Identification of Potent and Selective Acetylcholinesterase/Butyrylcholinesterase Inhibitors by Virtual Screening.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) play important roles in human neurodegenerative disorders such as Alzheimer's disease. In this study, machine learning methods were applied to develop quantitative structure-activity relationship models for the prediction of novel AChE and BChE inhibitors based on data from quantitative high-throughput screening assays. The models were used to virtually screen an in-house collection of ∼360K compounds. The optimal models achieved good performance with area under the receiver operating characteristic curve values ranging from 0.83 ± 0.03 to 0.87 ± 0.01 for the prediction of AChE/BChE inhibition activity and selectivity. Experimental validation showed that the best-performing models increased the assay hit rate by several folds. We identified 88 novel AChE and 126 novel BChE inhibitors, 25% (AChE) and 53% (BChE) of which showed potent inhibitory effects (IC50 < 5 µM). In addition, structure-activity relationship analysis of the BChE inhibitors revealed scaffolds for chemistry design and optimization. In conclusion, machine learning models were shown to efficiently identify potent and selective inhibitors against AChE and BChE and novel structural series for further design and development of potential therapeutics against neurodegenerative disorders.

High-Throughput Neurite Outgrowth Assay Using GFP-Labeled iPSC-Derived Neurons.

The potential neurotoxicity from an increasing number of drugs and untested environmental chemicals creates a need to develop reliable and efficient in vitro methods for identifying chemicals that may adversely affect the nervous system. An important process in neurodevelopment is neurite outgrowth, which can be affected by developmental neurotoxicity. Currently, neurite outgrowth assays rely mainly on staining, which requires multiple sample processing steps, particularly washing steps, that may introduce variation and limit throughput. Here, we describe a neurite outgrowth assay that uses induced pluripotent stem cell (iPSC)-derived human cortical glutamatergic neurons and/or spinal motor neurons labeled with green fluorescent protein (GFP) to test compounds in a high-content and high-throughput format. This method enables live and time-lapse imaging of GFP-labeled neurons using an assay plate that is continuously imaged at multiple times after chemical treatment. In this article, we describe how to thaw frozen GFP-labeled neurons, culture them, treat them with a compound of interest, and analyze neurite outgrowth using a high-content imaging platform. In this assay, GFP-labeled iPSC-derived human neurons represent a promising tool for identifying and prioritizing compounds with potential developmental neurotoxicity for further hazard characterization. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Thawing and seeding of iPSC-derived neurons Basic Protocol 2: Compound plate preparation and treatment of neurons Basic Protocol 3: High-content imaging and analysis.

Fatality assessment and variant risk monitoring for COVID-19 using three new hospital occupancy related metrics.

BACKGROUND: Though case fatality rate (CFR) is widely used to reflect COVID-19 fatality risk, its use is limited by large temporal and spatial variation. Hospital mortality rate (HMR) is also used to assess the severity of COVID-19, but HMR data is not directly available globally. Alternative metrics are needed for COVID-19 severity and fatality assessment. METHODS: We introduce new metrics for COVID-19 fatality risk measurements/monitoring and a new mathematical model to estimate average hospital length of stay for deaths (Ldead) and discharges (Ldis). Multiple data sources were used for our analyses. FINDINGS: We propose three, new metrics: hospital occupancy mortality rate (HOMR), ratio of total deaths to hospital occupancy (TDHOR), and ratio of hospital occupancy to cases (HOCR), for dynamic assessment of COVID-19 fatality risk. Estimated Ldead and Ldis for 501,079 COVID-19 hospitalizations in 34 US states between 7 August 2020 and 1 March 2021 were 18·2(95%CI:17·9-18·5) and 14·0(95%CI:13·9-14·0) days, respectively. We found the dramatic changes in COVID-19 CFR observed in 27 countries during early stages of the pandemic were mostly caused by undiagnosed cases. Compared to the first week of November 2021, the week mean HOCRs (mimics hospitalization-to-case ratio) for Omicron variant (58·6% of US new cases as of 25 December 2021) decreased 65·16% in the US as of 16 January 2022. INTERPRETATION: The new and reliable measurements described here could be useful for COVID-19 fatality risk and variant-associated risk monitoring. FUNDING: No specific funding was associated with the present study.

Acetylcholinesterase Inhibition Assays for High-Throughput Screening.

Acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh), a vital neurotransmitter that regulates muscle movement and brain function, including memory, attention, and learning. Inhibition of AChE activity can cause a variety of adverse health effects and toxicity. Identifying AChE inhibitors quickly and efficiently warrants developing AChE inhibition assays in a quantitative, high-throughput screening (qHTS) platform. In this chapter, protocols for multiple homogenous AChE inhibition assays used in a qHTS system are provided. These AChE inhibition assays include a (1) human neuroblastoma (SH-SY5Y) cell-based assay with fluorescence or colorimetric detection; (2) human recombinant AChE with fluorescence or colorimetric detection; and (3) combination of human recombinant AChE and liver microsomes with colorimetric detection, which enables detection of test compounds requiring metabolic activation to become AChE inhibitors. Together, these AChE assays can help identify, prioritize, and predict chemical hazards in large compound libraries using qHTS systems.

Cell-Based Imaging Assay for Detection of Phospholipidosis.

Accumulation of lysosomal phospholipids in cells exposed to cationic amphiphilic drugs is characteristic of drug-induced phospholipidosis. The morphological hallmark of phospholipidosis is the appearance of unicentric or multicentric-lamellar bodies when viewed under an electron microscope (EM). The EM method, the gold standard of detecting cellular phospholipidosis, has downsides, namely, low-throughput, high-costs, and unsuitability for screening a large chemical library. This chapter describes a cell-based high-content phospholipidosis assay using the LipidTOX reagent in a high-throughput screening (HTS) platform. This assay has been optimized and validated in HepG2 and HepRG cells, and miniaturized into a 1536-well plate, thus can be used for high-throughput screening (HTS) to identify chemical compounds that induce phospholipidosis.

Identification of Compounds for Butyrylcholinesterase Inhibition.

Butyrylcholinesterase (BChE) is a nonspecific cholinesterase enzyme that hydrolyzes choline-based esters. BChE plays a critical role in maintaining normal cholinergic function like acetylcholinesterase (AChE) through hydrolyzing acetylcholine (ACh). Selective BChE inhibition has been regarded as a viable therapeutic approach in Alzheimer's disease. As of now, a limited number of selective BChE inhibitors are available. To identify BChE inhibitors rapidly and efficiently, we have screened 8998 compounds from several annotated libraries against an enzyme-based BChE inhibition assay in a quantitative high-throughput screening (qHTS) format. From the primary screening, we identified a group of 125 compounds that were further confirmed to inhibit BChE activity, including previously reported BChE inhibitors (e.g., bambuterol and rivastigmine) and potential novel BChE inhibitors (e.g., pancuronium bromide and NNC 756), representing diverse structural classes. These BChE inhibitors were also tested for their selectivity by comparing their IC50 values in BChE and AChE inhibition assays. The binding modes of these compounds were further studied using molecular docking analyses to identify the differences between the interactions of these BChE inhibitors within the active sites of AChE and BChE. Our qHTS approach allowed us to establish a robust and reliable process to screen large compound collections for potential BChE inhibitors.

Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition.

BACKGROUND: Inhibition of acetylcholinesterase (AChE), a biomarker of organophosphorous and carbamate exposure in environmental and occupational human health, has been commonly used to identify potential safety liabilities. So far, many environmental chemicals, including drug candidates, food additives, and industrial chemicals, have not been thoroughly evaluated for their inhibitory effects on AChE activity. AChE inhibitors can have therapeutic applications (e.g., tacrine and donepezil) or neurotoxic consequences (e.g., insecticides and nerve agents). OBJECTIVES: The objective of the current study was to identify environmental chemicals that inhibit AChE activity using in vitro and in silico models. METHODS: To identify AChE inhibitors rapidly and efficiently, we have screened the Toxicology in the 21st Century (Tox21) 10K compound library in a quantitative high-throughput screening (qHTS) platform by using the homogenous cell-based AChE inhibition assay and enzyme-based AChE inhibition assays (with or without microsomes). AChE inhibitors identified from the primary screening were further tested in monolayer or spheroid formed by SH-SY5Y and neural stem cell models. The inhibition and binding modes of these identified compounds were studied with time-dependent enzyme-based AChE inhibition assay and molecular docking, respectively. RESULTS: A group of known AChE inhibitors, such as donepezil, ambenonium dichloride, and tacrine hydrochloride, as well as many previously unreported AChE inhibitors, such as chelerythrine chloride and cilostazol, were identified in this study. Many of these compounds, such as pyrazophos, phosalone, and triazophos, needed metabolic activation. This study identified both reversible (e.g., donepezil and tacrine) and irreversible inhibitors (e.g., chlorpyrifos and bromophos-ethyl). Molecular docking analyses were performed to explain the relative inhibitory potency of selected compounds. CONCLUSIONS: Our tiered qHTS approach allowed us to generate a robust and reliable data set to evaluate large sets of environmental compounds for their AChE inhibitory activity. https://doi.org/10.1289/EHP6993.

Evaluation of chemical compounds that inhibit neurite outgrowth using GFP-labeled iPSC-derived human neurons.

Due to the increasing number of drugs and untested environmental compounds introduced into commercial use, there is recognition for a need to develop reliable and efficient screening methods to identify compounds that may adversely impact the nervous system. One process that has been implicated in neurodevelopment is neurite outgrowth; the disruption of which can result in adverse outcomes that persist later in life. Here, we developed a green fluorescent protein (GFP) labeled neurite outgrowth assay in a high-content, high-throughput format using induced pluripotent stem cell (iPSC) derived human spinal motor neurons and cortical glutamatergic neurons. The assay was optimized for use in a 1536-well plate format. Then, we used this assay to screen a set of 84 unique compounds that have previously been screened in other neurite outgrowth assays. This library consists of known developmental neurotoxicants, environmental compounds with unknown toxicity, and negative controls. Neurons were cultured for 40 h and then treated with compounds at 11 concentrations ranging from 1.56 nM to 92 µM for 24 and 48 h. Effects of compounds on neurite outgrowth were evaluated by quantifying total neurite length, number of segments, and maximum neurite length per cell. Among the 84 tested compounds, neurite outgrowth in cortical neurons and motor neurons were selectively inhibited by 36 and 31 compounds, respectively. Colchicine, rotenone, and methyl mercuric (II) chloride inhibited neurite outgrowth in both cortical and motor neurons. It is interesting to note that some compounds like parathion and bisphenol AF had inhibitory effects on neurite outgrowth specifically in the cortical neurons, while other compounds, such as 2,2',4,4'-tetrabromodiphenyl ether and caffeine, inhibited neurite outgrowth in motor neurons. The data gathered from these studies show that GFP-labeled iPSC-derived human neurons are a promising tool for identifying and prioritizing compounds with developmental neurotoxicity potential for further hazard characterization.

Simple and rapid determination of dioxin in fish and sea food using a highly sensitive reporter cell line, CBG 2.8D.

Food, especially animal origin food is the main source of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), and dioxin-like polychlorinated biphenyls (dl-PCBs) for human exposure. So, a simple, rapid and cheap bioassay method is needed for determination of dioxins in food samples. In this study, we used a new highly sensitive reporter cell line to determine the concentration of dioxins in 33 fish and seafood samples. The samples were extracted by shaking with water/isopropanol (1:1 v/v) and hexane and cleaned-up by a multi layered silica gel column and an alumina column, then analyzed using CBG 2.8D cell line. We compared the results obtained from the CBG 2.8D cell assay to those obtained from conventional High-Resolution Gas Chromatography-High Resolution Mass Spectrometry (HRGC-HRMS) analysis. Good correlations were observed between these two methods (r2=0.93). While the slope of regression line was 1.76, the bioanalytical equivalent (BEQ) values were 1.76 folds higher than WHO-TEQ values and the conversion coefficient was 0.568 (the reciprocal of 1.76). In conclusion, CBG 2.8D cell assay was an applicable method to determine dioxins levels in fish and sea food samples.

Review of high-content screening applications in toxicology.

High-content screening (HCS) technology combining automated microscopy and quantitative image analysis can address biological questions in academia and the pharmaceutical industry. Various HCS experimental applications have been utilized in the research field of in vitro toxicology. In this review, we describe several HCS application approaches used for studying the mechanism of compound toxicity, highlight some challenges faced in the toxicological community, and discuss the future directions of HCS in regards to new models, new reagents, data management, and informatics. Many specialized areas of toxicology including developmental toxicity, genotoxicity, developmental neurotoxicity/neurotoxicity, hepatotoxicity, cardiotoxicity, and nephrotoxicity will be examined. In addition, several newly developed cellular assay models including induced pluripotent stem cells (iPSCs), three-dimensional (3D) cell models, and tissues-on-a-chip will be discussed. New genome-editing technologies (e.g., CRISPR/Cas9), data analyzing tools for imaging, and coupling with high-content assays will be reviewed. Finally, the applications of machine learning to image processing will be explored. These new HCS approaches offer a huge step forward in dissecting biological processes, developing drugs, and making toxicology studies easier.

Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides.

The inhibition of acetylcholinesterase (AChE) has pharmaceutical applications as well as potential neurotoxic effects. The in vivo metabolites of some chemicals including organophosphorus pesticides can become more potent AChE inhibitors compared to their parental compounds. To account for the effects of biotransformation, we have developed and characterized a high-throughput screening method for identifying AChE inhibitors that become active or more potent following xenobiotic metabolism. In this study, an enzyme-based assay was developed in 1536-well plates using recombinant human AChE combined with human or rat liver microsomes. The AChE activity was measured by two methods with different readouts: colorimetric and fluorescent. The assay exhibited exceptional performance characteristics including large assay signal window, low well-to-well variability and high reproducibility. The performance of the assays with microsomes was characterized by testing a group of known AChE inhibitors including parent compounds and their metabolites. Large potency differences between the parent compounds and the metabolites were observed in the assay with microsome addition. Both assay readouts were required for maximal sensitivity. These results demonstrate that this platform is a promising method to profile large numbers of chemicals that require metabolic activation for inhibiting AChE activity.

Systems Modeling of Developmental Vascular Toxicity.

The more than 80,000 chemicals in commerce present a challenge for hazard assessments that toxicity testing in the 21st century strives to address through high-throughput screening (HTS) assays. Assessing chemical effects on human development adds an additional layer of complexity to the screening, with a need to capture complex and dynamic events essential for proper embryo-fetal development. HTS data from ToxCast/Tox21 informs systems toxicology models, which incorporate molecular targets and biological pathways into mechanistic models describing the effects of chemicals on human cells, 3D organotypic culture models, and small model organisms. Adverse Outcome Pathways (AOPs) provide a useful framework for integrating the evidence derived from these in silico and in vitro systems to inform chemical hazard characterization. To illustrate this formulation, we have built an AOP for developmental toxicity through a mode of action linked to embryonic vascular disruption (Aop43). Here, we review the model for quantitative prediction of developmental vascular toxicity from ToxCast HTS data and compare the HTS results to functional vascular development assays in complex cell systems, virtual tissues, and small model organisms. ToxCast HTS predictions from several published and unpublished assays covering different aspects of the angiogenic cycle were generated for a test set of 38 chemicals representing a range of putative vascular disrupting compounds (pVDCs). Results boost confidence in the capacity to predict adverse developmental outcomes from HTS in vitro data and model computational dynamics for in silico reconstruction of developmental systems biology. Finally, we demonstrate the integration of the AOP and developmental systems toxicology to investigate the unique modes of action of two angiogenesis inhibitors.

Development and Application of a Novel Bioassay System for Dioxin Determination and Aryl Hydrocarbon Receptor Activation Evaluation in Ambient-Air Samples.

Airborne persistent toxic substances are associated with health impacts resulting from air pollution, for example, dioxins, dioxin-like polychlorinated biphenyls, and certain polycyclic aromatic hydrocarbons (PAHs), which activate aryl hydrocarbon receptors (AhR) and thereby produce adverse outcomes. Thus, a bioassay for evaluating AhR activation is required for risk assessment of ambient-air samples, and for this purpose, we developed a new and sensitive recombinant mouse hepatoma cell line, CBG2.8D, in which a novel luciferase-reporter plasmid containing two copies of a newly designed dioxin-responsive domain and a minimal promoter derived from a native gene were integrated. The minimal detection limit for 2,3,7,8-tetrachlorodibenzo- p-dioxin with this assay system was 0.1 pM. We used CBG2.8D to determine dioxin levels in 45 ambient-air samples collected in Beijing. The measured bioanalytical equivalent (BEQ) values were closely correlated with the toxic equivalent values obtained from chemical analysis. In haze ambient-air samples, the total activation of aryl hydrocarbon receptors (TAA) was considerably higher than the BEQ of dioxin-rich fractions, according to the results of the cell-based bioassay. Notably, the haze samples contained abundant amounts of PAHs, whose relative toxicity equivalent was correlated with the TAA; this finding suggests that PAHs critically contribute to the AhR-related biological impacts of haze ambient-air samples.

Identification of Angiogenesis Inhibitors Using a Co-culture Cell Model in a High-Content and High-Throughput Screening Platform.

Angiogenesis is an important hallmark of cancer, contributing to tumor formation and metastasis. In vitro angiogenesis models for analyzing tube formation serve as useful tools to study these processes. However, current in vitro co-culture models using primary cells have limitations in usefulness and consistency. Therefore, in the present study, an in vitro co-culture assay system was optimized in a 1536-well format for high-throughput screening using human telomerase reverse transcriptase (hTERT)-immortalized mesenchymal stem cells and aortic endothelial cells. The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC) library containing 2816 drugs was evaluated using the in vitro co-culture assay. From the screen, 35 potent inhibitors (IC50 ≤1 µM) were identified, followed by 15 weaker inhibitors (IC50 1-50 µM). Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study, including thimerosal and podofilox. Among the inhibitors, some compounds were proved to be involved in the hypoxia-inducible factor-1α (HIF-1α) and the nuclear factor-kappa B (NF-κB) pathways. The co-culture model developed by using hTERT-immortalized cell lines described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening.

Why are most phospholipidosis inducers also hERG blockers?

Recent reports have noted that a number of compounds that block the human Ether-à-go-go related gene (hERG) ion channel also induce phospholipidosis (PLD). To explore a hypothesis explaining why most PLD inducers are also hERG inhibitors, a modeling approach was undertaken with data sets comprised of 4096 compounds assayed for hERG inhibition and 5490 compounds assayed for PLD induction. To eliminate the chemical domain effect, a filtered data set of 567 compounds tested in quantitative high-throughput screening (qHTS) format for both hERG inhibition and PLD induction was constructed. Partial least squares (PLS) modeling followed by 3D-SDAR mapping of the most frequently occurring bins and projection on to the chemical structure suggested that both adverse effects are driven by similar structural features, namely two aromatic rings and an amino group forming a three-center toxicophore. Non-parametric U-tests performed on the original 3D-SDAR bins indicated that the distance between the two aromatic rings is the main factor determining the differences in activity; at distances of up to about 5.5 Å, a phospholipidotic compound would also inhibit hERG, while at longer distances, a sharp reduction of the PLD-inducing potential leaves only a well-pronounced hERG blocking effect. The hERG activity itself diminishes after the distance between the centroids of the two aromatic rings exceeds 12.5 Å. Further comparison of the two toxicophores revealed that the almost identical aromatic rings to amino group distances play no significant role in distinguishing between PLD and hERG activity. The hypothesis that the PLD toxicophore appears to be a subset of the hERG toxicophore explains why about 80% of all phospholipidotic chemicals (the remaining 20% are thought to act via a different mechanism) also inhibit the hERG ion channel. These models were further validated in large-scale qHTS assays testing 1085 chemicals for their PLD-inducing potential and 1570 compounds for hERG inhibition. After removal of the modeling and experimental inconclusive compounds, the area under the receiver-operating characteristic (ROC) curve was 0.92 for the PLD model and 0.87 for the hERG model. Due to the exceptional ability of these models to recognize safe compounds (negative predictive values of 0.99 for PLD and 0.94 for hERG were achieved), their use in regulatory settings might be particularly useful.

Development and Application of Human Renal Proximal Tubule Epithelial Cells for Assessment of Compound Toxicity.

Kidney toxicity is a major problem both in drug development and clinical settings. It is difficult to predict nephrotoxicity in part because of the lack of appropriate in vitro cell models, limited endpoints, and the observation that the activity of membrane transporters which plays important roles in nephrotoxicity by affecting the pharmacokinetic profile of drugs is often not taken into account. We developed a new cell model using pseudo-immortalized human primary renal proximal tubule epithelial cells. This cell line (SA7K) was characterized by the presence of proximal tubule cell markers as well as several functional properties, including transporter activity and response to a few well-characterized nephrotoxicants. We subsequently evaluated a group of potential nephrotoxic compounds in SA7K cells and compared them to a commonly used human immortalized kidney cell line (HK-2). Cells were treated with test compounds and three endpoints were analyzed, including cell viability, apoptosis and mitochondrial membrane potential. The results showed that most of the known nephrotoxic compounds could be detected in one or more of these endpoints. There were sensitivity differences in response to several of the chemicals between HK-2 and SA7K cells, which may relate to differences in expressions of key transporters or other components of nephrotoxicity pathways. Our data suggest that SA7K cells appear as promising for the early detection of renal toxicants.

Identification of acetylcholinesterase inhibitors using homogenous cell-based assays in quantitative high-throughput screening platforms.

Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of acetylcholine, a neurotransmitter associated with muscle movement, cognition, and other neurobiological processes. Inhibition of AChE activity can serve as a therapeutic mechanism, but also cause adverse health effects and neurotoxicity. In order to efficiently identify AChE inhibitors from large compound libraries, homogenous cell-based assays in high-throughput screening platforms are needed. In this study, a fluorescent method using Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) and the Ellman absorbance method were both developed in a homogenous format using a human neuroblastoma cell line (SH-SY5Y). An enzyme-based assay using Amplex Red was also optimized and used to confirm the potential inhibitors. These three assays were used to screen 1368 compounds, which included a library of pharmacologically active compounds (LOPAC) and 88 additional compounds from the Tox21 program, at multiple concentrations in a quantitative high-throughput screening (qHTS) format. All three assays exhibited exceptional performance characteristics including assay signal quality, precision, and reproducibility. A group of inhibitors were identified from this study, including known (e.g. physostigmine and neostigmine bromide) and potential novel AChE inhibitors (e.g. chelerythrine chloride and cilostazol). These results demonstrate that this platform is a promising means to profile large numbers of chemicals that inhibit AChE activity.

Transporter-Mediated Nuclear Entry of Jasmonoyl-Isoleucine Is Essential for Jasmonate Signaling.

To control gene expression by directly responding to hormone concentrations, both animal and plant cells have exploited comparable mechanisms to sense small-molecule hormones in nucleus. Whether nuclear entry of these hormones is actively transported or passively diffused, as conventionally postulated, through the nuclear pore complex, remains enigmatic. Here, we identified and characterized a jasmonate transporter in Arabidopsis thaliana, AtJAT1/AtABCG16, which exhibits an unexpected dual localization at the nuclear envelope and plasma membrane. We show that AtJAT1/AtABCG16 controls the cytoplasmic and nuclear partition of jasmonate phytohormones by mediating both cellular efflux of jasmonic acid (JA) and nuclear influx of jasmonoyl-isoleucine (JA-Ile), and is essential for maintaining a critical nuclear JA-Ile concentration to activate JA signaling. These results illustrate that transporter-mediated nuclear entry of small hormone molecules is a new mechanism to regulate nuclear hormone signaling. Our findings provide an avenue to develop pharmaceutical agents targeting the nuclear entry of small molecules.

Effects of Acute Low-Dose Exposure to the Chlorinated Flame Retardant Dechlorane 602 and Th1 and Th2 Immune Responses in Adult Male Mice.

BACKGROUND: Although the chlorinated flame retardant Dechlorane (Dec) 602 has been detected in food, human blood, and breast milk, there is limited information on potential health effects, including possible immunotoxicity. OBJECTIVES: We determined the immunotoxic potential of Dec 602 in mice by examining the expression of phenotypic markers on thymocyte and splenic lymphocyte subsets, Th1/Th2 transcription factors, and the production of cytokines and antibodies. METHODS: Adult male C57BL/6 mice were orally exposed to environmentally relevant doses of Dec 602 (1 and 10 µg/kg body weight per day) for 7 consecutive days. Thymocyte and splenic CD4 and CD8 subsets and splenocyte apoptosis were examined by flow cytometric analysis. Cytokine expression was measured at both the mRNA and the protein levels. Levels of the transcription factors Th1 (T-bet and STAT1) and Th2 (GATA3) were determined using quantitative real-time polymerase chain reaction (qPCR). Serum levels of immunoglobulins IgG1, IgG2a, IgG2b and IgE were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Splenic CD4+ and CD8+ T cell subsets were decreased compared with vehicle controls, and apoptosis was significantly increased in splenic CD4+ T cells. Expression (mRNA and protein) of Th2 cytokines [interleukin (IL)-4, IL-10, and IL-13] increased, and that of Th1 cytokines [IL-2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α] decreased. The Th2 transcriptional factor GATA3 increased, whereas the Th1 transcriptional factors T-bet and STAT1 decreased. As additional indicators of the Th2-Th1 imbalance, production of IgG1 was significantly increased, whereas IgG2a was reduced. CONCLUSIONS: To our knowledge, we are the first to report evidence of the effects of Dec 602 on immune function in mice, with findings indicating that Dec 602 exposure favored Th2 responses and reduced Th1 function. CITATION: Feng Y, Tian J, Xie HQ, She J, Xu SL, Xu T, Tian W, Fu H, Li S, Tao W, Wang L, Chen Y, Zhang S, Zhang W, Guo TL, Zhao B. 2016. Effects of acute low-dose exposure to the chlorinated flame retardant dechlorane 602 and Th1 and Th2 immune responses in adult male mice. Environ Health Perspect 124:1406-1413; http://dx.doi.org/10.1289/ehp.1510314.