Utility of a multimodal computer-based assessment format for assessment with a higher degree of reliability and validity.

Multiple choice questions (MCQs) suffer from cueing, item quality and factual knowledge testing. This study presents a novel multimodal test containing alternative item types in a computer-based assessment (CBA) format, designated as Proxy-CBA. The Proxy-CBA was compared to a standard MCQ-CBA, regarding validity, reliability, standard error of measurement, and cognitive load, using a quasi-experimental crossover design. Biomedical students were randomized into two groups to sit a 65-item formative exam starting with the MCQ-CBA followed by the Proxy-CBA (group 1, n = 38), or the reverse (group 2, n = 35). Subsequently, a questionnaire on perceived cognitive load was taken, answered by 71 participants. Both CBA formats were analyzed according to parameters of the Classical Test Theory and the Rasch model. Compared to the MCQ-CBA, the Proxy-CBA had lower raw scores (p < 0.001, η2 = 0.276), higher reliability estimates (p < 0.001, η2 = 0.498), lower SEM estimates (p < 0.001, η2 = 0.807), and lower theta ability scores (p < 0.001, η2 = 0.288). The questionnaire revealed no significant differences between both CBA tests regarding perceived cognitive load. Compared to the MCQ-CBA, the Proxy-CBA showed increased reliability and a higher degree of validity with similar cognitive load, suggesting its utility as an alternative assessment format.

An in vitro model for hypertrophic adipocytes: Time-dependent adipocyte proteome and secretome changes under high glucose and high insulin conditions.

Obesity is the consequence of a positive energy balance and characterized by enlargement of the adipose tissue, which in part is due to hyperplasia and hypertrophy of the adipocytes. Not much is known about the transition of normal mature adipocytes to the hypertrophic state, which in vivo is very hard to study. Here, we have maintained mature human SGBS cells as a surrogate for adipocytes, changes of morphological and molecular metabolism of the adipocytes were monitored over the first 4 days and the last 4 days. In total, 393 cellular proteins and 246 secreted proteins were identified for further analysis. During the first 4 days of high glucose and insulin, the adipocytes seemed to prefer pyruvate as energy source, whereas beta-oxidation was down-regulated supporting lipid loading. Over time, lipid droplet fusion instead of lipid uptake became relatively important for growth of lipid droplets during the last 4 days. Moreover, ECM production shifted towards ECM turnover by the up-regulation of proteases over eight days. The present in vitro system provides insight into the metabolic changes of adipocytes under conditions of high glucose and insulin, which may help to understand the process of in vivo adipocyte hypertrophy during the development of obesity.

Glucose Restriction Plus Refeeding in Vitro Induce Changes of the Human Adipocyte Secretome with an Impact on Complement Factors and Cathepsins.

Adipose tissue is a major endocrine organ capable of secreting adipokines with a role in whole-body metabolism. Changes in the secretome profile during the development of obesity is suspected to contribute to the risk of health complications such as those associated with weight regain after weight loss. However, the number of studies on weight regain is limited and secretome changes during weight regain have hardly been investigated. In an attempt to generate leads for in vivo studies, we have subjected human Simpson Golabi Behmel Syndrome adipocytes to glucose restriction (GR) followed by refeeding (RF) as an in vitro surrogate for weight regain after weight loss. Using LC-MS/MS, we compared the secreted protein profile after GR plus RF with that of normal feeding (NF) to assess the consequences of GR plus RF. We identified 338 secreted proteins of which 49 were described for the first time as being secreted by adipocytes. In addition, comparison between NF and GR plus RF showed 39 differentially secreted proteins. Functional classification revealed GR plus RF-induced changes of enzymes for extracellular matrix modification, complement system factors, cathepsins, and several proteins related to Alzheimer's disease. These observations can be used as clues to investigate metabolic consequences of weight regain, weight cycling or intermittent fasting.

Weight loss-induced stress in subcutaneous adipose tissue is related to weight regain.

Initial successful weight loss is often followed by weight regain after the dietary intervention. Compared with lean people, cellular stress in adipose tissue is increased in obese subjects. However, the relation between cellular stress and the risk for weight regain after weight loss is unclear. Therefore, we determined the expression levels of stress proteins during weight loss and weight maintenance in relation to weight regain. In vivo findings were compared with results from in vitro cultured human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. In total, eighteen healthy subjects underwent an 8-week diet programme with a 10-month follow-up. Participants were categorised as weight maintainers or weight regainers (WR) depending on their weight changes during the intervention. Abdominal subcutaneous adipose tissue biopsies were obtained before and after the diet and after the follow-up. In vitro differentiated SGBS adipocytes were starved for 96 h with low (0·55 mm) glucose. Levels of stress proteins were determined by Western blotting. WR showed increased expressions of ß-actin, calnexin, heat shock protein (HSP) 27, HSP60 and HSP70. Changes of ß-actin, HSP27 and HSP70 are linked to HSP60, a proposed key factor in weight regain after weight loss. SGBS adipocytes showed increased levels of ß-actin and HSP60 after 96 h of glucose restriction. The increased level of cellular stress proteins in the adipose tissue of WR probably resides in the adipocytes as shown by in vitro experiments. Cellular stress accumulated in adipose tissue during weight loss may be a risk factor for weight regain.

Calorie restriction-induced changes in the secretome of human adipocytes, comparison with resveratrol-induced secretome effects.

Obesity is characterized by dysfunctional white adipose tissue (WAT) that ultimately may lead to metabolic diseases. Calorie restriction (CR) reduces the risk for age and obesity-associated complications. The impact of CR on obesity has been examined with human intervention studies, which showed alterations in circulating adipokines. However, a direct effect of CR on the human adipocyte secretome remains elusive. Therefore, the effect of a 96h low glucose CR on the secretion profile of in vitro cultured mature human SGBS adipocytes was investigated by using proteomics technology. Low-glucose CR decreased the adipocyte triglyceride contents and resulted in an altered secretion profile. Changes in the secretome indicated an improved inflammatory phenotype. In addition, several adipocyte-secreted proteins related to insulin resistance showed a reversed expression after low-glucose CR. Furthermore, 6 novel CR-regulated adipocyte-secreted proteins were identified. Since resveratrol (RSV) mimics CR we compared results from this study with data from our previous RSV study on the SGBS adipocyte secretome. The CR and RSV adipocyte secretomes partly differed from each other, although both treatment strategies lead to secretome changes indicating a less inflammatory phenotype. Furthermore, both treatments induced SIRT1 expression and resulted in a reversed expression of detrimental adipokines associated with metabolic complications.

Hypoxia-mimetic effects in the secretome of human preadipocytes and adipocytes.

White adipose tissue (WAT) regulates energy metabolism by secretion of proteins with endocrine and paracrine effects. Dysregulation of the secretome of obesity-associated enlarged WAT may lead to obesity-related disorders. This can be caused by hypoxia as a result of poorly vascularized WAT. The effect of hypoxia on the secretome of human (pre)adipocytes is largely unknown. Therefore, we investigated the effect of CoCl2, a hypoxia mimetic, on the secretome of human SGBS (pre)adipocytes by a proteomics approach combined with bioinformatic analysis. In addition, regulation of protein secretion was examined by protein turnover experiments. As such, secretome changes were particularly associated with protein down-regulation and extracellular matrix protein dysregulation. The observed up-regulation of collagens in adipocytes may be essential for cell survival while down-regulation of collagens in preadipocytes may indicate a disturbed differentiation process. These CoCl2-induced changes reflect WAT dysfunction that ultimately may lead to obesity-associated complications. In addition, 9 novel adipocyte secreted proteins were identified from which 6 were regulated by CoCl2. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000162.

Resveratrol-induced changes of the human adipocyte secretion profile.

Enlarged white adipose tissue (WAT) is a feature of obesity and leads to changes in its paracrine and endocrine function. Dysfunction of WAT cells is associated with obesity-associated disorders like type 2 diabetes and cardiovascular diseases. Resveratrol (RSV), a natural polyphenolic compound, mimics beneficial effects of calorie restriction. As such, RSV seems a promising therapeutic target for obesity-associated disorders. The effect of RSV on the human adipokine profile is still elusive. Therefore, a proteomic study together with bioinformatical analysis was performed to investigate the effect of RSV on the secretion profile of mature human SGBS adipocytes. RSV incubation resulted in elevated basal glycerol release and reduced intracellular TG content. This increased intracellular lipolysis was accompanied by profound changes in the adipocyte secretion profile. Extracellular matrix proteins were down-regulated while processing proteins were mostly up-regulated after RSV treatment. Interestingly, RSV induced secretion of proteins protective against cellular stress and proteins involved in the regulation of apoptosis. Furthermore, we found a RSV-induced up-regulation of adiponectin and ApoE accompanied by a down-regulation of PAI-1 and PEDF secretion which may improve anti-inflammatory processes and increased insulin sensitivity. These effects may contribute to alleviate obesity-induced metabolic complications. In addition, two novel RSV-regulated adipocyte-secreted proteins were identified.

Reorganization of the nuclear lamina and cytoskeleton in adipogenesis.

A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the 'linker of nucleus and cytoskeleton' (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy.

Identification of novel human adipocyte secreted proteins by using SGBS cells.

Adipose tissue is an endocrine organ secreting different types of proteins, known as adipokines. These adipokines play important roles in homeostasis and metabolism. Adipocyte differentiation leads to a change in adipokine secretion profile which is probably involved in disruption of homeostasis. Many adipokines have been identified but species differences and limitations of human adipose tissue material urged the need for better model systems. Here we used a human cell strain derived from a Simpson Golabi Behmel syndrome (SGBS) patient. SGBS cells have already been used in functional studies on adipocytes but not in a proteomic search for adipokines. In this study, 2D-MS/MS and nLC-MALDI-MS/MS were applied to investigate secretion profiles of SGBS adipokines. A total of 80 secreted proteins were identified; 6 proteins are novel adipocyte secreted proteins, 20 proteins have not been detected before in human adipose material and 23 additional proteins previously detected in visceral adipose tissue have been found here secreted by SGBS-cells of subcutaneous origin. It can be concluded that SGBS cells are both a valid human cell model for adipocyte secretion profiling and for searching for novel human (pre)adipocytes secreted proteins.

Adipocyte abundances of CES1, CRYAB, ENO1 and GANAB are modified in-vitro by glucose restriction and are associated with cellular remodelling during weight regain.

Long-term weight loss maintenance is a problem of overweight and obesity. Changes of gene expression during weight loss (WL) by calorie restriction (CR) are linked to the risk of weight regain (WR). However, detailed information on genes/proteins involved in the mechanism is still lacking. Therefore, we developed an in-vitro model system for glucose restriction (GR) and refeeding (RF) to uncover proteome differences between GR with RF vs normal feeding, of which we explored the relation with WR after WL. Human Simpson-Golabi-Behmel Syndrome cells were subjected to changing levels of glucose to mimic the condition of CR and RF. Proteome profiling was performed by liquid chromatography tandem mass spectrometry. This in-vitro model revealed 44 proteins differentially expressed after GR and RF versus feeding including proteins of the focal adhesions. Four proteins showed a persistent up- or down-regulation: liver carboxylesterase (CES1), mitochondrial superoxide dismutase [Mn] (SOD2), alpha-crystallin B-chain (CRYAB), alpha-enolase (ENO1). In-vivo weight loss-induced RNA expression changes linked CES1, CRYAB and ENO1 to WR. Moreover, of these 44 proteins, CES1 and glucosidase II alpha subunit (GANAB) during follow up correlated with WR. Correlation clustering of in-vivo protein expression data indicated an interaction of these proteins with structural components of the focal adhesions and cytoplasmic filaments in the adipocytes.

The secretory function of adipocytes in the physiology of white adipose tissue.

White adipose tissue, previously regarded as a passive lipid storage site, is now viewed as a dynamic tissue. It has the capacity to actively communicate by sending and receiving different types of signals. An overview of these signals, the external modulators that affect adipose tissue and the secreted signaling molecules, the adipokines, is presented. The secretory function is highlighted in relation to energy metabolism, inflammation and the extracellular matrix and placed in the context of adipose tissue biology. We observe that the endocrine function of adipocytes receives much attention, while its paracrine and autocrine functions are underestimated. Also, we provide examples that species specificity should not be neglected. We conclude that adipose tissue primarily is an energy storage organ, well supported by its secretory function.

Comparative proteomic analysis of cell lines and scrapings of the human intestinal epithelium.

BACKGROUND: In vitro models are indispensable study objects in the fields of cell and molecular biology, with advantages such as accessibility, homogeneity of the cell population, reproducibility, and growth rate. The Caco-2 cell line, originating from a colon carcinoma, is a widely used in vitro model for small intestinal epithelium. Cancer cells have an altered metabolism, making it difficult to infer their representativity for the tissue from which they are derived. This study was designed to compare the protein expression pattern of Caco-2 cells with the patterns of intestinal epithelial cells from human small and large intestine. HT-29 intestinal cells, Hep G2 liver cells and TE 671 muscle cells were included too, the latter two as negative controls. RESULTS: Two-dimensional gel electrophoresis was performed on each tissue and cell line protein sample. Principal component and cluster analysis revealed that global expression of intestinal epithelial scrapings differed from that of intestinal epithelial cell lines. Since all cultured cell lines clustered together, this finding was ascribed to an adaptation of cells to culture conditions and their tumor origin, and responsible proteins were identified by mass spectrometry. When investigating the profiles of Caco-2 cells and small intestinal cells in detail, a considerable overlap was observed. CONCLUSION: Numerous proteins showed a similar expression in Caco-2 cells, HT-29 cells, and both the intestinal scrapings, of which some appear to be characteristic to human intestinal epithelium in vivo. In addition, several biologically significant proteins are expressed at comparable levels in Caco-2 cells and small intestinal scrapings, indicating the usability of this in vitro model. Caco-2 cells, however, appear to over-express as well as under-express certain proteins, which needs to be considered by scientists using this cell line. Hence, care should be taken to prevent misinterpretation of in vitro obtained findings when translating them to the in vivo situation.

Differentiation stage-dependent preferred uptake of basolateral (systemic) glutamine into Caco-2 cells results in its accumulation in proteins with a role in cell-cell interaction.

Glutamine is an essential amino acid for enterocytes, especially in states of critical illness and injury. In several studies it has been speculated that the beneficial effects of glutamine are dependent on the route of supply (luminal or systemic). The aim of this study was to investigate the relevance of both routes of glutamine delivery to in vitro intestinal cells and to explore the molecular basis for proposed beneficial glutamine effects: (a) by determining the relative uptake of radiolabelled glutamine in Caco-2 cells; (b) by assessing the effect of glutamine on the proteome of Caco-2 cells using a 2D gel electrophoresis approach; and (c) by examining glutamine incorporation into cellular proteins using a new mass spectrometry-based method with stable isotope labelled glutamine. Results of this study show that exogenous glutamine is taken up by Caco-2 cells from both the apical and the basolateral side. Basolateral uptake consistently exceeds apical uptake and this phenomenon is more pronounced in 5-day-differentiated cells than in 15-day-differentiated cells. No effect of exogenous glutamine supply on the proteome was detected. However, we demonstrated that exogenous glutamine is incorporated into newly synthesized proteins and this occurred at a faster rate from basolateral glutamine, which is in line with the uptake rates. Interestingly, a large number of rapidly labelled proteins is involved in establishing cell-cell interactions. In this respect, our data may point to a molecular basis for observed beneficial effects of glutamine on intestinal cells and support results from studies with critically ill patients where parenteral glutamine supplementation is preferred over luminal supplementation.

Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene.

The GRU (glucocorticoid-response unit) within the distal enhancer of the gene encoding carbamoyl-phosphate synthase, which comprises REs (response elements) for the GR (glucocorticoid receptor) and the liver-enriched transcription factors FoxA (forkhead box A) and C/EBP (CCAAT/enhancer-binding protein), and a binding site for an unknown protein denoted P3, is one of the simplest GRUs described. In this study, we have established that the activity of this GRU depends strongly on the positioning and spacing of its REs. Mutation of the P3 site within the 25 bp FoxA-GR spacer eliminated GRU activity, but the requirement for P3 could be overcome by decreasing the length of this spacer to < or =12 bp, by optimizing the sequence of the REs in the GRU, and by replacing the P3 sequence with a C/EBPbeta sequence. With spacers of < or =12 bp, the activity of the GRU depended on the helical orientation of the FoxA and GR REs, with highest activities observed at 2 and 12 bp respectively. Elimination of the 6 bp C/EBP-FoxA spacer also increased GRU activity 2-fold. Together, these results indicate that the spatial positioning of the transcription factors that bind to the GRU determines its activity and that the P3 complex, which binds to the DNA via a 75 kDa protein, functions to facilitate interaction between the FoxA and glucocorticoid response elements when the distance between these transcription factors means that they have difficulties contacting each other.

The (patho)physiological functions of the MRP family.

The identification of certain members of the large superfamily of ATP binding cassette transport proteins such as MDR1 -P-glycoprotein and the multidrug resistance protein MRP1 as ATP-dependent drug efflux pumps has been a major contribution in our understanding of the multidrug resistance phenotype of cancer cells. Importantly, both transport proteins that exhibit only low structural homology have a very different substrate specificity but confer resistance to a similar spectrum of natural product chemotherapeutic drugs. In contrast to the drug transporter MDR1, MRP1 mainly transports anionic Phase II-conjugates. In addition MRP1-mediated drug resistance is highly dependent on high intracellular glutathione levels which may be linked to the apparent physiological involvement of MRP1 in glutathione-related cellular processes. This review summarizes the current knowledge about functional aspects of MRP1 and its five recently cloned homologues MRP2-MRP6 and discusses their substrate specificities and cellular localization with emphasis on drug resistance. Copyright 2000 Harcourt Publishers Ltd.

Lutein Leads to a Decrease of Factor D Secretion by Cultured Mature Human Adipocytes.

Purpose. Complement plays an important role in the pathogenesis of age related macular degeneration (AMD) and trials are currently being conducted to investigate the effect of complement inhibition on AMD progression. We previously found that the plasma level of factor D (FD), which is the rate limiting enzyme of the complement alternative pathway, was significantly decreased following lutein supplementation. FD is synthesized by adipose tissue, which is also the main storage site of lutein. In view of these findings we tested the hypothesis whether lutein could affect FD synthesis by adipocytes. Methods. A cell line of mature human adipocytes was incubated with 50 µg/mL lutein for 24 and 48 h, whereafter FD mRNA and protein expression were measured. Results. Lutein significantly inhibited adipocyte FD mRNA expression and FD protein release into adipocyte culture supernatants. Conclusions. Our earlier observations showing that a daily lutein supplement in individuals with early signs of AMD lowered the level of circulating FD might be caused by blocking adipocyte FD production.

Application of proteomics technology in adipocyte biology.

Obesity and its associated complications have reached epidemic proportions in Western-type societies. Concomitantly, the obesity incidence in developing countries is increasing. One hallmark of obesity is the differentiation of pre-adipocytes into mature triglyceride-loaded adipocytes present in subcutaneous and visceral adipose tissue depots. This may ultimately lead to dysfunctional adipose tissue together with detrimental changes in the profiles of (pre-)adipocyte-secreted proteins, known as adipokines. Obesity-induced alterations in adipokine profiles contribute to the development of obesity-associated disorders. Consequently, the interest in the molecular events responsible for adipose tissue modifications during weight gain and weight loss as well as in the aetiology of obesity-associated disorders is growing. Molecular mechanisms involved in pre-adipocyte differentiation and alterations in adipokine profiles have been examined at the gene and protein level by high-throughput technologies. Independent proteomics studies have contributed significantly to further insight into adipocyte biology, particularly with respect to adipokine profiling. In this review novel findings obtained with adipo-proteomics studies are highlighted and the relevance of proteomics technologies to further understand molecular aspects of adipocyte biology is discussed.

Screening for drug-induced hepatotoxicity in primary mouse hepatocytes using acetaminophen, amiodarone, and cyclosporin a as model compounds: an omics-guided approach.

Drug-induced hepatotoxicity is a leading cause of attrition for candidate pharmaceuticals in development. New preclinical screening methods are crucial to predict drug toxicity prior to human studies. Of all in vitro hepatotoxicity models, primary human hepatocytes are considered as 'the gold standard.' However, their use is hindered by limited availability and inter-individual variation. These barriers may be overcome by using primary mouse hepatocytes. We used differential in gel electrophoresis (DIGE) to study large-scale protein expression of primary mouse hepatocytes. These hepatocytes were exposed to three well-defined hepatotoxicants: acetaminophen, amiodarone, and cyclosporin A. Each hepatotoxicant induces a different hepatotoxic phenotype. Based on the DIGE results, the mRNA expression levels of deregulated proteins from cyclosporin A-treated cells were also analyzed. We were able to distinguish cyclosporin A from controls, as well as acetaminophen and amiodarone-treated samples. Cyclosporin A induced endoplasmic reticulum (ER) stress and altered the ER-Golgi transport. Moreover, liver carboxylesterase and bile salt sulfotransferase were differentially expressed. These proteins were associated with a protective adaptive response against cyclosporin A-induced cholestasis. The results of this study are comparable with effects in HepG2 cells. Therefore, we suggest both models can be used to analyze the cholestatic properties of cyclosporin A. Furthermore, this study showed a conserved response between primary mouse hepatocytes and HepG2 cells. These findings collectively lend support for use of omics strategies in preclinical toxicology, and might inform future efforts to better link preclinical and clinical research in rational drug development.

Proteomics in the search for mechanisms and biomarkers of drug-induced hepatotoxicity.

The safety assessment for pharmaceuticals includes in vivo repeated dose toxicity tests in laboratory animals. These in vivo studies often generate false negative results and unexpected toxicity. The appearance of this unexpected toxicity is one of the major reasons for the drawback of a drug from the market. The liver is often a target organ in toxicology since it is responsible for the metabolism and elimination of chemical compounds. Therefore, there is need for new screening methods which classify hepatotoxic compounds earlier in development. This will lead to safer drugs and a more efficient drug discovery process. Furthermore, these new screening methods are preferably in vitro test systems, aiming at reducing the use of laboratory animals. In this review the possibilities of proteomics and its promising results for improving current predictive and mechanistic toxicological studies are described. Biomarkers or protein panels for hepatotoxic mechanisms, which reflect the in vivo situation, need to be identified to allow a better toxicity screening. Therefore, in vivo studies and in vitro cell models are discussed and evaluated with regard to the protein expression of their metabolic enzymes, their similarities with liver, their use for analyzing toxicological mechanisms and hepatotoxicity screening. Studies in which proteomics are combined with other omics-technologies are also presented. The results from these integrated data analyses can be used for the development of improved panels of biomarkers for toxicity screening.

Proteomics investigations of drug-induced hepatotoxicity in HepG2 cells.

Unexpected hepatotoxicity is one of the major reasons of drugs failing in clinical trials. This emphasizes the need for new screening methods that address toxicological hazards early in the drug discovery process. Here, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxic compounds. Drug-induced hepatotoxicity is mainly divided in hepatic steatosis, cholestasis, or necrosis. For each class, a compound was selected, respectively amiodarone, cyclosporin A, and acetaminophen. The changes in protein expressions in HepG2, after exposure to these test compounds, were studied using quantitative two-dimensional differential gel electrophoresis. Identification of differentially expressed proteins was performed by Maldi-TOF/TOF MS and liquid chromatography-tandem mass spectrometry. In this study, 254 differentially expressed protein spots were detected in a two-dimensional proteome map from which 86 were identified, showing that the proteome of HepG2 cells is responsive to hepatotoxic compounds. cyclosporin A treatment was responsible for most differentially expressed proteins and could be discriminated in the hierarchical clustering analysis. The identified differential proteins show that cyclosporin A may induce endoplasmic reticulum (ER) stress and disturbs the ER-Golgi transport, with an altered vesicle-mediated transport and protein secretion as result. Moreover, the differential protein pattern seen after cyclosporin A treatment can be related to cholestatic mechanisms. Therefore, our findings indicate that the HepG2 in vitro cell system has distinctive characteristics enabling the assessment of cholestatic properties of novel compounds at an early stage of drug discovery.