Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

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Abstracts

International Journal of Toxicology ◽

10.1080/10915810802571781 ◽

2008 ◽

Vol 27 (6) ◽

pp. 405-405

Author(s):

David J. Dix

Keyword(s):

Risk Assessment ◽

High Throughput ◽

Environmental Protection Agency ◽

High Throughput Screening ◽

Human Health Risk ◽

Environmental Chemicals ◽

In Vitro Assays ◽

The Impact ◽

Toxicity Pathways

The U.S. Environmental Protection Agency (EPA), National Toxicology Program (NTP), and National Institutes of Health (NIH) Chemical Genomics Center (NCGC) have complementary research programs designed to improve chemical toxicity evaluations by developing high throughput screening (HTS) methods that evaluate the impact of environmental chemicals on key toxicity pathways. These federal partners are coordinating an extension of the EPA’s ToxCast program, the NTP’s HTS initiative, and the NCGC’s Molecular Libraries Initiative into a collaborative research program focused on identifying toxicity pathways and developing in vitro assays to characterize the ability of chemicals to perturb those pathways. The goal is to develop new paradigm for high throughput toxicity testing that collects mechanistic and quantitative data from in vitro assays measuring chemical modulation of biological processes involved in the progression to toxicity. As toxicity pathways are identified, the in vitro assays can be optimized for comparison to in vivo animal studies, and for predicting effects in humans. Subsequent computational modeling of toxicity pathway responses and appropriate chemical dosimetry will need to be developed to make these predictions relevant for human health risk assessment. This work was reviewed by EPA and approved for publication but does not necessarily reflect official Agency policy. Index Terms: Toxicogenomics, High Throughput Screening/Testing, EPA ToxCast, Chemical Risk Assessment

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Development of a Miniaturized 384-Well High Throughput Screen for the Detection of Substrates of Cytochrome P450 2D6 and 3A4 Metabolism

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710100600205 ◽

2001 ◽

Vol 6 (2) ◽

pp. 91-99 ◽

Cited By ~ 14

Author(s):

Ilona Kariv ◽

Mark P. Fereshteh ◽

Kevin R. Oldenburg

Keyword(s):

Drug Metabolism ◽

High Throughput ◽

High Throughput Screening ◽

Biologically Active ◽

In Vitro Assays ◽

Main Concern ◽

Assay Sensitivity ◽

Cyp Enzymes ◽

Assay Optimization

The identification of a large number of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with druglike properties early during the lead prioritization and development process. One of the major steps in lead prioritization is the assessment of drug metabolism mediated by the cytochrome P450 (CYP) enzymes to evaluate the potential drug-drug interactions. CYP2D6 and CYP3A4 comprise the main human CYP enzymes involved in drug metabolism. The recent availability of specific CYP cDNA expression systems and the development of specific fluorescent probes have accelerated the ability to develop robust in vitro assays in HTS format. The aim of this study was to optimize conditions for the CYP2D6 and CYP3A4 HTS assays and subsequently adapt those assays to a miniaturized 384-well format. Assay conversion to a miniaturized format presents certain difficulties, such as robustness of the signal and of compound delivery. Thus the assay optimization involved the comparison of different substrates to identify those most suitable for use in a miniaturized format. Because of current technical limitations in liquid dispensing of nanoliter volumes, assay sensitivity to organic solvents also provides a main concern during assay miniaturization. Therefore, compound activity from redissolved dry films and from DMSO stocks directly delivered into assay buffer was compared. The data indicate that compound activity was comparable in both formats. The data support the conclusion that CYP2D6 and CYP3A4 in vitro metabolism assays can be successfully performed in 384-well plate format and the substrate potencies, as evaluated by the IC50 values, determined.

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Comparison of in Vitro Models for the Prediction of Compound Absorption across the Human Intestinal Mucosa

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104267162 ◽

2004 ◽

Vol 9 (7) ◽

pp. 598-606 ◽

Cited By ~ 45

Author(s):

Silvia Miret ◽

Leo Abrahamse ◽

Els M. de Groene

Keyword(s):

Cell Differentiation ◽

High Throughput ◽

High Throughput Screening ◽

In Vitro Models ◽

Assay System ◽

In Vitro Assays ◽

Cell Models ◽

Artificial Membrane ◽

Permeability Assay

Several in vitro assays have been developed to evaluate the gastrointestinal absorption of compounds. Our aim was to compare 3 of these methods: 1) the bio-mimetic artificial membrane permeability assay (BAMPA) method, which offers a high-throughput, noncellular approach to the measurement of passive transport; 2) the traditional Caco-2 cell assay, the use of which as a high-throughput tool is limited by the long cell differentiation time (21 days); and 3) The BioCoat™ high-throughput screening Caco-2 Assay System, which reduces Caco-2 cell differentiation to 3 days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations ( r = 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat™ HTS Caco-2 Assay System does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21-day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption.

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Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish

International Journal of Molecular Sciences ◽

10.3390/ijms22168401 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8401

Author(s):

Valentina Naef ◽

Maria Marchese ◽

Asahi Ogi ◽

Gianluca Fichi ◽

Daniele Galatolo ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Molecular Mechanisms ◽

Motor Impairment ◽

Model Organism ◽

Cell Degeneration ◽

Vertebrate Model ◽

Therapeutic Compounds

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs−/− larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.

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Development of High-Throughput Screening Assays for Inhibitors of ETS Transcription Factors

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555218798571 ◽

2018 ◽

Vol 24 (1) ◽

pp. 77-85

Author(s):

Simon L. Currie ◽

Steven L. Warner ◽

Hariprasad Vankayalapati ◽

Xiaohui Liu ◽

Sunil Sharma ◽

...

Keyword(s):

Prostate Cancer ◽

Transcription Factors ◽

High Throughput ◽

High Throughput Screening ◽

In Vitro Assays ◽

Ets Transcription Factors ◽

Virtual Screen ◽

Prostate Cells ◽

Screening Assays

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

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Development of a Microplate-Based Scintillation Proximity Assay for MraY Using a Modified Substrate

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104272007 ◽

2005 ◽

Vol 10 (2) ◽

pp. 149-156 ◽

Cited By ~ 12

Author(s):

S. M. Solapure ◽

P. Raphael ◽

C. N. Gayathri ◽

S. P. Barde ◽

B. Chandrakala ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Antibacterial Agents ◽

Specific Activity ◽

Wild Type ◽

Scintillation Proximity Assay ◽

Peptidoglycan Synthesis ◽

Homogeneous Assay ◽

Radioactive Substrate

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.

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A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

Nucleic Acids Research ◽

10.1093/nar/gkaa1213 ◽

2020 ◽

Author(s):

Yuru Wang ◽

Christopher D Katanski ◽

Christopher Watkins ◽

Jessica N Pan ◽

Qing Dai ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Method ◽

Targeted Mutagenesis ◽

Rna Repair ◽

Dna And Rna ◽

Modified Dna ◽

Dna Substrates ◽

Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

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High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

Nature Communications ◽

10.1038/s41467-021-23954-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Zhou Fang ◽

Junjian Chen ◽

Ye Zhu ◽

Guansong Hu ◽

Haoqian Xin ◽

...

Keyword(s):

Antimicrobial Activity ◽

High Throughput ◽

High Throughput Screening ◽

Rational Design ◽

Click Reaction ◽

Reaction Times ◽

Great Promise ◽

Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

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