Low concentration of anti‐7,8‐dihydroxy‐9,10‐epoxy‐7,8,9,10‐tetrahydrobenzo[a]pyrene induces alterations of extracellular protein profile of exposed epithelial cells

7,8‐Dihydroxy‐9,10‐epoxy‐7,8,9,10‐tetrahydrobenzo[a]pyrene (BPDE) exposure induces adduct formation and oxidative damage on DNA, and consequently triggers complicated stress responses, including such responses as signaling pathway activation, cell cycle arrest, DNA repair, translesion DNA synthesis and mutagenesis. In the present study, 2‐DE and MALDI‐TOF MS were employed to analyze the differential extracellular protein patterns of human amniotic epithelial cells (FL cells) after exposure to 5 nM BPDE and control. As a result, one protein spot that appeared in the culture medium of BPDE treatment group was successfully identified as 14‐3‐3ζ, and three up‐regulated protein spots were identified as annexin A3, annexin V and hydroxypyruvate isomerase homolog. Among them, 14‐3‐3ζ was further detected in some pleural fluid specimens also. These results demonstrate that BPDE exposure can induce alterations of extracellular protein profiles of exposed cells, which may be served as a starting point for searching candidate biomarkers for benzo[a]pyrene exposure.     

dUTP Pyrophosphatase, its appearance in extracellular compartment may serve as a potential biomarker for N-methyl-N'-nitro-N-nitrosoguanidine exposure in mammalian cells

The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a model chemical widely used for studying the molecular events induced by the widespread environmental N-nitroso alkylating carcinogen. Many studies have focused on understanding MNNG-induced mutagenesis and carcinogenesis. However, the search for specific indicators of MNNG exposure is still underway. In this study, we analyzed the proteins in culture medium of human amnion epithelial cells (FL cells) exposed to MNNG by 2-DE followed by MALDI-TOF MS, in the hope of finding a specific protein marker suitable for MNNG risk assessment. Image visualization and statistical analysis indicated that 12 spots appeared and 4 spots up-regulated after MNNG exposure. Most of them were identified by MS. These proteins include nuclear isoform of dUTP pyrophosphatase (DUT-N), phosphoglycerate mutase 1, heparan sulfate proteoglycan perlecan, etc., which are involved in multiple cellular functions. Interestingly, 2-DE and MS analyses of cell lysate exposed to MNNG revealed that DUT-N was down-regulated. The appearance of DUT-N in culture medium and its down-regulation in cell lysate was confirmed by Western blot. These data suggest that these proteins, especially DUT-N, could be used as candidate biomarkers for monitoring MNNG exposure.     

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 5 mg/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.     

Proteome profile changes during transdifferentiation of NRP-152 rat prostatic basal epithelial cells

NRP-152 is an androgen responsive, non-tumorigenic cell line, which shows basal epithelial cell characteristics under normal growth conditions. It has been noted that NRP-152 undergoes morphological and cytoskeletal changes toward its luminal counterpart NRP-154 when it is grown under growth restrictive conditions. We have extensively investigated the details of protein change of NRP-152 during transdifferentiation using proteomic techniques. NRP-152 cells were cultured under normal and growth restrictive media conditions for 3, 5 days. NRP-154 cells were normally cultured. Protein samples were submitted to 2D gel electrophoresis and silver stained. Protein patterns on the gels were comparatively analysed using Melanie III software. Protein spots exhibiting significant changes in NRP-152 cells during the time course were excised and subjected to in-gel tryptic digestion. After 6 days of growth restrictive conditions in NRP-152, the cells were morphologically changed resembling luminal phenotype. Of the 35 protein spots that were up-reglated, 20 proteins from 21 spots were identified by peptide mass fingerprinting and, of 21 proteins spots that were down-regulated, 10 proteins from 12 spots were identified as landmark proteins. Our study confirmed that basal NRP-152 cells were proportionally transdifferentiated into luminal featuring cells according to the duration of growth restrictive culture conditions. This suggests that human prostatic basal epithelial cells may be changed into luminal cells under certain conditions. Proteomic approach enabled us to identify 30 proteins involved in this differentiation with a single experiment. These proteins will be subjected to further functional studies to evaluate their possible roles related to cellular differentiation. These data strongly support that proteomics is a very powerful approach for studying physiologic and pathologic cellular changes such as differentiation and carcinogenesis.     

Differential expression profile of membrane proteins in zebrafish (Danio rerio) brain exposed to methyl parathion

Methyl parathion (MP) is a widely used organophosphorus pesticide, which has been related to a broad spectrum of toxic effects on environmental organisms. The present study investigated the changes in the protein profile of enriched membrane fraction from zebrafish (Danio rerio) brain exposed to three concentrations (0.5, 1 and 2 mg/L) of MP. 2-DE revealed that the abundance of 21 protein spots was significantly changed by MP stress. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database search, 16 protein spots were identified as membrane proteins, among which 8 were down-regulated, while 8 were up-regulated. These proteins are mainly involved in oxidative stress response, signal transduction, metabolism, protein synthesis and degradation, neuroplasticity and regeneration as well as synaptic transmission. These results may aid our understanding of the mechanism of MP-induced neurotoxicity and provide the possibility of the establishment of candidate biomarkers of MP.     

Induction of a cell-survival adaptive response in MRC-5 cells by hydroquinone

Although it is known that some cell types exhibit an adaptive response to low levels of cytotoxic agents, its molecular mechanism is still unclear and it has yet to be established whether this is a universal phenomenon that occurs in all cell types in response to exposure to every chemical. Hydroquinone is a synthetically produced as well as naturally occurring chemical. Human exposure to hydroquinone is predominantly through diet, cigarette smoke and occupational contact. Here, we asked whether exposure of human lung embryonic MRC-5 fibroblasts to low doses of hydroquinone leads to a cell-survival adaptive response. We further examined the possible mechanisms of an adaptive response using proteomics. We found that exposure of MRC-5 cells to low levels of hydroquinone resulted in adaptation to further exposure to lethal doses of hydroquinone at the cell-survival level, measured using the alamarBlue assay, lactate dehydrogenase leakage assay and Annexin V-FITC/PI staining. To determine the polypeptide products involved in the adaptive response, two-dimensional electrophoresis combined with mass spectrometry was performed. Twenty-three protein spots were significantly changed during the adaptive response. Among them, 21 protein spots were identified by peptide mass fingerprinting and/or peptide sequence analysis by MALDI-TOF-TOF. The identified proteins included proteins involved in energy metabolism, protein folding, redox regulation, cell structure and cell signaling. Our data suggest that the hydroquinone-induced adaptive response is a complex process involving in a modulation of diverse cellular functions, and that the redox regulation might be a common mechanism during the adaptive response.     

Induction of a cell-survival adaptive response in MRC-5 cells by hydroquinone

Although it is known that some cell types exhibit an adaptive response to low levels of cytotoxic agents, its molecular mechanism is still unclear and it has yet to be established whether this is a universal phenomenon that occurs in all cell types in response to exposure to every chemical. Hydroquinone is a synthetically produced as well as naturally occurring chemical. Human exposure to hydroquinone is predominantly through diet, cigarette smoke and occupational contact. Here, we asked whether exposure of human lung embryonic MRC-5 fibroblasts to low doses of hydroquinone leads to a cell-survival adaptive response. We further examined the possible mechanisms of an adaptive response using proteomics. We found that exposure of MRC-5 cells to low levels of hydroquinone resulted in adaptation to further exposure to lethal doses of hydroquinone at the cell-survival level, measured using the alamarBlue assay, lactate dehydrogenase leakage assay and Annexin V-FITC/PI staining. To determine the polypeptide products involved in the adaptive response, two-dimensional electrophoresis combined with mass spectrometry was performed. Twenty-three protein spots were significantly changed during the adaptive response. Among them, 21 protein spots were identified by peptide mass fingerprinting and/or peptide sequence analysis by MALDI-TOF-TOF. The identified proteins included proteins involved in energy metabolism, protein folding, redox regulation, cell structure and cell signaling. Our data suggest that the hydroquinone-induced adaptive response is a complex process involving in a modulation of diverse cellular functions, and that the redox regulation might be a common mechanism during the adaptive response.     

Proteomic evaluation of cadmium toxicity on the midge Chironomus riparius Meigen larvae

Heavy-metal pollution of aquatic ecosystems is a widespread phenomenon after industrial consumption. Whether aquatic organisms are adapted to the heavy-metal pollutants or not, such environmental stress causes changes in physiological responses. In this study, the aquatic midge, Chironomus riparius Meigen, was used to find changes of expression of proteins in relation to cadmium exposure. Dose-response relationships between cadmium concentrations and mortality of 3rd instar midge larvae were observed and the protein levels were compared using PD-Quest after 2-DE. Comparing the intensity of protein spots, 21 proteins decreased and 18 proteins increased in response to cadmium treatment. With increased proteins, three enzymes such as S-adenosylmethionine decarboxylase, O-methyltransferase, and aspartokinase were involved in the glutathione biosynthesis and a key enzyme regulating fatty acid biosynthesis, oleyl-acyl carrier protein thioesterase was also identified. According to the functional classification of decreased levels of proteins, they were involved in energy production, protein fate, nucleotide biosynthesis, cell division, transport and binding, signal transduction, and fatty acid and phospholipid metabolism in the cell. In addition, phenol hydroxylase, thioesterase, zinc metalloprotease, and aspartate kinase were newly expressed after cadmium exposure at the concentration of the LC(10 )value. Therefore, these proteins seem to be potential biomarkers for cadmium exposure in the aquatic ecosystems.     

Cisplatin treatment leads to changes in nuclear protein and microRNA expression

Using a proteomic approach, we have previously shown that exposure to different concentrations of cisplatin during a 12-h period can lead to changes in nuclear protein expression and alternative splicing in HeLa cells. To further shed light on the DNA damage response (DDR) induced by cisplatin, we examined the nuclear proteome profiles of HeLa cells treated with 5μM cisplatin for different times (2, 12, and 24h). Two-dimensional electrophoresis (2-DE) identified 98 differentially expressed proteins in cisplatin-treated cells as compared to control cells. Among them, 54 spots (55%) were down-regulated and 44 spots (45%) were up-regulated. 51 spots were subjected to Matrix-assisted-laser-desorption-ionization Time-of-flight/time-of-flight Mass spectrometry (MALDI-TOF/TOF MS) identification, and 40 spots were identified. Among these, 22 proteins were located in nucleus. These proteins were involved in stress response, cell cycle and division, apoptosis, mRNA processing, transport, splicing and microRNA (miRNA) maturation. The changed expression of Annexin A1 and Lamin B1 were confirmed by Western blot. The role of Annexin A1 in the response to cisplatin-induced DNA damage was further analyzed, and it was shown that after Annexin A1 knockdown, cisplatin-induced DNA damage was significantly increased. In addition, the changed expression of several miRNAs was also observed by quantitative real-time PCR (qRT-PCR). Taken together, these data indicate that cisplatin-induced DDR is a complex process, and that those proteins identified by proteomics can lead to new directions for a better understanding of this process.     

Proteome changes in human bronchoalveolar cells following styrene exposure indicate involvement of oxidative stress in the molecular‐response mechanism

Styrene is a volatile organic compound that is widely used as an intermediate in many industrial settings. There are known adverse health effects at environmentally significant concentrations, but little is known about the molecular effect of exposure to styrene at sub‐acute toxic concentrations. We exposed human lung epithelial cells, at a wide range of concentrations (1 mg/m³–10 g/m³), to styrene and analyzed the effects on the proteome level by 2‐DE, where 1380 proteins spots were detected and 266 were identified unambiguously by MS. A set of 16 protein spots were found to be significantly altered due to exposure to styrene at environmentally significant concentrations of 1–10 mg/m³ (0.2–2.3 ppm). Among these, superoxide dismutase as well as biliverdin reductase A could be correlated with the molecular pathway of oxidative stress, while eukaryotic translation initiation factor 5A‐1, ezrin, lamin B2 and voltage‐dependent anion channel 2 have been reported to be involved in apoptosis. Treatment with styrene also caused the formation of styrene oxide–protein adducts, specifically for thioredoxin reductase 1. These results underline the relevance of oxidative stress as a primary molecular response mechanism of lung epithelial cells to styrene exposure at indoor‐relevant concentrations.     

Downregulation of Cellular Protective Factors of Rumen Epithelium in Goats Fed High Energy Diet

Energy-rich diets can challenge metabolic and protective functions of the rumen epithelial cells, but the underlying factors are unclear. This study sought to evaluate proteomic changes of the rumen epithelium in goats fed a low, medium, or high energy diet. Expression of protein changes were compared by two-dimensional differential gel electrophoresis followed by protein identification with matrix assisted laser desorption ionisation tandem time-of-flight mass spectrometry. Of about 2,000 spots commonly detected in all gels, 64 spots were significantly regulated, which were traced back to 24 unique proteins. Interestingly, the expression profiles of several chaperone proteins with important cellular protective functions such as heat shock cognate 71 kDa protein, peroxiredoxin-6, serpin H1, protein disulfide-isomerase, and selenium-binding protein were collectively downregulated in response to high dietary energy supply. Similar regulation patterns were obtained for some other proteins involved in transport or metabolic functions. In contrast, metabolic enzymes like retinal dehydrogenase 1 and ATP synthase subunit beta, mitochondrial precursor were upregulated in response to high energy diet. Lower expressions of chaperone proteins in the rumen epithelial cells in response to high energy supply may suggest that these cells were less protected against the potentially harmful rumen toxic compounds, which might have consequences for rumen and systemic health. Our findings also suggest that energy-rich diets and the resulting acidotic insult may render rumen epithelial cells more vulnerable to cellular damage by attenuating their cell defense system, hence facilitating the impairment of rumen barrier function, typically observed in energy-rich fed ruminants.     

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.     

Proteomic analysis reveals different protein changes during endothelin-1- or leukemic inhibitory factor-induced hypertrophy of cardiomyocytes in vitro

Proteomic analyses are being increasingly used to identify protein changes accompanying changes in cellular function. An advantage of this approach is that it is largely unbiased by prior assumptions on the importance of each protein in the process under investigation. Here we have evaluated the protein changes that accompany the enlargement, or hypertrophy, of cardiomyocytes in culture. We have taken the additional step of comparing the changes that accompany a concentric hypertrophic phenotype stimulated by endothelin-1 exposure and an eccentric hypertrophic phenotype stimulated by leukemic inhibitory factor exposure. Following separation of the protein extracts by two-dimensional gel electrophoresis and staining with colloidal Coomassie Brilliant Blue, we identified 15 protein spots representing 12 proteins that changed in response to endothelin-1. In comparison, 17 protein spots representing 17 proteins changed in response to leukemic inhibitory factor, and 35 protein spots representing 28 proteins did not change under these conditions. Importantly the well established marker of cardiac pathology, atrial natriuretic factor, was identified as a protein up-regulated by both endothelin-1 and leukemic inhibitory factor (2.4+/-0.8- and 2.2+/-0.3-fold, respectively). However, nine of the observed protein changes occurred for only endothelin-1, whereas 11 of the changes occurred only with leukemic inhibitory factor exposure. These two different stimuli are therefore able to elicit unique changes in the protein expression profile of cardiac myocytes. This is consistent with the differences in morphologies noted as well as the different signaling pathways utilized by these different stimuli.     

DIGE-based protein expression analysis of B[a]P-exposed hepatoma cells reveals a complex stress response including alterations in oxidative stress, cell cycle control, and cytoskeleton motility at toxic and subacute concentrations

Although the effects of high concentrations of the carcinogen benzo[a]pyrene (B[a]P) have been studied extensively, little is known about its effects at subacute toxic concentrations, which are typical for environmental pollutants. We exposed murine Hepa1c1c7 cells to a toxic concentration (5 μM) and a subacute concentration (50 nM) of B[a]P over a period of 2-24 h to differentiate between acute and pseudochronic effects and conducted a time-course analysis of B[a]P-influenced protein expression by DIGE. In total, a set of 120 spots were found to be significantly altered due to B[a]P exposure of which 112 were subsequently identified by mass spectrometry. Clustering and principal component analysis were conducted to identify sets of proteins responding in a concerted manner to the exposure. Our results indicate an immediate response to the contaminant at the protein level and demonstrate that B[a]P exposure alters the cellular response by disturbing proteins involved in oxidative stress, cell cycle regulation, apoptosis, and cytoskeleton organization. Furthermore, network analysis of protein-protein interactions revealed a complex network of interacting, B[a]P-regulated proteins mostly belonging to the cytoskeleton organization and several signal transduction pathways.     

Calcium oxalate dihydrate crystal induced changes in glycoproteome of distal renal tubular epithelial cells

Calcium oxalate dihydrate (COD) crystals can adhere onto the apical surface of renal tubular epithelial cells. This process is associated with crystal growth and aggregation, resulting in kidney stone formation. Glycoproteins have been thought to play roles in response to crystal adhesion. However, components of the glycoproteome that are involved in this cellular response remain largely unknown. Our present study therefore aimed to identify altered glycoproteins upon COD crystal adhesion onto tubular epithelial cells representing distal nephron, the initiating site of kidney stone formation. Madin-Darby Canine Kidney (MDCK) cells were maintained in culture medium with or without COD crystals for 48 h (n = 5 flasks per group). Cellular proteins were extracted, resolved by 2-DE and visualized by SYPRO Ruby total protein stain, whereas glycoproteins were detected by Pro-Q Emerald glycoprotein dye. Spot matching and quantitative intensity analysis revealed 16 differentially expressed glycoprotein spots, whose corresponding total protein levels were not changed by COD crystal adhesion. These altered glycoproteins were successfully identified by Q-TOF MS and/or MS/MS analyses, and potential glycosylation sites were identified by the GlycoMod tool. For example, glycoforms of three proteasome subunits (which have a major role in regulating cell-cell dissociation) were up-regulated, whereas a glycoform of actin-related protein 3 (ARP3) (which plays an important role in cellular integrity) was down-regulated. These coordinated changes implicate that COD crystal adhesion induced cell dissociation and declined cellular integrity in the distal nephron. Our findings provide some novel insights into the pathogenic mechanisms of kidney stone disease at the molecular level, particularly cell-crystal interactions.