l of three ions, Cd, Cr, and Ni, with and without Pb. Soluble proteins were resolved by two-dimensional gel electrophoresis. Four proteins were differentially expressed and very abundant in the leaf samples after plants were exposed to all these four metals. The first protein spot contained two proteins: chitinase and a chloroplast drought-induced stress protein CDSP-34. The second spot contained a thaumatin-like protein. Two proteins in spot 3 were identified as heat-shock cognate 70-1 and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Several peptides were identified in spot 4 but none could be matched to any sequence in the NCBI database.     

Comparative proteomic analysis of Cd-responsive proteins in wheat roots

Cadmium (Cd) is a major environmental toxicant to plant cells due to its potential inhibitory effects on many physiological processes. To gain a comprehensive understanding of plant response to Cd, wheat seedlings were exposed to a range of Cd concentrations (10, 100 and 200 μM) for 1 week and a combination of physiological and proteomic approaches were used to evidence Cd effects and to access the plant response to Cd toxicity. Root and shoot elongation was decreased, whereas the H₂O₂ and malondialdehyde content in wheat seedlings was increased significantly at higher Cd concentration. Protein profiles analyzed by two-dimensional electrophoresis revealed that 46 protein spots showed 1.5-fold change in protein abundance following Cd exposure; 31 protein spots were up-regulated and 15 protein spots were down-regulated; 25 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As expected, most of the up-regulated proteins are involved in heavy metal detoxification and antioxidant processes. Enzyme activity analysis revealed that ascorbate peroxidase and glutathione S-transferase activity was stimulated by Cd treatment. Abundance changes of these proteins, together with their putative functions provide us a new insight that can lead to an integrated understanding of the molecular basis of Cd responses in plants.     

Dietary Ca inhibits waterborne Cd uptake in Cd-exposed rainbow trout, Oncorhynchusmykiss

The effects of chronic exposure to waterborne Cd and elevated dietary Ca, alone and in combination, were examined in juvenile rainbow trout, Oncorhynchusmykiss. Fish were chronically exposed to 0.05 (control) or 2.56 μg/l Cd [as Cd(NO₃)₂·4H₂O] and were fed 2% body mass/day of control (29.6 mg Ca/g) or Ca-supplemented trout food (52.8 mg Ca/g as CaCl₂·2H₂O). Cd accumulated mainly in gill, liver, and kidney. Waterborne Cd inhibited unidirectional Ca uptake from water into the gill and induced hypocalcemia in the plasma on day 40. Waterborne Cd also induced an elevated Ca concentration on day 20 in the gill tissue of trout fed the Ca-supplemented diet and a decreased Ca concentration on day 35 in the gills of trout fed the control diet. Dietary Ca protected against Cd accumulation in gill, liver, and kidney, but did not protect against the inhibition of Ca uptake into the gill or plasma hypocalcemia. When fed Ca-supplemented diet and exposed to waterborne Cd, fish showed 35% mortality, compared to 0–2% in control fish and in the Cd-exposed fish with normal Ca in the diet. Growth, on the other hand, was not affected by any treatment.     

Metal accumulation and metallothionein induction in the spotted dogfish Scyliorhinus canicula

Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts. Accumulation rates can be high, which despite the fact that normal background levels for metals in the marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals of the present study were to examine differences in accumulation rates and toxicity of a range of metals at equimolar concentrations (10 µM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this purpose, we exposed the dogfish to Ni (587 µg/L), Cd (1124 µg/L), Pb (2072 µg/L), Cu (635 µg/L), and Ag (1079 µg/L and two additional exposures at 10 µg/L and 1 µg/L) for one week and measured total metal accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.