The interplay of calponin, wnt signaling, and cytoskeleton protein governs transgenerational phenotypic abnormalities in drosophila exposed to zinc oxide nanoparticles.

ZnO nanoparticles (ZnO NPs) are widely used engineered nanomaterials. Due to induced genotoxicity, increased oxidative stress, and teratogenicity, these NPs have been reported to be toxic. In the present study, we emphasise the role of vital proteins in regulating ZnO NP-induced abnormal phenotypes, particularly the deformed thorax and single wing in the Drosophila melanogaster progeny fed on 0.1-10 mM ZnO NPs. To understand how protein expression regulates this particular phenotype on ZnO NPs exposure, toxicoproteomics profile of control and abnormal phenotype flies was generated using LC/MS/MS. Gene ontology enrichment studies of proteomics data were carried out using CLUEGO and STRAP software. The bioinformatics tool STRING was used to generate a protein-protein interaction map of key proteins of enrichment analysis. Following ZnO NP exposure, the differential expression of key proteins of the Wnt pathway was prominent. Altered expression of various proteins of the Wnt pathway (CaMKII), cytoskeleton (Actin), and calponin resulted in developmental defects in drosophila progeny. In addition, immunohistology studies showed a significant deviation in the expression of wingless protein of ZnO NPs treated larvae in comparison to control. According to these findings, the interaction of the wnt pathway and cytoskeletal proteins with ZnO NPs caused developmental abnormalities in the subsequent generation of drosophila, highlighting the transgenerational toxic effects of these nanoparticles.

Identification of proteome markers for drug-induced liver injury in zebrafish embryos.

The zebrafish embryo (ZFE) is a promising alternative non-rodent model in toxicology, and initial studies suggested its applicability in detecting hepatic responses related to drug-induced liver injury (DILI). Here, we hypothesize that detailed analysis of underlying mechanisms of hepatotoxicity in ZFE contributes to the improved identification of hepatotoxic properties of compounds and to the reduction of rodents used for hepatotoxicity assessment. ZFEs were exposed to nine reference hepatotoxicants, targeted at induction of steatosis, cholestasis, and necrosis, and effects compared with negative controls. Protein profiles of the individual compounds were generated using LC-MS/MS. We identified differentially expressed proteins and pathways, but as these showed considerable overlap, phenotype-specific responses could not be distinguished. This led us to identify a set of common hepatotoxicity marker proteins. At the pathway level, these were mainly associated with cellular adaptive stress-responses, whereas single proteins could be linked to common hepatotoxicity-associated processes. Applying several stringency criteria to our proteomics data as well as information from other data sources resulted in a set of potential robust protein markers, notably Igf2bp1, Cox5ba, Ahnak, Itih3b.2, Psma6b, Srsf3a, Ces2b, Ces2a, Tdo2b, and Anxa1c, for the detection of adverse responses.

A novel particulate matter sampling and cell exposure strategy based on agar membrane for cytotoxicity study.

Laboratories use different strategies to sample and extract atmospheric particulate matter (PM), some of which can be very complicated. Due to the absence of a standard protocol, it is difficult to compare the results of PM toxicity assessment across different laboratories. Here, we proposed a novel PM sampling and cell exposure strategy based on agar membrane. The agar membrane, prepared by a simple freeze-drying method, has a relatively flat surface and porous interior. We demonstrated that the agar membrane was a reliable substitute material for PM sampling. Then the PM on the agar membranes was directly extracted with the culture medium by vortex method, and the PM on the polytetrafluoroethylene (PTFE) filters was extracted with water by the traditional ultrasonic method for comparison. The extraction efficiency was evaluated and in vitro cytotoxicity assays were carried out to investigate the toxic effects of PM extracted with two strategies on macrophage cells. The results showed that the PM extracted from agar membranes induced higher cytotoxicity and more differentially expressed proteins. Overall, the novel PM sampling-cell exposure strategy based on the agar membrane is easy to operate, biocompatible and comparable, and has low disturbance, could be an alternative sampling and extraction method for PM toxicity assessment.

Proteomic analysis suggests that monoterpenes in lemongrass disrupt Ca2+ homeostasis in Haemaphysalis longicornis leading to mitochondrial depolarization and cytotoxicity.

Complex mixtures of bioactive ingredients in plant essential oils present complex chemistries which involve different modes of action. An increasing body of scientific reports has recently focused on the acaricidal activities of plant essential oils attributed to their monoterpene components, but information about their underlying molecular mechanism of action is scarce. Here, after the chemical analysis of lemongrass oil, a proteomic analysis of the ovary, salivary gland, and midgut of Haemaphysalis longicornis exposed to Cymbopogon citratus (lemongrass) essential oil was performed via data-independent acquisition mass spectrometry (DIA-MS) technology to further elucidate the molecular mechanisms involved. Pathway analysis reveals the activation of metabolic pathways mediated by oxidoreductases and transferases. Furthermore, the upregulation of various calcium-associated proteins and the upregulation of cytochrome c1, cytochrome c oxidase polypeptide IV, and programmed cell death protein 6-like isoform X1 suggest a cytotoxic mode of action via the formation of reactive oxygen species (ROS), mitochondrial Ca2+ overload, mitochondrial uncoupling, and depolarization, and ATP depletion leading to either apoptotic or necrotic death. Morphological alterations observed after the RNAi of a major detoxification enzyme (glutathione S-transferase) merit further investigation. Hence, the cytotoxic mode of action exhibited by C. citratus oil could be vital for the development of eco-friendly acaricide.

Advantages of omics technology for evaluating cadmium toxicity in zebrafish.

In the last decade, several advancements have been made in omics technologies and they have been applied extensively in diverse research areas. Especially in toxicological research, omics technology can efficiently and accurately generate relevant data on the molecular dynamics associated with adverse outcomes. Toxicomics is defined as the combination of toxicology and omics technologies and encompasses toxicogenomics, toxicoproteomics, and toxicometabolomics. This paper reviews the trend of applying omics technologies to evaluate cadmium (Cd) toxicity in zebrafish (D. rerio). Cd is a toxic heavy metal posing several environmental concerns; however, it is being used widely in everyday life. Zebrafish embryos and larvae are employed as standard models for many toxicity tests because they share 71.4% genetic homology with humans. This study summarizes the toxicity of Cd on the nerves, liver, heart, skeleton, etc. of zebrafish and introduces detailed omics techniques to understand the results of the toxicomic studies. Finally, the trend of toxicity evaluation in the zebrafish model of Cd based on omics technology is presented.

Proteomics in systems toxicology.

Proteins are the ultimate product of gene expression. As they hinge between gene transcription and phenotype, they offer a more realistic perspective of toxicopathic effects, responses and even susceptibility to insult than targeting genes and mRNAs while dodging some inter-individual variability that hinders measuring downstream endpoints like metabolites or enzyme activity. Toxicologists have long focused on proteins as biomarkers but the advent of proteomics shifted risk assessment from narrow single-endpoint analyses to whole-proteome screening, enabling deriving protein-centric adverse outcome pathways (AOPs), which are pivotal for the derivation of Systems Biology informally named Systems Toxicology. Especially if coupled pathology, the identification of molecular initiating events (MIEs) and AOPs allow predictive modeling of toxicological pathways, which now stands as the frontier for the next generation of toxicologists. Advances in mass spectrometry, bioinformatics, protein databases and top-down proteomics create new opportunities for mechanistic and effects-oriented research in all fields, from ecotoxicology to pharmacotoxicology.

Weight of evidence approach using a TK gene mutation assay with human TK6 cells for follow-up of positive results in Ames tests: a collaborative study by MMS/JEMS.

BACKGROUND: Conflicting results between bacterial mutagenicity tests (the Ames test) and mammalian carcinogenicity tests might be due to species differences in metabolism, genome structure, and DNA repair systems. Mutagenicity assays using human cells are thought to be an advantage as follow-up studies for positive results in Ames tests. In this collaborative study, a thymidine kinase gene mutation study (TK6 assay) using human lymphoblastoid TK6 cells, established in OECD TG490, was used to examine 10 chemicals that have conflicting results in mutagenicity studies (a positive Ames test and a negative result in rodent carcinogenicity studies). RESULTS: Two of 10 test substances were negative in the overall judgment (20% effective as a follow-up test). Three of these eight positive substances were negative after the short-term treatment and positive after the 24 h treatment, despite identical treatment conditions without S9. A toxicoproteomic analysis of TK6 cells treated with 4-nitroanthranilic acid was thus used to aid the interpretation of the test results. This analysis using differentially expressed proteins after the 24 h treatment indicated that in vitro specific oxidative stress is involved in false positive response in the TK6 assay. CONCLUSIONS: The usefulness of the TK6 assay, by current methods that have not been combined with new technologies such as proteomics, was found to be limited as a follow-up test, although it still may help to reduce some false positive results (20%) in Ames tests. Thus, the combination analysis with toxicoproteomics may be useful for interpreting false positive results raised by 24 h specific reactions in the assay, resulting in the more reduction (> 20%) of false positives in Ames test.

Comparison of quantitation methods in proteomics to define relevant toxicological information on AhR activation of HepG2 cells by BaP.

The application of quantitative proteomics provides a new and promising tool for standardized toxicological research. However, choosing a suitable quantitative method still puzzles many researchers because the optimal method needs to be determined. In this study, we investigated the advantages and limitations of two of the most commonly used global quantitative proteomics methods, namely label-free quantitation (LFQ) and tandem mass tags (TMT). As a case study, we exposed hepatocytes (HepG2) to the environmental contaminant benzo[a]pyrene (BaP) using a concentration of 2 µM. Our results revealed that both methods yield a similar proteome coverage, in which for LFQ a wider range of fold changes was observed but with less significant p-values compared to TMT. We detected 37 and 47 significantly enriched pathways by LFQ and TMT, respectively, with 17 overlapping pathways. To define the minimally required effort in proteomics as a benchmark, we artificially reduced the LFQ, and TMT data sets stepwise and compared the pathway enrichment. Thereby, we found that fewer proteins are necessary for detecting significant enrichment of pathways in TMT compared to LFQ, which might be explained by the higher reproducibility of the TMT data that was observed. In summary, we showed that the TMT approach is the preferable one when investigating toxicological questions because it offers a high reproducibility and sufficient proteome coverage in a comparably short time.

Artificial intelligence and machine learning for protein toxicity prediction using proteomics data.

Instead of only focusing on the targeted drug delivery system, researchers have a great interest in developing peptide-based therapies for the procurement of numerous class of diseases. The main idea behind this is to anchor the properties of the receptor to design peptide-based therapeutics. As these macromolecules have distinct physicochemical properties over small molecules, it becomes an obligatory field for the treatment of diseases. For this, various in silico models have been developed to speculate the proteins by virtue of the application of machine learning and artificial intelligence. By analysing the properties and structural alert of toxic proteins, researchers aim to dissert some of the mechanisms of protein toxicity from which therapeutic insights may be drawn. Numerous models already exist worldwide emphasizing themselves as leading paramount for toxicity prediction in protein macromolecules. Few of them comparatively compete with the other predictive protein toxicity models and convincingly give a high-performance result in terms of accuracy. But their foundation is quite ambiguous, and varying approaches are found at the level of toxicoproteomic data utilization while building a machine learning model. In this review work, we present the contribution of artificial intelligence and machine learning approaches in prediction of protein toxicity using proteomics data.

Toxicoproteomics Disclose Pesticides as Downregulators of TNF-α, IL-1ß and Estrogen Receptor Pathways in Breast Cancer Women Chronically Exposed.

Deleterious effects have been widely associated with chronic pesticide exposure, including cancer development. In spite of several known consequences that pesticides can trigger in the human body, few is known regarding its impact on breast cancer women that are chronically exposed to such substances during agricultural work lifelong. In this context, the present study performed a high-throughput toxicoproteomic study in association with a bioinformatics-based design to explore new putative processes and pathways deregulated by chronic pesticide exposure in breast cancer patients. To reach this goal, we analyzed comparatively non-depleted plasma samples from exposed (n = 130) and non-occupationally exposed (n = 112) women diagnosed with breast cancer by using a label-free proteomic tool. The list of proteins differentially expressed was explored by bioinformatics and the main pathways and processes further investigated. The toxicoproteomic study revealed that women exposed to pesticides exhibited mainly downregulated events, linked to immune response, coagulation and estrogen-mediated events in relation to the unexposed ones. Further investigation shown that the identified deregulated processes and pathways correlated with significant distinct levels tumor necrosis factor alpha and interleukin 1 beta in the blood, and specific clinicopathological characteristics pointed out by bioinformatics analysis as adipose-trophic levels, menopause and intratumoral clots formation. Altogether, these findings reinforce pesticides as downregulators of several biological process and highlight that these compounds can be linked to poor prognosis in breast cancer.

Cytotoxicity and toxicoproteomic analyses of human lung epithelial cells exposed to extracts of atmospheric particulate matters on PTFE filters using acetone and water.

Differences of cytotoxicity associated with exposure to different extracts of atmospheric particulate matters (PMs) are still not well characterized by in vitro toxicoproteomics. In this study, in vitro cytotoxicity assays and toxicoproteomic analyses were carried out to investigate toxic effects of PM collected using polytetrafluoroethylene (PTFE) filters extracted with acetone for PM2.1 and water for PM2.1 and PM10 on A549 human lung epithelial cells. The cytotoxicity assays based on cell viability, cell apoptosis and reactive oxygen species generation indicated that PM2.1 extracted with acetone had the highest toxicity. iTRAQ labeling and LC-MS/MS analyses indicated that the number of differentially expressed proteins in A549 cells affected by PM2.1 extracted with acetone was noticeably higher than that of the other two groups. Hierarchical cluster analyses showed that the influences of the extracts of PM2.1 and PM10 using water on the proteome of A549 cells were similar, whereas significantly different from the effect of PM2.1 extracted with acetone. Pathways analyses indicated that PM2.1 extracted with acetone influenced the expression of proteins involved in 14 pathways including glycolysis/gluconeogenesis, pentose phosphate pathway, proteasome, etc. PM2.1 extracted with water affected the expression of proteins involved in 3 pathways including non-homologous end-joining, ribosome and endocytosis. However, PM10 extracted with water affected the expression of proteins involved in only spliceosome pathway. The extracts of PM using different extractants to detach PM from PTFE filters influenced the cytotoxic effects of PM and the proteome of A549 cells. Therefore, extractants should be assessed carefully before the investigations on cytotoxicity to improve the compatibility of experimental results among research teams.

[Clinical Pharmaceutical Research Based on New Proteome Analysis Based on Chromatographic Separation].

Comprehensive identification of antigens in immune complexes (IC-antigens) is beneficial to provide insights into pathophysiology and could form the basis for novel diagnostic and treatment strategies for many immune-related diseases. Immune complexome analysis is a method for comprehensively identifying and profiling IC-antigens in biological fluids (such as serum and cerebrospinal fluid). We applied this strategy to the analysis of circulating ICs in autoimmune diseases (rheumatoid arthritis, Sjögren's syndrome, systemic scleroderma, and systemic lupus erythematosus), infectious diseases, and cancers. Fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS) consists of fluorogenic derivatization of proteins, followed by HPLC of the derivatized proteins, isolation of the proteins differentially expressed in a certain group, enzymatic digestion of the isolated proteins followed by LC-tandem MS using a database-searching algorithm for protein identification. We have applied this method to understand the cardioprotective effect of pre-administration of docetaxel in adriamycin/docetaxel combination anti-cancer therapy, and the cellular processes that are affected by non-steroidal anti-inflammatory drugs (NSAIDs) in mouse stomach tissue during ulcer formation.

Leveraging proteomics to compare submerged versus air-liquid interface carbon nanotube exposure to a 3D lung cell model.

With the emerging concern over the potential toxicity associated with carbon nanotube inhalation exposure, several in vitro methods have been developed to evaluate cellular responses. Since the major concern for adverse effects by carbon nanotubes is inhalation, various lung cell culture models have been established for toxicity testing, thus creating a wide variation of methodology. Limited studies have conducted side-by-side comparisons of common methods used for carbon nanotube hazard testing. The aim of this work was to use proteomics to evaluate global cellular response, including pro-inflammatory and pro-fibrotic mediators, of a 3D lung model composed of macrophages, epithelial cells, and fibroblasts which mimics the human alveolar epithelial tissue barrier. The cells were exposed to Mitsui 7 (M-7) multi-walled carbon nanotubes (MWCNT) under submerged and air-liquid interface (ALI) conditions and discovery proteomics identified 3500 proteins. The M-7 ALI exposure compared to control was found to increase expression in proteins related to oxidative stress that were not found to be enriched in submerged exposure. Comparison of MWCNT exposure methods, M-7 ALI exposure versus M-7 submerged exposure, yielded protein enrichment in pathways known to be associated with carbon nanotube exposure stress response, such as acute phase response signaling and NRF2-mediated oxidative stress response. This study demonstrates a comparison of commonly deployed carbon nanotube exposure methods. These data should be considered by the nanotoxicology community when interpreting or cross comparing in vitro exposure results.

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.

A matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry-based toxicoproteomic screening method to assess in vitro particle potencies.

Knowledge of biological reactivity and underlying toxicity mechanisms of airborne particulate matter (PM) is central to the characterization of the risk associated with these pollutants. An integrated screening platform consisting of protein profiling of cellular responses and cytotoxic analysis was developed in this study for the estimation of PM potencies. Mouse macrophage (J774A.1) and human lung epithelial cells (A549) were exposed in vitro to Ottawa urban particles (EHC6802) and two reference mineral particles (TiO2 and SiO2 ). Samples from the in vitro exposure experiment were tested following an integrated classical cytotoxicity/toxicoproteomic assessment approach for cellular viability (CellTiter Blue®, lactate dehydrogenase) and proteomic analyses. Cellular proteins were pre-fractionated by molecular weight cut-off filtration, digested enzymatically and were analyzed by matrix-assisted laser desorption ionization-time-of-flight-time-of-flight-mass spectrometry for protein profiling and identification. Optimization of detergent removal, pre-fractionation strategies and enzymatic digestion procedures led to increased tryptic peptide (m/z) signals with reduced sample processing times, for small total protein contents. Proteomic analyses using this optimized procedure identified statistically significant (P < 0.05) PM dose-dependent changes at the molecular level. Ranking of PM potencies based on toxicoproteomic analysis were in line with classical cytotoxicity potency-based ranking. The high content toxicoproteomic approach exhibited the potential to add value to risk characterization of environmental PM exposures by complementing and validating existing cytotoxicity testing strategies.

Investigation of nonalcoholic fatty liver disease-induced drug metabolism by comparative global toxicoproteomics.

Non-alcoholic fatty liver disease (NAFLD) includes conditions such as steatosis, non-alcoholic steatohepatitis, and ultimately hepatocellular carcinoma. Although the pathology of NAFLD is well-established, NAFLD-induced drug metabolism mediated by cytochrome P450 (CYP) in the liver has remained largely unexplored. Therefore, we investigated NAFLD-induced drug metabolism mediated by CYP by quantitative toxicoproteomics analysis. After administration of a methionine-choline deficient (MCD) diet to induce development of NAFLD, tandem mass tags-based liquid chromatography-tandem mass spectrometry analysis was conducted to investigate the dynamics of hepatic proteins. A total of 1295 proteins were identified, of which 934 were quantified by proteomic analysis. Among these proteins, 21 proteins were up-regulated and 51 proteins were down-regulated by the MCD diet. Notably, domain annotation enrichment using InterPro indicated that proteins related to CYPs were significantly decreased. When we investigated CYP activity using in vivo and in vitro CYP cocktail assays, most CYPs were significantly decreased, whereas CYP2D was not changed after administration of the MCD diet. In conclusion, we identified significantly altered levels of CYPs and their activities induced by the MCD diet and confirmed the NAFLD-induced drug metabolism by pharmacokinetic analysis.

Toxicoproteomic assessment of liver responses to acute pyrrolizidine alkaloid intoxication in rats.

A toxicoproteomic study was performed on liver of rats treated with retrorsine (RTS), a representative hepatotoxic pyrrolizidine alkaloid at a toxic dose (140 mg/kg) known to cause severe acute hepatotoxicity. By comparing current data with our previous findings in mild liver lesions of rats treated with a lower dose of RTS, seven proteins and three toxicity pathways of vascular endothelial cell death, which was further verified by observed sinusoidal endothelial cell losses, were found uniquely associated with retrorsine-induced hepatotoxicity. This toxicoproteomic study of acute pyrrolizidine alkaloid intoxication lays a foundation for future investigation to delineate molecular mechanisms of pyrrolizidine alkaloid-induced hepatotoxicity.

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.

Differential impacts of individual and combined exposures of deoxynivalenol and zearalenone on the HepaRG human hepatic cell proteome.

Numerous surveys have highlighted the natural co-occurrence of deoxynivalenol (DON) and zearalenone (ZEA) mycotoxins in food and feed. Nevertheless, data regarding cellular mechanisms involved in response to their individual and simultaneous exposures are lacking. In this study, in order to analyze how low mycotoxin doses could impact cellular physiology and homeostasis, proteomic profiles of proliferating human hepatic cells (HepaRG) exposed for 1h and 24h to low DON and ZEA cytotoxicity levels (0.2 and 20µM respectively), alone or in combination, were analyzed by LC-MS/MS. Proteome analyses of mycotoxin-treated cells identified 4000 proteins with about 1.4% and 3.7% of these proteins exhibiting a significantly modified abundance compared to controls after 1h or 24h, respectively. Analysis of the Gene Ontology biological process annotations showed that cell cycle, proliferation and/or development as well as on DNA metabolic processes were affected for most treatments. Overall, different proteins, and thus biological processes, were impacted depending on the considered mycotoxin and exposure duration. Finally, despite the important proteome changes observed following 24h exposure to both mycotoxins, only the uptake of ZEA by the cells was suggested by the mycotoxin quantification in cell supernatants. BIOLOGICAL SIGNIFICANCE: This study investigated the proteomic changes that occurred after DON and ZEA (individually and in combination) short exposures at low cytotoxicity levels in proliferating HepaRG cells using LC-MS/MS. The obtained results showed that the cellular response is time- and mycotoxin or mixture-dependent. In particular, after 1h exposure, the DON+ZEA combination led to more proteomic changes than DON or ZEA alone, whereas the opposite was observed after 24h. In addition, the significant cellular response to stress induced by ZEA after 24h exposure seemed to be reduced when combined with DON. Thus, these results supported a possible mitigation by the hepatocytes when exposed to the mycotoxin mixture for a long duration. These findings represent an essential step to further explore adaptive cell response to mycotoxin exposure using with more complex incubation kinetics and combining different "omics" tools. Moreover, as mycotoxin quantification in cell supernatants showed different behaviors for DON and ZEA, this also raises the question about how mycotoxins actually trigger the cell response.

Effect of enrofloxacin on the proteome of earthworms.

The environmental and human health risks of veterinary drugs are becoming public health issues. Enrofloxacin (EF) is an extensively used animal-specific antibacterial agent that leaves drug residues in the environment. This study investigated the proteomic response of the earthworm Eisenia fetida to EF exposure. Earthworms were exposed to EF in soil at 1-500mg·kg-1, and samples were collected at intervals during a 28 day period. The extracted proteins were separated by two dimensional electrophoresis to detect differentially expressed proteins (DEPs) in EF-exposed earthworms. In total, 35 unique DEPs were found. These proteins were subjected to MALDI-TOF/TOF-MS analysis and identified through comparison of their mass spectra with those in protein databases. The DEPs were grouped on the basis of their function, into metabolism, stress-related, transport, transcription, and predicted/hypothetical protein categories. Knowledge of proteins that are induced or repressed by EF in earthworms could provide insight into mechanisms of sub-clinical physiological effects of xenobiotic residues in the environment, and may also help understand synergy between pollutants. As several DEPs in E. fetida showed similarity to human protein sequences, E. fetida has potential as an indicator species to assess the environmental and biological risks of drug residues.