Browsing by Author "Ryde, Ian T"

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    PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.
    (Curr Protoc Toxicol, 2016-02-01) Gonzalez-Hunt, Claudia P; Rooney, John P; Ryde, Ian T; Anbalagan, Charumathi; Joglekar, Rashmi; Meyer, Joel N
    Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays.
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    Resveratrol Protects Against Hydroquinone-Induced Oxidative Threat in Retinal Pigment Epithelial Cells.
    (Investigative ophthalmology & visual science, 2020-04) Neal, Samantha E; Buehne, Kristen L; Besley, Nicholas A; Yang, Ping; Silinski, Peter; Hong, Jiyong; Ryde, Ian T; Meyer, Joel N; Jaffe, Glenn J

    Purpose

    Oxidative stress in retinal pigment epithelial (RPE) cells is associated with age-related macular degeneration (AMD). Resveratrol exerts a range of protective biologic effects, but its mechanism(s) are not well understood. The aim of this study was to investigate how resveratrol could affect biologic pathways in oxidatively stressed RPE cells.

    Methods

    Cultured human RPE cells were treated with hydroquinone (HQ) in the presence or absence of resveratrol. Cell viability was determined with WST-1 reagent and trypan blue exclusion. Mitochondrial function was measured with the XFe24 Extracellular Flux Analyzer. Expression of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit was evaluated by qPCR. Endoplasmic reticulum stress protein expression was measured by Western blot. Potential reactions between HQ and resveratrol were investigated using high-performance liquid chromatography mass spectrometry with resveratrol and additional oxidants for comparison.

    Results

    RPE cells treated with the combination of resveratrol and HQ had significantly increased cell viability and improved mitochondrial function when compared with HQ-treated cells alone. Resveratrol in combination with HQ significantly upregulated HO-1 mRNA expression above that of HQ-treated cells alone. Resveratrol in combination with HQ upregulated C/EBP homologous protein and spliced X-box binding protein 1. Additionally, new compounds were formed from resveratrol and HQ coincubation.

    Conclusions

    Resveratrol can ameliorate HQ-induced toxicity in RPE cells through improved mitochondrial bioenergetics, upregulated antioxidant genes, stimulated unfolded protein response, and direct oxidant interaction. This study provides insight into pathways through which resveratrol can protect RPE cells from oxidative damage, a factor thought to contribute to AMD pathogenesis.