Toxicology in the Fast Lane: Application of High-Throughput Bioassays to Detect Modulation of Key Enzymes and Receptors

BackgroundLegislation at state, federal, and international levels is requiring rapid evaluation of the toxicity of numerous chemicals. Whole-animal toxicologic studies cannot yield the necessary throughput in a cost-effective fashion, leading to a critical need for a faster and more cost-effective toxicologic evaluation of xenobiotics.ObjectivesWe tested whether mechanistically based screening assays can rapidly provide information on the potential for compounds to affect key enzymes and receptor targets, thus identifying those compounds requiring further in-depth analysis.MethodsA library of 176 synthetic chemicals was prepared and examined in a high-throughput screening (HTS) manner using nine enzyme-based and five receptor-based bioassays.ResultsAll the assays have high Z' values, indicating good discrimination among compounds in a reliable fashion, and thus are suitable for HTS assays. On average, three positive hits were obtained per assay. Although we identified compounds that were previously shown to inhibit a particular enzyme class or receptor, we surprisingly discovered that triclosan, a microbiocide present in personal care products, inhibits carboxylesterases and that dichlone, a fungicide, strongly inhibits the ryanodine receptors.ConclusionsConsidering the need to rapidly screen tens of thousands of anthropogenic compounds, our study shows the feasibility of using combined HTS assays as a novel approach toward obtaining toxicologic data on numerous biological end points. The HTS assay approach is very useful to quickly identify potentially hazardous compounds and to prioritize them for further in-depth studies

Primary irritant and delayed-contact hypersensitivity reactions to the freshwater cyanobacterium Cylindrospermopsis raciborskii and its associated toxin cylindrospermopsin

BACKGROUND: Freshwater cyanobacteria are common inhabitants of recreational waterbodies throughout the world; some cyanobacteria can dominate the phytoplankton and form blooms, many of which are toxic. Numerous reports in the literature describe pruritic skin rashes after recreational or occupational exposure to cyanobacteria, but there has been little research conducted on the cutaneous effects of cyanobacteria. Using the mouse ear swelling test (MEST), we sought to determine whether three toxin-producing cyanobacteria isolates and the purified cyanotoxin cylindrospermopsin produced delayed-contact hypersensitivity reactions. METHODS: Between 8 and 10 female Balb/c mice in each experiment had test material applied to depilated abdominal skin during the induction phase and 10 or 11 control mice had vehicle only applied to abdominal skin. For challenge (day 10) and rechallenge (day 17), test material was applied to a randomly-allocated test ear; vehicle was applied to the other ear as a control. Ear thickness in anaesthetised mice was measured with a micrometer gauge at 24 and 48 hours after challenge and rechallenge. Ear swelling greater than 20% in one or more test mice is considered a positive response. Histopathology examination of ear tissues was conducted by independent examiners. RESULTS: Purified cylindrospermopsin (2 of 9 test mice vs. 0 of 5 control mice; p = 0.51) and the cylindrospermopsin-producing cyanobacterium C. raciborskii (8 of 10 test mice vs. 0 of 10 control mice; p = 0.001) were both shown to produce hypersensitivity reactions. Irritant reactions were seen on abdominal skin at induction. Two other toxic cyanobacteria (Microcystis aeruginosa and Anabaena circinalis) did not generate any responses using this model. Histopathology examinations to determine positive and negative reactions in ear tissues showed excellent agreement beyond chance between both examiners (κ = 0.83). CONCLUSION: The irritant properties and cutaneous sensitising potential of cylindrospermopsin indicate that these toxicological endpoints should be considered by public health advisors and reservoir managers when setting guidelines for recreational exposure to cyanobacteria

Cd(II) and Pb(II) complexes of the polyether ionophorous antibiotic salinomycin

<p>Abstract</p> <p>Background</p> <p>The natural polyether ionophorous antibiotics are used for the treatment of coccidiosis in poultry and ruminants. They are effective agents against infections caused by Gram-positive microorganisms. On the other hand, it was found that some of these compounds selectively bind lead(II) ions in <it>in vivo </it>experiments, despite so far no Pb(II)-containing compounds of defined composition have been isolated and characterized. To assess the potential of polyether ionophores as possible antidotes in the agriculture, a detailed study on their <it>in vitro </it>complexation with toxic metal ions is required. In the present paper we report for the first time the preparation and the structure elucidation of salinomycin complexes with ions of cadmium(II) and lead(II).</p> <p>Results</p> <p>New metal(II) complexes of the polyether ionophorous antibiotic salinomycin with Cd(II) and Pb(II) ions were prepared and structurally characterized by IR, FAB-MS and NMR techniques. The spectroscopic information and elemental analysis data reveal that sodium salinomycin (SalNa) undergoes a reaction with heavy metal(II) ions to form [Cd(Sal)₂(H₂O)₂] (<b>1</b>) and [Pb(Sal)(NO₃)] (<b>2</b>), respectively. Abstraction of sodium ions from the cavity of the antibiotic is occurring during the complexation reaction. Salinomycin coordinates with cadmium(II) ions as a bidentate monoanionic ligand through the deprotonated carboxylic moiety and one of the hydroxyl groups to yield <b>1</b>. Two salinomycin anions occupy the equatorial plane of the Cd(II) center, while two water molecules take the axial positions of the inner coordination sphere of the metal(II) cation. Complex <b>2 </b>consists of monoanionic salinomycin acting in polydentate coordination mode in a molar ratio of 1: 1 to the metal ion with one nitrate ion for charge compensation.</p> <p>Conclusion</p> <p>The formation of the salinomycin heavy metal(II) complexes indicates a possible antidote activity of the ligand in case of chronic/acute intoxications likely to occur in the stock farming.</p

Analysis of MicroRNA Expression in Embryonic Developmental Toxicity Induced by MC-RR

As cynobacterial blooms frequently occur in fresh waters throughout the world, microcystins (MCs) have caused serious damage to both wildlife and human health. MCs are known to have developmental toxicity, however, the possible molecular mechanism is largely unknown. This is the first toxicological study to integrate post-transcriptomic, proteomic and bioinformatics analysis to explore molecular mechanisms for developmental toxicity of MCs in zebrafish. After being microinjected directly into embryos, MC-RR dose-dependently decreased survival rates and increased malformation rates of embryos, causing various embryo abnormalities including loss of vascular integrity and hemorrhage. Expressions of 31 microRNAs (miRNAs) and 78 proteins were significantly affected at 72 hours post-fertilisation (hpf). Expressions of miR-430 and miR-125 families were also significantly changed. The altered expressions of miR-31 and miR-126 were likely responsible for the loss of vascular integrity. MC-RR significantly reduced the expressions of a number of proteins involved in energy metabolism, cell division, protein synthesis, cytoskeleton maintenance, response to stress and DNA replication. Bioinformatics analysis shows that several aberrantly expressed miRNAs and proteins (involved in various molecular pathways) were predicted to be potential MC-responsive miRNA-target pairs, and that their aberrant expressions should be the possible molecular mechanisms for the various developmental defects caused by MC-RR

Effects of leucine supplemented diet on intestinal absorption in tumor bearing pregnant rats

BACKGROUND: It is known that amino acid oxidation is increased in tumor-bearing rat muscles and that leucine is an important ketogenic amino acid that provides energy to the skeletal muscle. METHODS: To evaluate the effects of a leucine supplemented diet on the intestinal absorption alterations produced by Walker 256, growing pregnant rats were distributed into six groups. Three pregnant groups received a normal protein diet (18% protein): pregnant (N), tumor-bearing (WN), pair-fed rats (Np). Three other pregnant groups were fed a diet supplemented with 3% leucine (15% protein plus 3% leucine): leucine (L), tumor-bearing (WL) and pair-fed with leucine (Lp). Non pregnant rats (C), which received a normal protein diet, were used as a control group. After 20 days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. RESULTS: Tumor-bearing pregnant rats showed impairment in food intake, body weight gain and muscle protein content, which were less accentuated in WL than in WN rats. These metabolic changes led to reduction in both fetal and tumor development. Leucine absorption slightly increased in WN group. In spite of having a significant decrease in leucine and methionine absorption compared to L, the WL group has shown a higher absorption rate of methionine than WN group, probably due to the ingestion of the leucine supplemented diet inducing this amino acid uptake. Glucose absorption was reduced in both tumor-bearing groups. CONCLUSIONS: Leucine supplementation during pregnancy in tumor-bearing rats promoted high leucine absorption, increasing the availability of the amino acid for neoplasic cells and, mainly, for fetus and host utilization. This may have contributed to the better preservation of body weight gain, food intake and muscle protein observed in the supplemented rats in relation to the non-supplemented ones

Skin sensitization in silico protocol

The assessment of skin sensitization has evolved over the past few years to include in vitro assessments of key events along the adverse outcome pathway and opportunistically capitalize on the strengths of in silico methods to support a weight of evidence assessment without conducting a test in animals. While in silico methods vary greatly in their purpose and format; there is a need to standardize the underlying principles on which such models are developed and to make transparent the implications for the uncertainty in the overall assessment. In this contribution, the relationship of skin sensitization relevant effects, mechanisms, and endpoints are built into a hazard assessment framework. Based on the relevance of the mechanisms and effects as well as the strengths and limitations of the experimental systems used to identify them, rules and principles are defined for deriving skin sensitization in silico assessments. Further, the assignments of reliability and confidence scores that reflect the overall strength of the assessment are discussed. This skin sensitization protocol supports the implementation and acceptance of in silico approaches for the prediction of skin sensitization

Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

BACKGROUND: Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. METHODS: In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF)-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF), which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. RESULTS: WF decreased the viability of C2C12 myotubes, especially at concentrations of 20-25 mug.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. CONCLUSION: These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model. © 2008 Yano et al; licensee BioMed Central Ltd

An interaction map of circulating metabolites, immune gene networks, and their genetic regulation

Background: Immunometabolism plays a central role in many cardiometabolic diseases. However, a robust map of immune-related gene networks in circulating human cells, their interactions with metabolites, and their genetic control is still lacking. Here, we integrate blood transcriptomic, metabolomic, and genomic profiles from two population-based cohorts (total N = 2168), including a subset of individuals with matched multi-omic data at 7-year follow-up. Results: We identify topologically replicable gene networks enriched for diverse immune functions including cytotoxicity, viral response, B cell, platelet, neutrophil, and mast cell/basophil activity. These immune gene modules show complex patterns of association with 158 circulating metabolites, including lipoprotein subclasses, lipids, fatty acids, amino acids, small molecules, and CRP. Genome-wide scans for module expression quantitative trait loci (mQTLs) reveal five modules with mQTLs that have both cis and trans effects. The strongest mQTL is in ARHGEF3 (rs1354034) and affects a module enriched for platelet function, independent of platelet counts. Modules of mast cell/basophil and neutrophil function show temporally stable metabolite associations over 7-year follow-up, providing evidence that these modules and their constituent gene products may play central roles in metabolic inflammation. Furthermore, the strongest mQTL in ARHGEF3 also displays clear temporal stability, supporting widespread trans effects at this locus. Conclusions: This study provides a detailed map of natural variation at the blood immunometabolic interface and its genetic basis, and may facilitate subsequent studies to explain inter-individual variation in cardiometabolic disease.Peer reviewe