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High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress

View Article: PubMed Central - PubMed

ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.


Related in: MedlinePlus

Concentration response curves (CRCs) of compounds active in the multiplexed ARE-bla/CTG assay in HepG2 cells. Metrazoline oxalate, idazoxan hydrochloride, nitrovin, and aurothioglucose activate the ARE/NF-E2-related factor 2 (Nrf2) pathway in HepG2 cells, as visualized by the blue/green ratio CRC (supplemental online data). (A, B): Little or no activity is seen in the CTG cell viability assay for metrazoline oxalate and idazoxan hydrochloride, indicating that these compounds are not toxic to HepG2 cells at the concentrations tested. Nonlinear regression analysis, with three-parameters least-squares fit. (C, D): Nitrovin and aurothioglucose are toxic at the highest concentrations, but activate the ARE/Nrf2 pathway at lower concentrations. Abbreviations: ARE-bla, antioxidant response element β-lactamase; CTG, CellTiter-Glo assay.
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Figure 6: Concentration response curves (CRCs) of compounds active in the multiplexed ARE-bla/CTG assay in HepG2 cells. Metrazoline oxalate, idazoxan hydrochloride, nitrovin, and aurothioglucose activate the ARE/NF-E2-related factor 2 (Nrf2) pathway in HepG2 cells, as visualized by the blue/green ratio CRC (supplemental online data). (A, B): Little or no activity is seen in the CTG cell viability assay for metrazoline oxalate and idazoxan hydrochloride, indicating that these compounds are not toxic to HepG2 cells at the concentrations tested. Nonlinear regression analysis, with three-parameters least-squares fit. (C, D): Nitrovin and aurothioglucose are toxic at the highest concentrations, but activate the ARE/Nrf2 pathway at lower concentrations. Abbreviations: ARE-bla, antioxidant response element β-lactamase; CTG, CellTiter-Glo assay.

Mentions: To begin to understand the mechanism of action behind the cytoprotection of the active compounds, we evaluated the activity of the compounds in an ARE-β-lactamase (ARE-bla) reporter gene cell line. Activation of the ARE/nuclear factor-E2-related factor 2 (ARE/Nrf2) pathway has been found to be cytoprotective in astrocytes under oxidative stress [52]. Activation of this Nrf2 pathway may also explain the protective nuclear phenotype induced by some compounds in the absence of H2O2, because xenobiotics induce Nrf2 [53]. Of the 34 compounds, we found 4 that directly activated ARE-β-lactamase expression in HepG2 cells: idazoxan hydrochloride, aurothioglucose, metrazoline oxalate, and nitrovin. Whereas idazoxan hydrochloride and metrazoline oxalate had a simple increase in ARE activation (BLA expression), the activity of both aurothioglucose and nitrovin in the ARE-bla assay was bell shaped, with cytotoxicity at higher concentrations of compounds (Fig. 6).


High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress
Concentration response curves (CRCs) of compounds active in the multiplexed ARE-bla/CTG assay in HepG2 cells. Metrazoline oxalate, idazoxan hydrochloride, nitrovin, and aurothioglucose activate the ARE/NF-E2-related factor 2 (Nrf2) pathway in HepG2 cells, as visualized by the blue/green ratio CRC (supplemental online data). (A, B): Little or no activity is seen in the CTG cell viability assay for metrazoline oxalate and idazoxan hydrochloride, indicating that these compounds are not toxic to HepG2 cells at the concentrations tested. Nonlinear regression analysis, with three-parameters least-squares fit. (C, D): Nitrovin and aurothioglucose are toxic at the highest concentrations, but activate the ARE/Nrf2 pathway at lower concentrations. Abbreviations: ARE-bla, antioxidant response element β-lactamase; CTG, CellTiter-Glo assay.
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Related In: Results  -  Collection

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Figure 6: Concentration response curves (CRCs) of compounds active in the multiplexed ARE-bla/CTG assay in HepG2 cells. Metrazoline oxalate, idazoxan hydrochloride, nitrovin, and aurothioglucose activate the ARE/NF-E2-related factor 2 (Nrf2) pathway in HepG2 cells, as visualized by the blue/green ratio CRC (supplemental online data). (A, B): Little or no activity is seen in the CTG cell viability assay for metrazoline oxalate and idazoxan hydrochloride, indicating that these compounds are not toxic to HepG2 cells at the concentrations tested. Nonlinear regression analysis, with three-parameters least-squares fit. (C, D): Nitrovin and aurothioglucose are toxic at the highest concentrations, but activate the ARE/Nrf2 pathway at lower concentrations. Abbreviations: ARE-bla, antioxidant response element β-lactamase; CTG, CellTiter-Glo assay.
Mentions: To begin to understand the mechanism of action behind the cytoprotection of the active compounds, we evaluated the activity of the compounds in an ARE-β-lactamase (ARE-bla) reporter gene cell line. Activation of the ARE/nuclear factor-E2-related factor 2 (ARE/Nrf2) pathway has been found to be cytoprotective in astrocytes under oxidative stress [52]. Activation of this Nrf2 pathway may also explain the protective nuclear phenotype induced by some compounds in the absence of H2O2, because xenobiotics induce Nrf2 [53]. Of the 34 compounds, we found 4 that directly activated ARE-β-lactamase expression in HepG2 cells: idazoxan hydrochloride, aurothioglucose, metrazoline oxalate, and nitrovin. Whereas idazoxan hydrochloride and metrazoline oxalate had a simple increase in ARE activation (BLA expression), the activity of both aurothioglucose and nitrovin in the ARE-bla assay was bell shaped, with cytotoxicity at higher concentrations of compounds (Fig. 6).

View Article: PubMed Central - PubMed

ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.


Related in: MedlinePlus