He-Qing Huang

Construction of nanometer cisplatin core-ferritin (NCC-F) and proteomic analysis of gastric cancer cell apoptosis induced with cisplatin released from the NCC-F

Both transmission electron microscopy (TEM) and fluorescence spectrometry were used to reveal the characteristics of both subunit disassociation and recombination in apo-pig pancreas ferritin (apoPPF) in an alkaline medium ranging reversibly from pH 7.0 to 13.0. The experimental results indicated that apoPPF could be completely disassociated into 24 free subunits at pH 13.0, and then these subunits could be quickly reassembled into the original apoPPF once the pH of the reactive medium was returned to pH7.0. This novel and simple method could be used to effectively construct a novel nanometer cisplatin core-PPF (NCC-PPF). The major characteristics of NCC-PPF were investigated using various analytical methods such as ultraviolet-spectrometry, circular dichroism spectrometry and TEM, which indicated that its molecular structure was still similar to that of the original apoPPF. Results from the inductively coupled plasma mass spectrometer (ICP-MS) method showed that 11.26 cisplatin (CDDP) molecules were successfully packaged within the NCC-PPF shell, indicating that each molecule of apoPPF had the ability to enwrap 11.26 CDDP molecules for constructing the NCC-PPF. Flow cytometry showed that NCC-PPF also had the ability to release CDDP for inducing the apoptosis of gastric cancer cells BGC823 (GCC), but this phenomenon could scarcely be observed using apoPPF. A differential proteomic technique using two-dimensional electrophoresis (2-DE) gels selected and identified the differential proteins from cell apoptosis in order to reveal the molecular pathway of GCC apoptosis by both NCC-PPF and free CDDP, giving 13 differential expression proteins. These differential proteins could be further classified into six groups, which were described as being involved in the regulation of apoptosis, RNA transcription, oxidative stress response, signal transduction, cell metabolism, and cytoskeleton changes. In addition, a real-time PCR method was used to prove the expression level of mRNA and to identify the reliability of the protein expression according to these differential proteins, which indicated that the mRNA level changes of six differential proteins corresponded to those of its differential protein expression in 2-DE gels. These studies played an important role in reasonably revealing the different pathways of GCC apoptosis induced with both the CDDP released by NCC-PPF and the free CDDP. We thus suggest that apoPPF has great potential for constructing a nanometer carrier filled with various drugs for application in clinical work.

Proteomic analysis of methyl parathion-responsive proteins in Sparus latus liver

The contamination of marine ecosystems by organophosphate pesticides is of great concern. The use of protein expression profiles may provide a good method to help us understand the methyl parathion (MP) toxicity to aquatic organisms. In this study, Sparus latus, was selected as the target organism. The toxicological effects of MP were investigated after 24 h exposure using proteomics to analyze their liver tissues. Certain enzyme activity parameters of the liver extracts were also examined, including CAT. After analyzing the proteomic profile of the liver using 2D gel electrophoresis, we found that the protein expression levels of 25 spots increased or decreased significantly in the exposed groups. Sixteen of the 25 protein spots were successfully identified using MALDI-TOF MS/MS. These proteins were roughly categorized into diverse functional classes such as cell redox homeostasis, metabolic processes and cytoskeleton system. These data demonstrated that proteomics was a powerful tool to provide valuable insights into the possible mechanisms of toxicity of MP contaminants in aquatic species. Additionally, these data may provide novel biomarkers for evaluation of MP contamination in the environment.

Proteomic changes in response to acute cadmium toxicity in gill tissue of Paralichthys olivaceus

In the present study, we developed a two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique for examining the response of the proteome from gill tissue of Paralichthys olivaceus (POGT) to acute cadmium (AC) toxicity. Approximately 700 protein spots were detected from the gill sample when applying a 600μg protein 2D-PAGE gel in the pH range 5.0-8.0, and approximately 400 of these were identified by peptide mass fingerprinting (PMF) and database search. Compared to a control sample, significant changes were visualized in 18 protein spots exposed to seawater cadmium acute toxicity at 10.0ppm for 24h. Among these spots, two were up-regulated, one was down-regulated, seven showed low expression, and eight showed high expression. The collected spots were further identified by PMF and database search. Ten of the 18 proteins identified on the 2D-PAGE gel, including heat shock protein 70 and calcium-binding protein, demonstrated a synchronous response to AC, and we suggest that the variable levels and trends of these spots on the gel might be utilized as biomarker profiles to investigate cadmium contamination levels in seawater and to evaluate the degree of risk of human fatalities. The experimental results emphasize that the application of multiple biomarkers has an advantage over single biomarkers for monitoring levels of heavy metal contamination in seawater.

Alterations of protein profile in zebrafish liver cells exposed to methyl parathion: A membrane proteomics approach

Methyl parathion (MP) is an extensively used organophosphorus pesticide, which has been associated with a wide spectrum of toxic effects on environmental organisms. The aim of this study is to investigate the alterations of membrane protein profiles in zebrafish liver (ZFL) cell line exposed to MP for 24 h using proteomic approaches. Two-dimensional gel electrophoresis revealed a total of 13 protein spots, whose expression levels were significantly altered by MP. These differential proteins were subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis, and nine proteins were identified to be membrane proteins, among which seven were up-regulated, while two were down-regulated. In addition, the mRNA levels corresponding to these differential membrane proteins were further analyzed by quantitative real-time PCR. And the differential expression of arginase-2 was specially validated via Western blotting. Regarding the physiological functions, these proteins are involved in molecular chaperon, cytoskeleton system, cell metabolism, signal transduction, transport and hormone receptor respectively, suggesting the complexity of MP-mediated toxicity to ZFL cell. These data could provide useful insights for better understanding the hepatotoxic mechanisms of MP and develop novel protein biomarkers for effectively monitoring MP contamination level in aquatic environment.

Effects of chronic tramadol exposure on the zebrafish brain: A proteomic study

Tramadol hydrochloride (TH), has become the most prescribed opioid worldwide. However, its neurotoxicity and abuse potential are not well documented. In the present study, TH administration induced abnormal behavior and body and brain mean weight loss. Two principal metabolites O- and N-desmethyltramadol were detected in the brain tissue, and N-desmethyltramadol was the main metabolite produced. A total of 30 differential protein spots were identified using semi-quantitative 2D-PAGE and proteomic analyses, and classified into 13 categories, in which subtypes of 14-3-3 proteins, creatine kinase, ATP synthase beta chain, and tubulin were identified at the separated location on the gels 3, 3, 4, and 11 times respectively. Many TH responsive proteins have functions related to oxidative stress, including 14-3-3 proteins, creatine kinase BB, ubiquitin carboxy-terminal hydrolase L-1, ATP synthase, synaptosome-associated protein, tubulin and actin. Irrespective of oxidative damage, other pathways affected include apoptosis, energy metabolism, signal disorders, and cytoskeletal structure. Ultrastructural observation of mitochondria showed a series of morphological changes in the case of TH exposure.

Purification, electrophoretic behavior, and kinetics of iron release of liver ferritin of Dasyatis akajei

From the liver of fish Dasyatis akajei, ferritin has been isolated by thermal denaturation and ammonium sulfate fractionation and then further purified by anion exchange chromatography and gel exclusion chromatography. The molecular weight of the liver ferritin of D. akajei (DALF) was measured to be 400 kDa by PAGE. Moreover, SDS-PAGE experimentation indicates that protein shell of DALF consists of the H and L subunits with molecular weight of 18 and 13 kDa, respectively. Using isoelectric focusing with pH ranging from 5.0 to 6.0, the ferritin purified by the PAGE exhibited three bands with different pI values in the gel slab. Diameters of the protein shell and iron core were also investigated by transmission electron microscope and determined to be 10–12 nm and 5–8 nm, respectively. A kinetic study of DALF reveals that the rate of self-regulation of the protein shell rather than the complex surface of the iron core plays an important role in forming a process for iron release with mixed orders.

Proteomic analysis of methyl parathion-responsive proteins in zebrafish (Danio rerio) brain.

Methyl parathion (MP), an organophosphorus pesticide used worldwide, has been associated with a wide spectrum of toxic effects on organisms in the environment. This study set out to analyze the alteration of protein profiles in MP-exposed zebrafish (Danio rerio) brain and find the proteins responsive to MP toxicity. Zebrafish were subjected to 1, 3 and 5mg/L MP and the proteomic changes in their brains were revealed using two-dimensional gel electrophoresis. Six protein spots were observed to be significantly changed by MP exposure. Among these, 4 spots were down-regulated, while 2 spots were up-regulated. These altered spots were excised from the gels and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry and database searching. The results indicate that these proteins were involved in binding, catalysis, regulation of energy metabolism and cell structure. These data may provide novel biomarkers for the evaluation of MP contamination and useful insights for understanding the mechanisms of MP toxicity.

Transferrin-cisplatin specifically deliver cisplatin to HepG2 cells in vitro and enhance cisplatin cytotoxicity

Cisplatin is a major broad-spectrum chemotherapeutic agent, however, its dose-dependent side effects limit the administration of large doses. Presently, developing a drug targeted delivery system is suggested as one of the most promising approaches to minimize the side effects of cisplatin. Here, we found that each human serum transferrin (HTf) has the potential to bind with over 22 cisplatins, and the complex of apo-HTf-cisplatin can specifically deliver cisplatin to HepG2 cells (human hepatocellular liver carcinoma cell line) in vitro, and facilitate HepG2 cells to apoptosis. Moreover, proteomics methods revealed that the abundances of 23 proteins in HepG2 cells were remarkably altered in response to cisplatin/apo-HTf-cisplatin exposure, and Realtime-PCR revealed that a number of important genes related to chemotherapeutic cytotoxicity and chemotherapeutic resistance are differentially transcribed between the HepG2 cells of cisplatin exposed and HTf-cisplatin exposed. The pathway analysis of the differentially expressed proteins and gene transcriptions indicated that those regulated proteins and gene transcriptions are involved in apoptosis regulation, transcription, cell cycle control, protein biosynthesis, energy metabolism, signal transduction, protein binding and other functions. It indicated that the cisplatin toxicity in HepG2 cell is diverse, the transport process has an effect on the cisplatin cytotoxicity, and the mechanism of the apoptosis of HepG2 cells induced by apo-HTf-cisplatin is different from that of cisplatin.

Application of capillary isoelectric focusing and peptide mass fingerprinting in carbohydrate-deficient transferrin detection

Carbohydrate-deficient transferrin (CDT) is a specific biomarker of alcohol abuse and is widely used in clinical diagnosis to detect and follow up excessive alcohol consumption. However, false %CDT results still exist in CDT detection, because of interference from genetic variants and the lack of standardization in CDT analysis. Therefore, it is still very important to find a method with high sensitivity and high accuracy for CDT detection. Here, we compared the detection sensitivity and accuracy of pI values based methods [isoelectric focusing on polyacrylamide gel electrophoresis (IEF-PAGE) and capillary isoelectric focusing (CIEF)] with hydrophobic characteristic based methods [reversed-phase high-performance liquid chromatography (RP-HPLC)] on CDT detection. Moreover, we investigated the potential of peptide mass fingerprinting (PMF), a method based on the mass spectrometry to identify human transferrin (HTf) variants including CDT isoforms and genetic variants, based on their specific peptide masses. Results indicated that PMF can identify HTf variants including CDT isoforms and genetic variants based on their specific peptides, and CIEF showed higher sensitivity detection of HTf variants than RP-HPLC and IEF-PAGE did. Accordingly, we suggest that PMF is suitable for identifying CDT with high accuracy, and CIEF has potential for detection of CDT and genetic variants with high sensitivity; moreover, they are both worth further investigation in clinical diagnosis.

Proteomic approach for identifying gonad differential proteins in the oyster (Crassostrea angulata) following food-chain contamination with HgCl2

UNLABELLED Hg discharged into the environmental waters can generally be bioaccumulated, transformed and transmited by living organisms, thus resulting in the formation of Hg-toxicity food chains. The pathway and toxicology of food chain contaminated with environmental Hg are rarely revealed by proteomics. Here, we showed that differential proteomics had the potential to understand reproduction toxicity mechanism in marine molluscs through the Hg-contaminated food chain. Hg bioaccumulation was found in every link of the HgCl2-Chlorella vulgaris-oyster-mice food chain. Morphological observations identified the lesions in both the oyster gonad and the mice ovary. Differential proteomics was used to study the mechanisms of Hg toxicity in the oyster gonad and to find some biomarkers of Hg contamination in food chain. Using 2-DE and MALDI-TOF/TOF MS, we identified 13 differential protein spots, of which six were up-regulated, six were down-regulated, while one was undecided. A portion of these differential proteins was further confirmed using real-time PCR and western blotting methods. Their major functions involved binding, protein translocation, catalysis, regulation of energy metabolism, reproductive functioning and structural molecular activity. Among these proteins, 14-3-3 protein, GTP binding protein, arginine kinase (AK) and 71kDa heat shock connate protein (HSCP 71) are considered to be suitable biomarkers of environmental Hg contamination. Furthermore, we established the gene correspondence, responding to Hg reproductive toxicity, between mouse and oyster, and then used real-time PCR to analyze mRNA differential expression of the corresponding genes in mice. The results indicated that the mechanism of Hg reproductive toxicity in mouse was similar to that in oyster. We suggest that the proteomics would be further developed in application research of food safety including toxicological mechanism. BIOLOGICAL SIGNIFICANCE It is well known that mercury (Hg) is one of the best toxic metal elements in nature. The research reports as previously described indicated that multiple mercury compounds can directly contaminate the aquatic animals by flowing of water body and through the diffusion of air. The pollution sources of the mercury compounds in marine water were mainly found from the pathways such as steam power plant and mineral exploitation which are located on the inshore. Of note, after being released into environmental waters, mercury compounds undergo the processes of bioaccumulation, transformation and transmission in living organisms, thus resulting in the multiple forms of Hg found in Hg-toxicity food chains, and among them, methyl mercury (MeHg) showing the high toxic characteristics is the main form of Hg. The abundant reports indicated that the metal salts were easily found within the various organs of the animals, but it is difficult to judge the level of its perniciousness according to its content only in vivo. Here, the algae to have been contaminated by the mercury compounds have the ability for contaminating both the fish and shellfish as food pathway quickly. If these fish and shellfish edible as food will be taken by human, they will further affect the human health badly. However, studies about their perniciousness are rarely reported, especially in using proteomics. The oysters as normal food are largely consumed in Southern China, especially in Xiamen City. Similarly, a pathway question that the contaminated oysters can effect on the human health such as cancer is unclear or poorly understood. Here, we showed that an analytical technology such as differential proteomics has potential to understand toxicity mechanism induced by Hg-contamination through the food pathway. It is for reason that the oyster proteomics including relative analytical methods have been used to reveal the contaminant level and to determine its perniciousness using toxic algae as food. Here, we also indicated that the research here shows great significance for both analysis of food safety and toxicology of the metal compounds. In addition, a few biomarkers have shown their strong potential for monitoring the level of Hg pollution in sea in the manuscript and gene correspondence between mouse and oyster, the two contiguous links of the Hg-contaminated food chain, was further investigated to better illustrate our finding in the analysis of food chain proteomics. Moreover, similar research work is rarely reported compared to the current proteomic development, showing that a lot of novel results by proteomic methods in the manuscript have strong potential for developing the new area of food chain proteomics.