Nisha Verma

Recent advances in the use of Sus scrofa (pig) as a model system for proteomic studies

Of the numerous animal models available for proteomic studies only a small number have been successfully used in understanding human biology. To date, rodents have been widely employed in proteomic and genomic studies but often these models do not truly mimic the relevant human conditions. On the other hand, the pig shows similarity in size, shape and physiology to human and has been used as a major mammalian model for many studies concerning xenotransplantation, cardiovascular diseases, blood dynamics, nutrition, general metabolic functions, digestive‐related disorders, respiratory diseases, diabetes, kidney and bladder diseases, organ‐specific toxicity, dermatology and neurological sequelae. With the substantially improved knowledge of the structure and function of the pig genome in the last two decades it has been found that this animal shares a high sequence and chromosomal structure homology with humans. Nevertheless, in comparison to other available model organisms, very little work has been devoted to pig proteomics until recently. Keeping this in mind, the present review will highlight some of the advantages and disadvantages of pig as a model system for proteomic studies.

Review on proteomic analyses of benzo[a]pyrene toxicity

Benzo[a]pyrene (BaP), a five‐ring polycyclic aromatic hydrocarbon, is a well‐recognized environmental pollutant. Coal‐processing waste products, petroleum sludge, asphalt, creosote, and tobacco smoke, all contain high levels of BaP. Exposure to BaP elicits many adverse biological effects, including tumor formation, immunosuppression, teratogenicity, and hormonal effects. In addition to the genetic damage caused by BaP exposure, several studies have indicated the disruption of protein–protein signaling pathways. However, contrary to the large number of studies on BaP‐induced DNA damage, only few data have been gathered on its effects at the protein level. This review highlights all proteomic studies to date used for assessing the toxicity of BaP and its metabolites in various organ systems. It will also give an overview on the role proteomics may play to elucidate the mechanisms underlying BaP toxicity.

CBB staining protocol with higher sensitivity and mass spectrometric compatibility

Various CBB‐based methods for staining proteins separated by 2‐D gel electrophoresis were compared with regard to sensitivity and resolution. A modified Kangs CBB staining protocol, which we have modified, includes phosphoric acid in a concentration of 8% instead of the original 2%. This proved to be the best approach. Protein amounts as low as 2 ng and ∼2300 spots in the gel can be detected by employing this protocol. The modified procedure takes less time to carry out. Moreover, this practice is more sensitive and resolves more protein spots than most protocols reported to date and is compatible with subsequent mass spectrometric analysis.

Precipitation by lanthanum ions: A straightforward approach to isolating phosphoproteins

Posttranslational modification (PTM) of proteins, particularly phosphorylation, is a key element in the regulation of cell functions. In many signal transduction processes, PTM is a pivotal step. Various analytical methods have been proposed for the identification of phosphoproteins; however, most of these methods require sophisticated equipment. Here we present an easily applicable method of phosphoprotein enrichment. This method is based on single-step precipitation by lanthanum chloride and allows subsequent protein identification by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF-MS). The method proved its suitability for the isolation of phosphoproteins from frozen tissue and cultured cells samples after cell lysis in various buffer systems (urea/thiourea and EGTA/EDTA). The tests revealed that the isolation of phosphoproteins can be achieved with high efficiency even from complex protein mixtures. Our results indicate that lanthanum-based enrichment of phosphoproteins can be a useful tool in phosphoproteomic studies.

Benzo[a]pyrene-mediated toxicity in primary pig bladder epithelial cells: A proteomic approach

UNLABELLED The studies described in this paper deal with a sequence of cellular events induced by the environmental toxicant benzo[a]pyrene (B[a]P) that were investigated in primary urinary bladder epithelia cells (PUBEC) from pigs by using a proteomic approach. Two-dimensional (2DE) gel electrophoresis unveiled the differences in protein expression between cells exposed to 0.5 μM B[a]P for 24 h and control cells. Twenty-five differentially expressed proteins involved in DNA repair, mitochondrial dysfunction, and apoptosis were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). These findings were supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by a time-dependent increase in histone H2A.x (H2AX) phosphorylation upon B[a]P exposure. On the other hand, the expression of voltage-dependent anion channel 2 (VDAC2), cathepsin D (CTSD), heat shock protein 27 (HSP27), and heat shock protein 70 (HSP70) hinted to apoptosis occurring through the intrinsic apoptotic mitochondrial pathway. Taken together, these data suggest that B[a]P is capable of inducing DNA damage in urinary bladder epithelial cells at low concentrations during a short exposure period, thus eventually leading to cell death by apoptosis. BIOLOGICAL SIGNIFICANCE Epidemiological studies have indicated PAHs as potential candidates for initiating bladder cancer development, although the precise risk is still unknown (Kaufman et al. (2009)). In recent years, the understanding of the metabolic capacity of urothelial cells has broadened continuously; i.e. a wide range of xenobiotic metabolizing cytochrome P450 enzymes (CYP) were detected in urothelial cells from humans and animals (Roos et al., 2006; Guhe et al., 1996), thus indicating that urothelial cells are not only passively exposed to reactive metabolites but also actively by intracellularly producing reactive intermediates that can induce cancer. Moreover, small quantities of non-metabolized B[a]P and its hydroxylated derivatives have been identified in blood and urine (Rossella et al. (2009)). Thus, it appears plausible that B[a]P, a highly lipophilic compound, is taken up by the urothelium and metabolically activated to carcinogenic intermediates in these cells. In our previous studies with primary uroepithelial cells isolated from freshly slaughtered pigs we demonstrated the ability of these cells for a strong uptake of B[a]P and its conversion to the oxidative metabolite (3-OH-B[a]P) (Verma et al. (2012)). The present study is a continuation of this previous work exhibiting the effects of B[a]P exposure on cellular functions of PUBEC. The results indicated caspase-dependent apoptosis induced by B[a]P due to DNA damage (possibly lethal double-strand breaks as indicated by H2AX phosphorylation). Taken together, these studies provide strong evidence for the ability of B[a]P to act as a bladder carcinogen.

Proteome and phosphoproteome of primary cultured pig urothelial cells

Epithelial tissue lining the inner side of the urinary bladder is the most common target for bladder cancer‐related diseases. Bladders of freshly slaughtered pigs were utilised for a comprehensive analysis of the proteome and phosphoproteome of bladder epithelial cells. Following protein separation by 2‐D gel electrophoresis and identification by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) the first proteome and phosphoproteome maps of pig urinary bladder epithelial cells (PUBEC) were established. A total of 120 selected protein spots were identified. By using the La3+ enrichment method further developed in our laboratory we identified 31 phosphoproteins with minimal contamination by non‐phosphopeptides. The 2‐DE map of pig urothelial cells may prove as a useful tool for studies on uroepithelial biology, and the analysed phosphoproteins expression pattern, together with the whole cell proteome, will be helpful for identifying the proteins involved in bladder‐related diseases.

Proteomic analysis of human bladder epithelial cells by 2D blue native SDS-PAGE reveals TCDD-induced alterations of calcium and iron homeostasis possibly mediated by nitric oxide.

A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity.

Gel‐based separation of phosphoproteins in samples stored in urea/thiourea after precipitation by lanthanum chloride

The recent introduction of the La3+ precipitation method for the enrichment of phosphoproteins allows a gel‐based analysis of these posttranslationally modified proteins. However, if this method is applied to cell lysates stored in urea‐containing lysis buffer for an extended period of time, incomplete phosphoprotein recovery is observed. We ascribe this effect to the presence of urea in the lysis buffer. To overcome this problem various strategies were tested, where cell lysates stored at least for one year were utilized. By applying an optimized protocol approximately 250 proteins could be observed following separation by 2DE.

Dose-Dependent Response to 3-Nitrobenzanthrone Exposure in Human Urothelial Cancer Cells

A product of incomplete combustion of diesel fuel, 3-nitrobenzanthrone (3-NBA), has been classified as a cancer-causing substance. It first gained attention as a potential urinary bladder carcinogen due to the presence of its metabolite in urine and formation of DNA adducts. The aim of the present study was to characterize the dose-response relationship of 3-NBA in human urothelial cancer cell line (RT4) exposed to concentrations ranging from 0.0003 μM (environmentally relevant) to 80 μM by utilizing toxicological and metabolomic approaches. We observed that the RT4 cells were capable of bioactivation of 3-NBA within 30 min of exposure. Activity measurements of various enzymes involved in the conversion of 3-NBA in RT4 cells demonstrated NAD(P)H:quinone oxidoreductase (NQO1) as the main contributor for its bioactivation. Moreover, cytotoxicity assessment exhibited an initiation of adaptive mechanisms at low dosages, which diminished at higher doses, indicating that the capacity of these mechanisms no longer suffices, resulting in increased levels of intracellular reactive oxygen species, reduced proliferation, and hyperpolarisation of the mitochondrial membrane. To characterize the underlying mechanisms of this cellular response, the metabolism of 3-NBA and metabolomic changes in the cells were analyzed. The metabolomic analysis of the cells (0.0003, 0.01, 0.08, 10, and 80 μM 3-NBA) showed elevated levels of various antioxidants at low concentrations of 3-NBA. However, at higher exposure concentrations, it appeared that the cells reprogrammed their metabolism to maintain the cell homeostasis via activation of pentose phosphate pathway (PPP).