Characterization of hepatic zonation in mice by mass-spectrometric and antibody-based proteomics approaches.

Periportal and perivenous hepatocytes show zonal heterogeneity in metabolism and signaling. Here, hepatic zonation in mouse liver was analyzed by non-targeted mass spectrometry (MS) and by the antibody-based DigiWest technique, yielding a comprehensive overview of protein expression in periportal and perivenous hepatocytes. Targeted immunoaffinity-based proteomics were used to substantiate findings related to drug metabolism. 165 (MS) and 82 (DigiWest) zonated proteins were identified based on the selected criteria for statistical significance, including 7 (MS) and 43 (DigiWest) proteins not identified as zonated before. New zonated proteins especially comprised kinases and phosphatases related to growth factor-dependent signaling, with mainly periportal localization. Moreover, the mainly perivenous zonation of a large panel of cytochrome P450 enzymes was characterized. DigiWest data were shown to complement the MS results, substantially improving possibilities to bioinformatically identify zonated biological processes. Data mining revealed key regulators and pathways preferentially active in either periportal or perivenous hepatocytes, with ß-catenin signaling and nuclear xeno-sensing receptors as the most prominent perivenous regulators, and several kinase- and G-protein-dependent signaling cascades active mainly in periportal hepatocytes. In summary, the present data substantially broaden our knowledge of hepatic zonation in mouse liver at the protein level.

NanoPASS: an easy-to-use user interface for nanoparticle dosimetry with the 3DSDD model.

Nanoparticles exhibit a specific diffusion and sedimentation behavior under cell culture conditions as used in nantoxicological in vitro testing. How a particular particle suspension behaves depends on the particular physicochemical characteristics of the particles and the cell culture system. Only a fraction of the nanoparticles applied to a cell culture will thus reach the cells within a given time frame. Therefore, dosimetric calculations are essential not only to determine the exact fraction of nanoparticles that has come into contact with the cells, but also to ensure experimental comparability and correct interpretation of results, respectively. Yet, the use of published dosimetry models is limited. Not the least because the correct application of these in silico tools usually requires bioinformatics knowledge, which often is perceived a hurdle. Moreover, not all models are freely available and accessible. In order to overcome this obstacle, we have now developed an easy-to-use interface for our recently published 3DSDD dosimetry model, called NanoPASS (NanoParticle Administration Sedimentation Simulator). The interface is freely available to all researchers. It will facilitate the use of in silico dosimetry in nanotoxicology and thus improve interpretation and comparability of in vitro results in the field.

Transcriptomic effect marker patterns of genotoxins - a comparative study with literature data.

Microarray approaches are frequently used experimental tools which have proven their value for example in the characterization of the molecular mode of action of toxicologically relevant compounds. In a regulatory context, omics techniques are still not routinely used, amongst others due to lacking standardization in experimental setup and data processing, and also due to issues with the definition of adversity. In order to exemplarily determine whether consensus transcript biomarker signatures for a certain toxicological endpoint can be derived from published microarray datasets, we here compared transcriptome data from human HepaRG hepatocarcinoma cells treated with different genotoxins, based on re-analyzed datasets extracted from the literature. Comparison of the resulting data show that even with similarly-acting compounds in the same cell line, considerable variation was observed with respect to the numbers and identities of differentially expressed genes. Greater concordance was observed when considering the whole data sets and biological functions associated with the genes affected. The present results highlight difficulties and possibilities in inter-experiment comparisons of omics data and underpin the need for future efforts towards improved standardization to facilitate the use of omics data in risk assessment. Existing omics datasets may nonetheless prove valuable in establishing biological context information essential for the development of adverse outcome pathways.

The use of 3D cultures of MCF-10A and MCF-12A cells by high content screening for effect-based analysis of non-genotoxic carcinogens.

The human breast epithelial cell lines MCF-10A and MCF-12A form well-differentiated acinus-like structures when grown in three-dimensional matrigel culture over a period of 20 days. In the present study, both cell lines were tested for their suitability to serve as an effect-based in vitro test system for non-genotoxic carcinogens. A software solution for automated Acinus Detection And Morphological Evaluation (ADAME) was developed to automatically acquire acinus images and to determine morphological parameters such as acinus size, lumen size, and acinus roundness. A number of test compounds were tested for their capacity to affect acinus formation and cellular differentiation. Human epidermal growth factor stimulated acinus growth for both cell lines whereas all-trans retinoic acid inhibited acinus growth. The strong estrogen 17ß-estradiol had no effect on acinus formation of estrogen receptor (ER)-negative MCF-10A cells, but yielded larger MCF-12A (ER-positive) acini. Thus, the parallel use of both cell lines allows the identification of estrogenic properties of a given test compound.

Proteomic analysis of 2-monochloropropanediol (2-MCPD) and 2-MCPD dipalmitate toxicity in rat kidney and liver in a 28-days study.

2-monochloropropanediol (2-MCPD), 3-monochloropropanediol (3-MCPD) and their fatty acid esters have recently been identified as heat-induced contaminants in fat- and salt-containing foodstuff. Toxicity of 3-MCPD has been studied previously in some detail. Disturbance of glycolysis and cellular redox functions appear to be involved in 3-MCPD toxicity. By contrast, only very few toxicological data are available for 2-MCPD or 2-MCPD esters, especially at the molecular level. This study was therefore aimed to provide a comprehensive overview of proteomic alterations induced in rat kidney and liver by 2-MCPD and 2-MCPD dipalmitate, a representative 2-MCPD fatty acid ester. Sub-toxic doses of 10 mg/kg body weight 2-MCPD, or equimolar doses of 2-MCPD dipalmitate were applied in a 28-day in vivo gavage oral toxicity study in male rats. Two-dimensional gel electrophoresis and mass-spectrometric protein identification using material from 5 animals per treatment group were employed together with bioinformatic data mining to obtain information about the molecular basis of the observed proteomic alterations. Obtained data indicate toxic consequences of 2-MCPD exposure in the kidney and provide evidence that 2-MCPD exerts its cellular effects in rat kidney by mechanisms different from 3-MCPD.

In silico genotoxicity and carcinogenicity prediction for food-relevant secondary plant metabolites.

Humans are exposed to thousands of different secondary plant metabolites which may have beneficial health effects, but numerous compounds may also have toxic potential. In the present study we have examined the genotoxic and carcinogenic potential of 609 food-relevant phytochemicals by using computer models for toxicity prediction. We developed a scoring method and combined the results of different models to increase the predictive power. A combination of the VEGA models SARpy, KNN, ISS, and CAESAR, and of the LAZAR model "Salmonella typhimurium" for genotoxicity prediction performed better than the single models regarding specificity and accuracy. Statistical evaluation of the combined model for carcinogenicity prediction was not possible due to the low number of substances suitable for model validation. The in silico results of the present exercise will be useful for priority setting purposes regarding future risk assessment of secondary plant metabolites. Based on our analysis, (-)-asimilobine, aloin, annoretine, chrysothrone, coptisine, elymoclavine, and thalicminine were predicted to be genotoxic with high probability and may therefore be selected for subsequent experimental genotoxicity testing. Moreover, the class of pyrrolizidine alkaloids is suggested to be a high priority subject for further studies as these substances have been predicted to be carcinogenic with high probability.

Proteomic effects of repeated-dose oral exposure to 2-monochloropropanediol and its dipalmitate in rat testes.

2- and 3-monochloropropanediol (2-MCPD) and their fatty acid esters are food contaminants which are concomitantly formed upon thermal treatment of foodstuff containing fats and salt. Exposure to 2- or 3-MCPD thus results, for example, from refined vegetable oils, in instant meals or infant formula, as well as in cereals or pastries. The molecular mechanisms of 2-MCPD toxicity are poorly understood. Here, we performed a comprehensive proteomic analysis of 2-MCDP-induced alterations in the testes from rats following oral administration of 10 mg/kg body weight per day 2-MCPD, or an equimolar dose of 2-MCPD dipalmitate as a representative 2-MCPD fatty acid ester. In the absence of overt histopathologically detectable toxicity, moderate alterations in cellular proteomic signatures were recorded. The observations are in line with the assumption that the molecular mechanisms of 2-MCPD and 3-MCPD toxicity differ. Observed proteomic alterations point towards effects of 2-MCPD on mitogen-dependent signaling and mitochondrial energy utilization. Presented data for the first time provide insight into proteomic effects of 2-MCPD in testicular tissue.

Identification and evaluation of potentially mutagenic and carcinogenic food contaminants.

Heat processing of food gives rise to a plethora of chemical compounds whose toxicological effects are largely unknown. Due to a general lack of experimental toxicological data, assessing the risks associated with the consumption of these substances remains a challenge. Computer models that allow for an in silico prediction of physicochemical and toxicological characteristics, may be able to fill current data gaps and facilitate the risk assessment of toxicologically uncharacterised chemicals, their transformation products and their biological metabolites. The overall aims of the present project were for the fellow: (i) to get acquainted with the application of computational toxicological analyses tools in risk assessment based on results and experiences from previous research performed at the German Federal Institute for Risk Assessment (BfR); and (ii) to apply the newly gained skills on historic and novel data using updated and additional in silico tools. The project contributed to the continuous further education of the fellow in the use of computational toxicology tools, corroborated findings related to the safety of heat-induced contaminants and laid the foundations for future collaborations between the fellow's home institution, the Institute of Marine Research (IMR) in Norway, and the BfR in Germany.

[Heat-induced contaminants in foodstuffs : Acrylamide, furan, and fatty acid esters of monochloropropanediols and glycidol]. / Erhitzungsbedingte Kontaminanten in Lebensmitteln : Acrylamid, Furan und Fettsäureester von Monochlorpropandiolen und Glycidol.

The production and preparation of foodstuffs may entail at high temperatures the generation of undesirable, potentially harmful compounds. Among the best investigated heat-induced contaminants are acrylamide, furan, and the fatty acid esters of glycidol and the monochloropropanediols. This article presents the main insights into the formation, toxicology, and exposure of these compounds. Acrylamide and glycidol were characterized as carcinogens with a genotoxic mechanism in animal experiments. Their content in foods should be minimized. For 3­monochloropropanediol (3-MCPD), a tolerable daily intake can be derived. In contrast, a complete risk assessment is currently not possible for furan and 2­MCPD owing to insufficient data.Many other heat-induced substances in foodstuffs were identified in addition to the compounds mentioned above, but for most no data on their toxicological properties and human exposure is available. Therefore, no risk assessment can currently be undertaken for these compounds. To prioritize this large number of compounds according to their possible hazard potential, it is reasonable to utilize computer modeling programs for the prediction of defined toxicological endpoints based on the molecular chemical structures. However, substances classed as a priority must be further investigated with regard to the toxicology and quantification of the food content of these compounds to allow a meaningful risk assessment.

Lanthanum chloride precipitation-based toxicoproteomic analysis of 3-monochloropropane-1,2-diol toxicity in rat kidney reveals involvement of extracellular signal-regulated kinase 2.

The heat-induced food contaminant 3-monochloropropane-1,2-diol (3-MCPD) and its fatty acid esters exert nephrotoxicity in rodents. Previous studies including a non-targeted toxicoproteomics approach using samples from a 28-day oral toxicity study in rats with 10 mg/kg body weight (b.w.) of 3-MCPD, an equimolar dose of 53 mg/kg b.w. 3-MCPD dipalmitate and a lower dose of 13.3 mg/kg b.w. of 3-MCPD dipalmitate, revealed substance-induced alterations in metabolic pathways, especially for glycolysis and energy metabolism. In order to obtain deeper insight into mechanisms of 3-MCPD toxicity, samples from the above-mentioned study were reanalyzed using a lanthanum chloride precipitation-based toxicoproteomics approach in order to increase the yield of phosphorylated proteins, crucial players in cellular signaling. A comparison of standard 2D-gel-based proteomics and lanthanum chloride precipitation was performed, thus providing a comprehensive case study on these two methods using in vivo effects of an important food toxicant in a primary target organ. While resulting in similar 2D-gel electrophoresis pherograms and spot counts, data analysis demonstrated that lanthanum precipitation yielded more significantly deregulated proteins thus considerably improving our knowledge on 3-MCPD-dependent proteomic alterations in the kidney. 3-MCPD-induced deregulation of the phosphorylated, active version of extracellular signal-regulated kinase 2 (ERK2) in rat kidney was demonstrated using mass spectrometry and immunohistochemistry. In summary, this paper for the first time links 3-MCPD effects to deregulation of the ERK/mitogen-activated protein kinase signaling pathway in rat kidney and demonstrates that lanthanum chloride precipitation is suited to support the gain of mechanistic knowledge on organ toxicity using 2D-gel-based proteomics.

Use of in silico models for prioritization of heat-induced food contaminants in mutagenicity and carcinogenicity testing.

Numerous Maillard reaction and lipid oxidation products are present in processed foods such as heated cereals, roasted meat, refined oils, coffee, and juices. Due to the lack of experimental toxicological data, risk assessment is hardly possible for most of these compounds. In the present study, an in silico approach was employed for the prediction of the toxicological endpoints mutagenicity and carcinogenicity on the basis of the structure of the respective compound, to examine (quantitative) structure-activity relationships for more than 800 compounds. Five software tools for mutagenicity prediction (T.E.S.T., SARpy, CAESAR, Benigni-Bossa, and LAZAR) and three carcinogenicity prediction tools (CAESAR, Benigni-Bossa, and LAZAR) were combined to yield so-called mutagenic or carcinogenic scores for every single substance. Alcohols, ketones, acids, lactones, and esters were predicted to be mutagenic and carcinogenic with low probability, whereas the software tools tended to predict a considerable mutagenic and carcinogenic potential for thiazoles. To verify the in silico predictions for the endpoint mutagenicity experimentally, twelve selected compounds were examined for their mutagenic potential using two different validated in vitro test systems, the bacterial reverse mutation assay (Ames test) and the in vitro micronucleus assay. There was a good correlation between the results of the Ames test and the in silico predictions. However, in the case of the micronucleus assay, at least three substances, 2-amino-6-methylpyridine, 6-heptenoic acid, and 2-methylphenol, were clearly positive although they were predicted to be non-mutagenic. Thus, software tools for mutagenicity prediction are suitable for prioritization among large numbers of substances, but these predictions still need experimental verification.

Comparative proteomic analysis of 2-MCPD- and 3-MCPD-induced heart toxicity in the rat.

The chlorinated propanols 2- and 3-monochloropropanediol (MCPD), and their fatty acid esters have gained public attention due to their frequent occurrence as heat-induced food contaminants. Toxic properties of 3-MCPD in kidney and testis have extensively been characterized. Other 3-MCPD target organs include heart and liver, while 2-MCPD toxicity has been observed in striated muscle, heart, kidney, and liver. Inhibition of glycolysis appears to be important in 3-MCPD toxicity, whereas mechanisms of 2-MCPD toxicity are still unknown. It is thus not clear whether toxicity by the two isomeric compounds is dependent on similar or dissimilar modes of action. A 28-day oral feeding study in rats was conducted using daily non-toxic doses of 2-MCPD or 3-MCPD [10 mg/kg body weight], or an equimolar (53 mg/kg body weight) or a lower (13.3 mg/kg body weight) dose of 2-MCPD dipalmitate. Comprehensive comparative proteomic analyses of substance-induced alterations in the common target organ heart revealed striking similarities between effects induced by 2-MCPD and its dipalmitate ester, whereas the degree of effect overlap between 2-MCPD and 3-MCPD was much less. The present data demonstrate that even if exerting effects in the same organ and targeting similar metabolic networks, profound differences between molecular effects of 2-MCPD and 3-MCPD exist thus warranting the necessity of separate risk assessment for the two substances. This study for the first time provides molecular insight into molecular details of 2-MCPD toxicity. Furthermore, for the first time, molecular data on 3-MCPD toxicity in the heart are presented.

2-Chloro-1,3-propanediol (2-MCPD) and its fatty acid esters: cytotoxicity, metabolism, and transport by human intestinal Caco-2 cells.

The food contaminants 3-chloro-1,2-propanediol (3-MCPD) and 3-MCPD fatty acid esters have attracted considerable attention in the past few years due to their toxic properties and their occurrence in numerous foods. Recently, significant amounts of the isomeric compounds 2-chloro-1,3-propanediol (2-MCPD) fatty acid esters have been detected in refined oils. Beside the interrogation which toxic effects might be related to the core compound 2-MCPD, the key question from the risk assessment perspective is again-as it was discussed for 3-MCPD fatty acid esters before-to which degree these esters are hydrolyzed in the gut, thereby releasing free 2-MCPD. Here, we show that free 2-MCPD but not 2-MCPD fatty acid esters were able to cross a monolayer of differentiated Caco-2 cells as an in vitro model for the human intestinal barrier. Instead, the esters were hydrolyzed by the cells, thereby releasing free 2-MCPD which was neither absorbed nor metabolized by the cells. Cytotoxicity assays revealed that free 2-MCPD as well as free 3-MCPD was not toxic to Caco-2 cells up to a level of 1 mM, whereas cellular viability was slightly decreased in the presence of a few 2-MCPD and 3-MCPD fatty acid esters at concentrations above 10 µM. The observed cytotoxic effects correlated well with the induction of caspase activity and might be attributed to the induction of apoptosis by free fatty acids which were released from the esters in the presence of Caco-2 cells.

Sulphate fertilization ameliorates long-term aluminum toxicity symptoms in perennial ryegrass (Lolium perenne).

Effects of the oxanion sulphate on plant aluminum (Al(3+)) detoxification mechanisms are not well understood. Therefore, holistic physiological and biochemical modifications induced by progressively increased doses of sulphate fertilization in the presence of long-term Al(3+) stress were investigated in the aluminum sensitive perennial ryegrass (Lolium perenne L. cvJumbo). Plant growth inhibition induced by Al(3+) was decreased in response to increasing doses of sulphate supply. Aluminum concentrations measured in roots of perennial ryegrass by atomic absorption spectrometry declined significantly with increasing sulphate concentrations. In parallel, we determined a rise of sulphur in shoots and roots of perennial ryegrass. Inclusion of up to 360 µM of sulphate enhanced cysteine and glutathione biosynthesis in Al(3+) (1.07 µM) treated plants. This increase of thiol-containing compounds favored all modifications in the glutathione redox balance, declining lipid peroxidation, decreasing the activity of superoxide dismutase, and modifying the expression of proteins involved in the diminution of Al(3+) toxicity in roots. In particular, proteome analysis by 1D-SDS-PAGE and LC-MS/MS allowed to identify up (e.g. vacuolar proton ATPase, proteosome ß subunit, etc) and down (Glyoxilase I, Ascorbate peroxidase, etc.) regulated proteins induced by Al(3+) toxicity symptoms in roots. Although, sulphate supply up to 480 µM caused a reduction in Al(3+) toxicity symptoms, it was not as efficient as compared to 360 µM sulphate fertilization. These results suggest that sulphate fertilization ameliorates Al(3+) toxicity responses in an intracellular specific manner within Lolium perenne.