Quantitative proteomics of heavy metal stress responses in Sydney rock oysters

Currently, there are few predictive biomarkers in key biomonitoring species, such as oysters, that can detect heavy metal pollution in coastal waterways. Several attributes make oysters superior to other organisms for positive biomonitoring of heavy metal pollution. In particular, they are filter feeders with a high capacity for bioaccumulation. In this study, we used two proteomics approaches, namely label-free shotgun proteomics based on SDS-PAGE gel separation and gas phase fractionation, to investigate the heavy metal stress responses of Sydney rock oysters. Protein samples were prepared from haemolymph of oysters exposed to 100 μg/L of PbCl(2), CuCl(2), or ZnCl(2) for 4 days in closed aquaria. Peptides were identified using a Bivalvia protein sequence database, due to the unavailability of a complete oyster genome sequence. Statistical analysis revealed 56 potential biomarker proteins, as well as several protein biosynthetic pathways to be greatly impacted by metal stress. These have the potential to be incorporated into bioassays for prevention and monitoring of heavy metal pollution in Australian oyster beds. The study confirms that proteomic analysis of biomonitoring species is a promising approach for assessing the effects of environmental pollution, and our experiments have provided insights into the molecular mechanisms underlying oyster stress responses.     

污染胁迫下的蚯蚓蛋白质组学研究进展

随着蛋白质组学的发展和每年有大量环境污染物进入土壤环境中,污染胁迫模式动物的相关生物标志物受到日益关注。蚯蚓,作为土壤中最大的无脊椎动物,是研究和评价土壤生态污染良好的模式动物。研究蚯蚓的蛋白质组学,对于寻找环境生态污染相关生物标志物和阐明生态毒理学机制有着十分重要的现实意义。目前已知的污染胁迫下蚯蚓蛋白质组学研究,提供了几个特定污染物胁迫蚯蚓的蛋白表达谱。这些蛋白涉及许多生物学过程,例如信号传导、糖酵解、能量代谢、分子伴侣和转录调节,提示了相关污染物可能的生态毒理学机制,有望成为潜在的生物标志物,用于有毒污染物的监测,但其特异性需要进一步试验的验证。对蚯蚓受污染胁迫的蛋白质组表达谱及潜在生物标志物进行简要综述。     

Proteomics as a route to identification of toxicity targets in environmental toxicology

Ecotoxicology describes a three-way relationship between ecosystems, chemical pollutants and living organisms. It is predicated on the fact that chemical pollution can exert toxic effects on organisms at the individual and population levels. These toxic effects may provide important information to supplement chemical analysis of environmental samples and aid in assessing the environmental quality of specific ecosystems. Traditionally, effects have been detected by means of biomarkers which, of necessity, were often molecules or processes known to be affected by pollutants. Proteomics provides a means of achieving high-throughput analysis of effects on protein populations and sub-populations with the potential to identify novel biomarkers. This review summarises the main approaches currently used in this area and assesses the potential of proteomics for identification of novel toxicity targets.     

环境污染物对水生生物产生氧化压力的分子生物标志物

为了能够建立一种简单、快速、准确的环境污染监测预警体系,人们进行了广泛的研究,其中有关环境污染物对分子生物标志物的影响已成为研究热点。生物体内的氧自由基和其它活性氧分子（ROS）对组织和细胞成分造成的伤害,称之为氧化压力,环境中的有毒物质能够对生物体产生不同程度的氧化压力。生物体内的强氧化剂或体外因素（如环境污染物）引起的强氧化物与抗氧化防御系统之间的平衡能够用于评估环境压力对生物体产生影响的程度,尤其适合于评估不同种化学物质引起氧化损伤的程度。这些抗氧化防御系统及其对氧化压力的敏感性在环境毒物学研究中占有非常重要的地位,大量研究结果表明：过渡金属、多环芳烃、有机氯和有机磷农药、多氯联苯、二氧芑和其它异型物质都能够对生物体产生氧化压力。这些有毒物质能够引起各种有害影响,如对膜脂、DNA和蛋白产生损伤;改变抗氧化酶的活性等。总结了这种氧化压力的研究进展情况,并讨论了这些分子生物标志物在水生生物中的应用。     

Proteomics approach and techniques in identification of reliable biomarkers for diseases

Biomarkers, also called biological markers, are indicators to identify a biological case or situation as well as detecting any presence of biological activities and processes. Proteins are considered as a type of biomarkers based on their characteristics. Therefore, proteomics approach is one of the most promising approaches in this field. The purpose of this review is to summarize the use of proteomics approach and techniques to identify proteins as biomarkers for different diseases. This review was obtained by searching in a computerized database. So, different researches and studies that used proteomics approach to identify different biomarkers for different diseases were reviewed. Also, techniques of proteomics that are used to identify proteins as biomarkers were collected. Techniques and methods of proteomics approach are used for the identification of proteins' activities and presence as biomarkers for different types of diseases from different types of samples. There are three essential steps of this approach including: extraction and separation of proteins, identification of proteins, and verification of proteins. Finally, clinical trials for new discovered biomarker or undefined biomarker would be on.     

A semiparametric modeling framework for potential biomarker discovery and the development of metabonomic profiles

Background  

The discovery of biomarkers is an important step towards the development of criteria for early diagnosis of disease status. Recently electrospray ionization (ESI) and matrix assisted laser desorption (MALDI) time-of-flight (TOF) mass spectrometry have been used to identify biomarkers both in proteomics and metabonomics studies. Data sets generated from such studies are generally very large in size and thus require the use of sophisticated statistical techniques to glean useful information. Most recent attempts to process these types of data model each compound's intensity either discretely by positional (mass to charge ratio) clustering or through each compounds' own intensity distribution. Traditionally data processing steps such as noise removal, background elimination and m/z alignment, are generally carried out separately resulting in unsatisfactory propagation of signals in the final model.     

Proteomic profiling of pancreatic cancer for biomarker discovery

Pancreatic cancer is a uniformly lethal disease that is difficult to diagnose at early stage and even more difficult to cure. In recent years, there has been a substantial interest in applying proteomics technologies to identify protein biomarkers for early detection of cancer. Quantitative proteomic profiling of body fluids, tissues, or other biological samples to identify differentially expressed proteins represents a very promising approach for improving the outcome of this disease. Proteins associated with pancreatic cancer identified through proteomic profiling technologies could be useful as biomarkers for the early diagnosis, therapeutic targets, and disease response markers. In this article, we discuss recent progress and challenges for applying quantitative proteomics technologies for biomarker discovery in pancreatic cancer.     

Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: antioxidants, lipid peroxidation and DNA damage

Oxidative stress can take place in marine bivalves under a series of environmental adverse conditions. The study of different systems related to oxidative stress in these organisms can give important information about their physiological status and also about environmental health. Bivalves have been proposed as good sentinel organisms in pollution monitoring studies through the analysis of biochemical biomarkers, and most of the biomarkers analyzed are those related to oxidative stress. However, it is very important to know how other environmental factors not associated to the presence of pollutants might affect these parameters. We have studied a series of mechanisms related to oxidative stress in mussels which inhabit the Brazilian coast, especially in Perna perna species, subjected to different stress conditions, such as the exposure to different contaminants in the laboratory and in the field, the exposure of mussels to air and re-submersion, simulating the tidal oscillations, and in mussels collected at different seasons. Both oxidative damage levels and antioxidant defense systems were strongly affected by the different environmental stress. This review summarizes the data obtained in some studies carried out in bivalves from the Brazilian coast.     

Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: Antioxidants, lipid peroxidation and DNA damage

Oxidative stress can take place in marine bivalves under a series of environmental adverse conditions. The study of different systems related to oxidative stress in these organisms can give important information about their physiological status and also about environmental health. Bivalves have been proposed as good sentinel organisms in pollution monitoring studies through the analysis of biochemical biomarkers, and most of the biomarkers analyzed are those related to oxidative stress. However, it is very important to know how other environmental factors not associated to the presence of pollutants might affect these parameters. We have studied a series of mechanisms related to oxidative stress in mussels which inhabit the Brazilian coast, especially in Perna perna species, subjected to different stress conditions, such as the exposure to different contaminants in the laboratory and in the field, the exposure of mussels to air and re-submersion, simulating the tidal oscillations, and in mussels collected at different seasons. Both oxidative damage levels and antioxidant defense systems were strongly affected by the different environmental stress. This review summarizes the data obtained in some studies carried out in bivalves from the Brazilian coast.     

Molecular epidemiology studies of carcinogenic environmental pollutants. Effects of polycyclic aromatic hydrocarbons (PAHs) in environmental pollution on exogenous and oxidative DNA damage

Exposure to high levels of environmental air pollution is known to be associated with an increased carcinogenic risk. The individual contribution to this risk derived from specific carcinogenic chemicals within the complex mixture of air pollution is less certain, but may be explored by the use of molecular epidemiological techniques. Measurements of biomarkers of exposure, of effect and of susceptibility provide information of potential benefit for epidemiological and cancer risk assessment. The application of such techniques has been mostly concerned in the past with the carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) that are associated with particulate matter in air pollution, and has showed clear evidence of genotoxic effects, such as DNA adducts, chromosome aberrations (CA) and ras oncogene overexpression, in environmentally exposed Czech and Polish populations. We are currently extending these studies by an investigation of populations exposed to environmental pollution in three European countries, Czech Republic, Slovak Republic and Bulgaria. This pays particular attention to PAHs, but also investigates the extent of radically induced (oxidative) DNA damage in the exposed populations. Policemen, bus drivers and controls, who carried personal monitors to determine their exposures to PAHs have been studied, and blood and urine were collected. Antioxidant and dietary status were assessed in these populations. Stationary monitors were also used for ambient air monitoring. Amongst the parameters studied in the biological samples were: (a) exposure biomarkers, such as PAH adducts with DNA, p53 and p21(WAF1) protein levels, (b) oxidative DNA damage, (c) the biological effect of the exposure by measurement of chromosome damage by fluorescence in situ hybridisation (FISH) or conventional methods, and (d) polymorphisms in carcinogen metabolising and DNA repair enzymes. Repair ability was also measured by the Comet assay. In vitro systems are being evaluated to characterise the genotoxicity of the organic compounds adsorbed to air particles.     

Hepatic proteome sensitivity in rainbow trout after chronically exposed to a human pharmaceutical verapamil

Verapamil (VRP), a cardiovascular pharmaceutical widely distributed and persistent in the aquatic environment, has potential toxicity to fish and other aquatic organisms. However, the molecular mechanisms that lead to these toxic effects are not well known. In the present study, proteomic analysis has been performed to investigate the protein patterns that are differentially expressed in liver of rainbow trout exposed to sublethal concentrations of VRP (0.5, 27.0, and 270 μg/liter) for 42 days. Two-dimensional electrophoresis coupled with MALDI-TOF/TOF mass spectrometry was employed to detect and identify the protein profiles. The analysis revealed that the expression of six hepatic acidic proteins were markedly altered in the treatment groups compared with the control group; three proteins especially were significantly down-regulated in fish exposed to VRP at environmental related concentration (0.5 μg/liter). These results suggested that the VRP induce mechanisms against oxidative stress (glucose-regulated protein 78 and 94 and protein disulfide-isomerase A3) and adaptive changes in ion transference regulation (calreticulin, hyperosmotic glycine-rich protein). Furthermore, for the first time, protein Canopy-1 was found to be significantly down-regulated in fish by chronic exposure to VRP at environmental related levels. Overall, our work supports that fish hepatic proteomics analysis serves as an in vivo model for monitoring the residual pharmaceuticals in aquatic environment and can provide valuable insight into the molecular events in VRP-induced toxicity in fish and other organisms.     

Trans-SILAC: sorting out the non-cell-autonomous proteome

Non-cell-autonomous proteins are incorporated into cells that form tight contacts or are invaded by bacteria, but identifying the full repertoire of transferred proteins has been a challenge. Here we introduce a quantitative proteomics approach to sort out non-cell-autonomous proteins synthesized by other cells or intracellular pathogens. Our approach combines stable-isotope labeling of amino acids in cell culture (SILAC), high-purity cell sorting and bioinformatics analysis to identify the repertoire of relevant non-cell-autonomous proteins. This 'trans-SILAC' method allowed us to discover many proteins transferred from human B to natural killer cells and to measure biosynthesis rates of Salmonella enterica proteins in infected human cells. Trans-SILAC should be a useful method to examine protein exchange between different cells of multicellular organisms or pathogen and host.     

Proteomic applications in ecotoxicology

Within the growing body of proteomics studies, issues addressing problems of ecotoxicology are on the rise. Generally speaking, ecotoxicology uses quantitative expression changes of distinct proteins known to be involved in toxicological responses as biomarkers. Unlike these directed approaches, proteomics examines how multiple expression changes are associated with a contamination that is suspected to be detrimental. Consequently, proteins involved in toxicological responses that have not been described previously may be revealed. Following identification of key proteins indicating exposure or effect, proteomics can potentially be employed in environmental risk assessment. To this end, bioinformatics may unveil protein patterns specific to an environmental stress that would constitute a classifier able to distinguish an exposure from a control state. The combined use of sets of marker proteins associated with a given pollution impact may prove to be more reliable, as they are based not only on a few unique markers which are measured independently, but reflect the complexity of a toxicological response. Such a proteomic pattern might also integrate some of the already established biomarkers of environmental toxicity. Proteomics applications in ecotoxicology may also comprise functional examination of known classes of proteins, such as glutathione transferases or metallothioneins, to elucidate their toxicological responses.     

微塑料污染对浮游生物影响研究进展

微塑料作为一种新型的环境污染物,大量存在于水环境中,给水生生物带来了极大的危害。浮游生物是水生食物链的基础,是水生生态系统物质循环和能量流动的重要环节;同时,浮游生物也是对各种环境污染物最敏感的类群。了解微塑料对浮游生物的影响是评价其生态风险的重要依据。本文介绍了环境中微塑料来源、特征及水生态系统微塑料污染现状,阐述了微塑料对水生生物的直接和间接危害,并重点聚焦于浮游植物和浮游动物,从个体、种群和群落的层次详细总结了微塑料的影响及其作用机制。最后,本文指出当前针对浮游生物微观基因和蛋白质组学,以及宏观种群和群落响应等方面的研究还非常缺乏,为今后开展微塑料危害研究提供参考。     

Micronuclei frequency in lymphocytes of individuals occupationally exposed to pesticides.

Pesticides have been widely used in developing countries over the years. A large amount of these remains in the environment and organisms. Pesticide pollution is detrimental to human health. The effects can be seen on a short or a long-term basis and the symptoms can vary from headache to cancer. Only a minority of studies focuses on their genotoxic effect. This study assesses the genotoxic effect of the pesticides used at banana-packaging plants with binucleate micronuclei assay using cultured lymphocytes. The studied population included 32 exposed and 37 unexposed women from Costa Rica. There is no significant difference between the two groups. However, women who worked at the packaging plant and had stillbirths or spontaneous abortions were 1.45 times more (alpha = 0.06) likely to have an increased micronuclei frequency than their coworkers who lacked those disorders; this may indicate genetic susceptibility. In vitro pesticides studies and susceptibility biomarkers are needed to identify subgroups with higher risks.     

Utility of proteomics to assess pollutant response of clams from the Doñana bank of Guadalquivir Estuary (SW Spain)

The utility of proteomics to assess pollutant response of Scrobicularia plana clams from three sites of Guadalquivir Estuary at the southern end of the National Park of Do?ana (SW Spain) has been studied. Protein expression profiles were analyzed by 2-DE in soluble fractions of S. plana gills. Nearly 2000 well-resolved spots were detected in silver-stained gels, with focused areas in the 4-6.5 pH range. Different protein expression signatures were found at each site, with the highest number of more intense spots in animals with the highest metal content. Nineteen more intense protein spots were analyzed out by nanospray-ion trap tandem mass spectrometry, de novo sequencing and a bioinformatics search for their possible identification. While sequence tags of 16 more intense protein spots were obtained, including several proteins induced by pollutant exposure of model organisms, only 2 proteins were unambiguously identified: hypoxanthine-guanine phosphoribosyltransferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Both enzymes were significantly higher in animals with the highest metal contents. Thus, we propose these two proteins, HPRT and G3PDH, as novel pollution biomarkers.