Meta-Analysis of Integrated Proteomic and Transcriptomic Data Discerns Structure-Activity Relationship of Carbon Materials with Different Morphologies.

The Fiber Pathogenicity Paradigm (FPP) establishes connections between fiber structure, durability, and disease-causing potential observed in materials like asbestos and synthetic fibers. While emerging nanofibers are anticipated to exhibit pathogenic traits according to the FPP, their nanoscale diameter limits rigidity, leading to tangling and loss of fiber characteristics. The absence of validated rigidity measurement methods complicates nanofiber toxicity assessment. By comprehensively analyzing 89 transcriptomics and 37 proteomics studies, this study aims to enhance carbon material toxicity understanding and proposes an alternative strategy to assess morphology-driven toxicity. Carbon materials are categorized as non-fibrous, high aspect ratio with shorter lengths, tangled, and rigid fibers. Mitsui-7 serves as a benchmark for pathogenic fibers. The meta-analysis reveals distinct cellular changes for each category, effectively distinguishing rigid fibers from other carbon materials. Subsequently, a robust random forest model is developed to predict morphology, unveiling the pathogenicity of previously deemed non-pathogenic NM-400 due to its secondary structures. This study fills a crucial gap in nanosafety by linking toxicological effects to material morphology, in particular regarding fibers. It demonstrates the significant impact of morphology on toxicological behavior and the necessity of integrating morphological considerations into regulatory frameworks.

Toxic and Genomic Influences of Inhaled Nanomaterials as a Basis for Predicting Adverse Outcome.

An immense variety of different types of engineered nanomaterials are currently being developed and increasingly applied to consumer products. Importantly, engineered nanomaterials may pose unexplored adverse health effects because of their small size. Particularly in occupational settings, the dustiness of certain engineered nanomaterials involves risk of inhalation and influences on lung function. These facts call for quick and cost-effective safety testing practices, such as that obtained through multiparametric high-throughput screening using cultured human lung cells. The predictive value of such in vitro-based testing depends partly on the effectiveness of coverage of the mechanisms underlying toxicity effects. The concept of adverse outcome pathways covers the array of causative effects starting from a molecular initiating event via cellular-, organ-, individual-, and population-level effects. Screening for adverse outcome pathway-related effects that drive the eventual toxic outcome provides a good basis for developing predictive testing methods and data-driven integrated testing strategies for hazard and risk assessment. Temporal and inherited genomic changes are likely to drive many adverse responses to engineered nanomaterials, such as multiwalled carbon nanotubes, of which one specific form has recently been evaluated as possibly carcinogenic. Here, we briefly describe current state-of-the-art strategies for analyzing and understanding genomic influences of engineered nanomaterial exposure, including the selected focus on lung disease, and strategies for using mechanistic knowledge to predict and prevent adverse outcome.

A Data Fusion Pipeline for Generating and Enriching Adverse Outcome Pathway Descriptions.

Increasing amounts of systems toxicology data, including omics results, are becoming publically available and accessible in databases. Data-driven and informatics-tool supported pipeline schemas for fitting such data into Adverse Outcome Pathway (AOP) descriptions could potentially aid the development of nonanimal-based hazard and risk assessment methods. We devised a 6-step workflow that integrated diverse types of toxicology data into a novel AOP scheme for pulmonary fibrosis. Mining of literature references and diverse data sources covering previous pathway descriptions and molecular results were coupled in a stepwise manner with informatics tools applications that enabled gene linkage and pathway identification in molecular interaction maps. Ultimately, a network of functional elements coupled 64 pulmonary fibrosis-associated genes into a novel, open-source AOP-linked molecular pathway, now available for commenting and improvements in WikiPathways (WP3624). Applying in silico-based knowledge extraction and modeling, the pipeline enabled screening and fusion of many different complex data types, including the integration of omics results. Overall, the taken, stepwise approach should be generally useful to construct novel AOP descriptions as well as to enrich developing AOP descriptions in progress.

A transcriptomics data-driven gene space accurately predicts liver cytopathology and drug-induced liver injury.

Predicting unanticipated harmful effects of chemicals and drug molecules is a difficult and costly task. Here we utilize a 'big data compacting and data fusion'-concept to capture diverse adverse outcomes on cellular and organismal levels. The approach generates from transcriptomics data set a 'predictive toxicogenomics space' (PTGS) tool composed of 1,331 genes distributed over 14 overlapping cytotoxicity-related gene space components. Involving ∼2.5 × 108 data points and 1,300 compounds to construct and validate the PTGS, the tool serves to: explain dose-dependent cytotoxicity effects, provide a virtual cytotoxicity probability estimate intrinsic to omics data, predict chemically-induced pathological states in liver resulting from repeated dosing of rats, and furthermore, predict human drug-induced liver injury (DILI) from hepatocyte experiments. Analysing 68 DILI-annotated drugs, the PTGS tool outperforms and complements existing tests, leading to a hereto-unseen level of DILI prediction accuracy.

Toward the Replacement of Animal Experiments through the Bioinformatics-driven Analysis of 'Omics' Data from Human Cell Cultures.

This paper outlines the work for which Roland Grafström and Pekka Kohonen were awarded the 2014 Lush Science Prize. The research activities of the Grafström laboratory have, for many years, covered cancer biology studies, as well as the development and application of toxicity-predictive in vitro models to determine chemical safety. Through the integration of in silico analyses of diverse types of genomics data (transcriptomic and proteomic), their efforts have proved to fit well into the recently-developed Adverse Outcome Pathway paradigm. Genomics analysis within state-of-the-art cancer biology research and Toxicology in the 21st Century concepts share many technological tools. A key category within the Three Rs paradigm is the Replacement of animals in toxicity testing with alternative methods, such as bioinformatics-driven analyses of data obtained from human cell cultures exposed to diverse toxicants. This work was recently expanded within the pan-European SEURAT-1 project (Safety Evaluation Ultimately Replacing Animal Testing), to replace repeat-dose toxicity testing with data-rich analyses of sophisticated cell culture models. The aims and objectives of the SEURAT project have been to guide the application, analysis, interpretation and storage of 'omics' technology-derived data within the service-oriented sub-project, ToxBank. Particularly addressing the Lush Science Prize focus on the relevance of toxicity pathways, a 'data warehouse' that is under continuous expansion, coupled with the development of novel data storage and management methods for toxicology, serve to address data integration across multiple 'omics' technologies. The prize winners' guiding principles and concepts for modern knowledge management of toxicological data are summarised. The translation of basic discovery results ranged from chemical-testing and material-testing data, to information relevant to human health and environmental safety.

YAP1 is a potential biomarker for cetuximab resistance in head and neck cancer.

OBJECTIVES: Targeted therapy against the epidermal growth factor receptor (EGFR) only variably represents a therapeutic advance in head and neck squamous cell carcinoma (HNSCC). This study addresses the need of biomarkers of treatment response to the EGFR-targeting antibody cetuximab (Erbitux®). MATERIALS AND METHODS: The intrinsic cetuximab sensitivity of HNSCC cell lines was assessed by a crystal violet assay. Gene copy number analysis of five resistant and five sensitive cell lines was performed using the Affymetrix SNP 6.0 platform. Quantitative real-time PCR was used for verification of selected copy number alterations and assessment of mRNA expression. The functional importance of the findings on the gene and mRNA level was investigated employing siRNA technology. The data was statistically evaluated using Mann-Whitney U-test and Spearman's correlation test. RESULTS: Analysis of the intrinsic cetuximab sensitivity of 32 HNSCC cell lines characterized five and nine lines as cetuximab sensitive or resistant, respectively. Gene copy number analysis of five resistant versus five sensitive cell lines identified 39 amplified protein-coding genes, including YAP1, in the genomic regions 11q22.1 or 5p13-15. Assessment using qPCR verified that YAP1 amplification associated with cetuximab resistance. Amplification of YAP1 correlated to higher mRNA levels, and RNA knockdown resulted in increased cetuximab sensitivity. Assessment of several independent clinical data sets in the public domain confirmed YAP1 amplifications in multiple tumor types including HNSCC, along with highly differential expression in a subset of HNSCC patients. CONCLUSION: Taken together, we provide evidence that YAP1 could represent a novel biomarker gene of cetuximab resistance in HNSCC cell lines.

The ToxBank Data Warehouse: Supporting the Replacement of In Vivo Repeated Dose Systemic Toxicity Testing.

The aim of the SEURAT-1 (Safety Evaluation Ultimately Replacing Animal Testing-1) research cluster, comprised of seven EU FP7 Health projects co-financed by Cosmetics Europe, is to generate a proof-of-concept to show how the latest technologies, systems toxicology and toxicogenomics can be combined to deliver a test replacement for repeated dose systemic toxicity testing on animals. The SEURAT-1 strategy is to adopt a mode-of-action framework to describe repeated dose toxicity, combining in vitro and in silico methods to derive predictions of in vivo toxicity responses. ToxBank is the cross-cluster infrastructure project whose activities include the development of a data warehouse to provide a web-accessible shared repository of research data and protocols, a physical compounds repository, reference or "gold compounds" for use across the cluster (available via wiki.toxbank.net), and a reference resource for biomaterials. Core technologies used in the data warehouse include the ISA-Tab universal data exchange format, REpresentational State Transfer (REST) web services, the W3C Resource Description Framework (RDF) and the OpenTox standards. We describe the design of the data warehouse based on cluster requirements, the implementation based on open standards, and finally the underlying concepts and initial results of a data analysis utilizing public data related to the gold compounds.

Requirement of apoptotic protease-activating factor-1 for bortezomib-induced apoptosis but not for Fas-mediated apoptosis in human leukemic cells.

Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)-deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.

MAML1 acts cooperatively with EGR1 to activate EGR1-regulated promoters: implications for nephrogenesis and the development of renal cancer.

Mastermind-like 1 (MAML1) is a transcriptional coregulator of activators in various signaling pathways, such as Notch, p53, myocyte enhancer factor 2C (MEF2C) and beta-catenin. In earlier studies, we demonstrated that MAML1 enhanced p300 acetyltransferase activity, which increased the acetylation of Notch by p300. In this study, we show that MAML1 strongly induced acetylation of the transcription factor early growth response-1 (EGR1) by p300, and increased EGR1 protein expression in embryonic kidney cells. EGR1 mRNA transcripts were also upregulated in the presence of MAML1. We show that MAML1 physically interacted with, and acted cooperatively with EGR1 to increase transcriptional activity of the EGR1 and p300 promoters, which both contain EGR1 binding sites. Bioinformatics assessment revealed a correlation between p300, EGR1 and MAML1 copy number and mRNA alterations in renal clear cell carcinoma and p300, EGR1 and MAML1 gene alterations were associated with increased overall survival. Our findings suggest MAML1 may be a component of the transcriptional networks which regulate EGR1 target genes during nephrogenesis and could also have implications for the development of renal cell carcinoma.

CD44 expression in oro-pharyngeal carcinoma tissues and cell lines.

Expression of CD44, a transmembrane hyaluronan-binding glycoprotein, is variably considered to have prognostic significance for different cancers, including oral squamous cell carcinoma. Although unclear at present, tissue-specific expression of particular isoforms of CD44 might underlie the different outcomes in currently available studies. We mined public transcriptomics databases for gene expression data on CD44, and analyzed normal, immortalized and tumour-derived human cell lines for splice variants of CD44 at both the transcript and protein levels. Bioinformatics readouts, from a total of more than 15,000 analyses, implied an increased CD44 expression in head and neck cancer, including increased expression levels relative to many normal and tumor tissue types. Also, meta-analysis of over 260 cell lines and over 4,000 tissue specimens of diverse origins indicated lower CD44 expression levels in cell lines compared to tissue. With minor exceptions, reverse transcribed polymerase chain reaction identified expression of the four main isoforms of CD44 in normal oral keratinocytes, transformed lines termed DT and HaCaT, and a series of paired primary and metastasis-derived cell lines from oral or pharyngeal carcinomas termed HN4/HN12, HN22/HN8 and HN30/HN31. Immunocytochemistry, Western blotting and flow cytometric assessments all confirmed the isoform expression pattern at the protein level. Overall, bioinformatic processing of large numbers of global gene expression analyses demonstrated elevated CD44 expression in head and neck cancer relative to other cancer types, and that the application of standard cell culture protocols might decrease CD44 expression. Additionally, the results show that the many variant CD44 exons are not fundamentally deregulated in a diverse range of cultured normal and transformed keratinocyte lines.

Differentiation-promoting culture of competent and noncompetent keratinocytes identifies biomarkers for head and neck cancer.

Aberrant contact-inhibited proliferation and differentiation induction couple with tumor severity, albeit with an imprecise association with prognosis. Assessment of contact inhibition and differentiation-promoting culture in this study of normal and immortalized oral keratinocytes (NOK and SVpgC2a, respectively) demonstrated elevated cloning ability and saturation density in the immortalized versus normal state, including consistent absence of differentiated morphological features. Transcriptomic analysis implicated 48 gene ontology categories, 8 molecular networks, and 10 key regulator genes in confluency-induced differentiation of NOK, all of which remained nonregulated in SVpgC2a. The SVpgC2a versus NOK transcriptome enriched 52 gene ontology categories altogether, 18 molecular networks, and 39 key regulator genes, several of which were associated with epithelial-mesenchymal transition. Assessment of the previously described gene sets relative to training data sets of head and neck squamous cell carcinoma samples, one including data on tumor differentiation and patient outcome and one present in the Human Gene Expression Map, identified four genes with association to poor survival (COX7A1, MFAP5, MPDU1, and POLD1). This gene set predicted poor outcome in an independent data set of 71 head and neck squamous cell carcinomas. The present study defines, for the first time to our knowledge, the broad gene spectrum that couples to induction, and loss, of oral keratinocyte differentiation. Bioinformatics assessments of the results relative to clinical data generated novel differentiation-related tumor biomarkers relevant to patient outcome.

Computational toxicology using the OpenTox application programming interface and Bioclipse.

BACKGROUND: Toxicity is a complex phenomenon involving the potential adverse effect on a range of biological functions. Predicting toxicity involves using a combination of experimental data (endpoints) and computational methods to generate a set of predictive models. Such models rely strongly on being able to integrate information from many sources. The required integration of biological and chemical information sources requires, however, a common language to express our knowledge ontologically, and interoperating services to build reliable predictive toxicology applications. FINDINGS: This article describes progress in extending the integrative bio- and cheminformatics platform Bioclipse to interoperate with OpenTox, a semantic web framework which supports open data exchange and toxicology model building. The Bioclipse workbench environment enables functionality from OpenTox web services and easy access to OpenTox resources for evaluating toxicity properties of query molecules. Relevant cases and interfaces based on ten neurotoxins are described to demonstrate the capabilities provided to the user. The integration takes advantage of semantic web technologies, thereby providing an open and simplifying communication standard. Additionally, the use of ontologies ensures proper interoperation and reliable integration of toxicity information from both experimental and computational sources. CONCLUSIONS: A novel computational toxicity assessment platform was generated from integration of two open science platforms related to toxicology: Bioclipse, that combines a rich scriptable and graphical workbench environment for integration of diverse sets of information sources, and OpenTox, a platform for interoperable toxicology data and computational services. The combination provides improved reliability and operability for handling large data sets by the use of the Open Standards from the OpenTox Application Programming Interface. This enables simultaneous access to a variety of distributed predictive toxicology databases, and algorithm and model resources, taking advantage of the Bioclipse workbench handling the technical layers.

Combining factors on protein and gene level to predict radioresponse in head and neck cancer cell lines.

BACKGROUND: Radiotherapy is the main therapy for head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems, highlighting the need for predictive markers. In this study, we evaluated selected proteins, mutations, and single nucleotide polymorphisms (SNPs) involved in apoptosis, cell proliferation, and DNA repair alone or combined as predictive markers for radioresponse in 42 HNSCC cell lines. METHODS: The expression of epidermal growth factor receptor, survivin, Bax, Bcl-2, Bcl-X(L) , cyclooxygenase-2 (COX-2), and heat shock protein 70 was analyzed by ELISA. Furthermore, mutations and SNPs in the p53 gene as well as SNPs in the MDM2, XRCC1, and XRCC3 genes were analyzed for their relation to radioresponse. To enable the evaluation of the predictive value of several factors combined, each cell line was allocated points based on the number of negative points (NNP) system, and the NNP sum was correlated with radioresponse. RESULTS: Survivin was the only factor that alone was significantly correlated with the intrinsic radiosensitivity (IR; r = 0.36, P = 0.02). The combination of survivin, Bax, Bcl-2, Bcl-X(L) , COX-2, and the p53 Arg72Pro polymorphism was found to most strongly correlate with radioresponse (r = 0.553, P < 0.001). CONCLUSION: These data indicate that the IR of 42 HNSCC cell lines can be predicted by a panel of factors on both the protein and gene levels. Moreover, among the investigated factors, survivin was the most promising biomarker of radioresponse.

Fibronectin 1 is a potential biomarker for radioresistance in head and neck squamous cell carcinoma.

Radiotherapy remains the backbone of head and neck cancer therapy but response is sometimes impeded by tumor radioresistance. Identifying predictive biomarkers of radiotherapy response is a crucial step towards personalized therapy. The aim of this study was to explore gene expression data in search of biomarkers predictive of the response to radiotherapy in head and neck squamous cell carcinoma (HNSCC). Microarray analysis was performed on five cell lines with various intrinsic radiosensitivity, selected from a panel of 29 HNSCC cell lines. The bioinformatics approach included Gene Ontology (GO) enrichment profiling and Ingenuity Pathway Analysis (IPA). The GO-analysis detected 16 deregulated categories from which development, receptor activity, and extracellular region represented the largest groups. Fourteen hub genes (CEBPA, CEBPB, CTNNB1, FN1, MYC, MYCN, PLAU, SDC4, SERPINE1, SP1, TAF4B, THBS1, TP53 and VLDLR) were identified from the IPA network analysis. The hub genes in the highest ranked network, (FN1, SERPINE1, THBS1 and VLDLR) were further subjected to qPCR analysis in the complete panel of 29 cell lines. Of these genes, high FN1 expression associated to high intrinsic radiosensitivity (p=0.047). In conclusion, gene ontologies and hub genes of importance for intrinsic radiosensitivity were defined. The overall results suggest that FN1 should be explored as a potential novel biomarker for radioresistance.

Formaldehyde dehydrogenase: beyond phase I metabolism.

Formaldehyde dehydrogenase, formally Class III alcohol dehydrogenase (ADH3), has recently been discovered to partially regulate nitrosothiol homeostasis by catalyzing the reduction of the endogenous nitrosylating agent S-nitrosoglutathione (GSNO). Several studies have implicated this enzyme, and in particular GSNO reduction, as playing an important role in conditions such as asthma, cardiovascular disease, and immune function. While ADH3 has received considerable attention in the biomedical literature where it is often referred to as GSNO reductase (GSNOR), ADH3-mediated GSNO reduction has received comparatively less attention in the environmental toxicology community. Herein, evidences for a role of ADH3 in cell signaling through thiol homeostasis is highlighted, underscoring that the enzyme functions more broadly than to metabolize formaldehyde.

Intrinsic differences in cisplatin sensitivity of head and neck cancer cell lines: Correlation to lysosomal pH.

BACKGROUND: Cisplatin treatment is beneficial for approximately 20% of patients with head and neck squamous cell carcinoma (HNSCC). Tools to predict the clinical outcome and evaluate intrinsic cisplatin sensitivity are, therefore, required. METHODS: Cisplatin sensitivity, lysosomal pH, and cell death pathway was studied in 5 HNSCC lines and compared with normal oral keratinocytes. RESULTS: We identified a linear relationship between lysosomal pH and cisplatin sensitivity. Reduced lysosomal acidification was correlated to decreased expression of the V(0)V(1)-ATPase B2 subunit, which is part of the lysosomal acidifying complex. Cisplatin caused apoptosis accompanied by lysosomal membrane permeabilization, and inhibition of lysosomal proteases (cathepsins) partly prevented cell death. CONCLUSION: Cisplatin-induced apoptosis of HNSCC is more efficient in cell lines with low lysosomal pH and is mediated by the release of lysosomal content. Lysosomal pH and expression of V(0)V(1)-ATPase subunits are possible future markers of intrinsic cisplatin sensitivity.

Commentary: mechanistic considerations for associations between formaldehyde exposure and nasopharyngeal carcinoma.

Occupational exposure to formaldehyde has been linked to nasopharyngeal carcinoma. To date, mechanistic explanations for this association have primarily focused on formaldehyde-induced cytotoxicity, regenerative hyperplasia and DNA damage. However, recent studies broaden the potential mechanisms as it is now well established that formaldehyde dehydrogenase, identical to S-nitrosoglutathione reductase, is an important mediator of cGMP-independent nitric oxide signaling pathways. We have previously described mechanisms by which formaldehyde can influence nitrosothiol homeostasis thereby leading to changes in pulmonary physiology. Considering evidences that nitrosothiols govern the Epstein-Barr virus infection cycle, and that the virus is strongly implicated in the etiology of nasopharyngeal carcinoma, studies are needed to examine the potential for formaldehyde to reactivate the Epstein-Barr virus as well as additively or synergistically interact with the virus to potentiate epithelial cell transformation.

High-throughput cell-based screening of 4910 known drugs and drug-like small molecules identifies disulfiram as an inhibitor of prostate cancer cell growth.

PURPOSE: To identify novel therapeutic opportunities for patients with prostate cancer, we applied high-throughput screening to systematically explore most currently marketed drugs and drug-like molecules for their efficacy against a panel of prostate cancer cells. EXPERIMENTAL DESIGN: We carried out a high-throughput cell-based screening with proliferation as a primary end-point using a library of 4,910 drug-like small molecule compounds in four prostate cancer (VCaP, LNCaP, DU 145, and PC-3) and two nonmalignant prostate epithelial cell lines (RWPE-1 and EP156T). The EC(50) values were determined for each cell type to identify cancer selective compounds. The in vivo effect of disulfiram (DSF) was studied in VCaP cell xenografts, and gene microarray and combinatorial studies with copper or zinc were done in vitro for mechanistic exploration. RESULTS: Most of the effective compounds, including antineoplastic agents, were nonselective and found to inhibit both cancer and control cells in equal amounts. In contrast, histone deacetylase inhibitor trichostatin A, thiram, DSF, and monensin were identified as selective antineoplastic agents that inhibited VCaP and LNCaP cell proliferation at nanomolar concentrations. DSF reduced tumor growth in vivo, induced metallothionein expression, and reduced DNA replication by downregulating MCM mRNA expression. The effect of DSF was potentiated by copper in vitro. CONCLUSIONS: We identified three novel cancer-selective growth inhibitory compounds for human prostate cancer cells among marketed drugs. We then validated DSF as a potential prostate cancer therapeutic agent. These kinds of pharmacologically well-known molecules can be readily translated to in vivo preclinical studies and clinical trials.

Medium-chain fatty acids and glutathione derivatives as inhibitors of S-nitrosoglutathione reduction mediated by alcohol dehydrogenase 3.

Alcohol dehydrogenase 3 (ADH3) has emerged as an important regulator of protein S-nitrosation in its function as S-nitrosoglutathione (GSNO) reductase. GSNO depletion is associated with various disease conditions, emphasizing the potential value of a specific ADH3 inhibitor. The present study investigated inhibition of ADH3-mediated GSNO reduction by various substrate analogues, including medium-chain fatty acids and glutathione derivatives. The observed inhibition type was non-competitive. Similar to the Michaelis constants for the corresponding omega-hydroxy fatty acids, the inhibition constants for fatty acids were in the micromolar range and showed a clear dependency on chain length with optimal inhibitory capacity for eleven and twelve carbons. The most efficient inhibitors found were undecanoic acid, dodecanoic acid and dodecanedioic acid, with no significant difference in inhibition constant. All glutathione-derived inhibitors displayed inhibition constants in the millimolar range, at least three orders of magnitudes higher than the Michaelis constants of the high-affinity substrates GSNO and S-hydroxymethylglutathione. The experimental results as well as docking simulations with GSNO and S-methylglutathione suggest that for ADH3 ligands with a glutathione scaffold, in contrast to fatty acids, a zinc-binding moiety is imperative for correct orientation and stabilization of the hydrophilic glutathione scaffold within a predominantly hydrophobic active site.