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U18666A Treatment Results in Cholesterol Accumulation, Reduced Na+, K+-ATPase Activity, and Increased Oxidative Stress in Rat Cortical Astrocytes

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Lipids

Abstract

The objective of this study was to determine the effect of U18666A, an inhibitor of cholesterol synthesis and its intracellular transport, on oxidative stress parameters in cortical astrocytes cultured from Wistar rats (0–3 days old). The cultures were incubated with U18666A (0.25 µg/mL) for 48 h, conditions that are considered ideal to mimic Niemann–Pick type C disease. A variety of indicators of oxidative stress were measured. U18666A treatment increased cholesterol 2-fold in treated compared to control astrocytes. Oxidative stress was significantly elevated in treated cells as demonstrated by a 1.7-fold increase in thiobarbituric acid reactive substances, a 60 % decrease is sulfhydral groups, and a 3.7-fold increase in carbonyl groups, indicative of increased lipid and protein oxidation following U18666A treatment. Consistent with these changes, both catalase and superoxide dismutase activities were significantly reduced nearly 50 % in treated compared to control astrocytes. Collectively, these change resulted in a 50 % reduction in Na+, K+-ATPase specific activity following U18666A treatment, suggesting a significant alteration in its plasma membrane environment. Overall, these changes indicate that U18666A treatment results in increased cholesterol levels and an increased level of oxidative stress in cortical astrocytes, consistent with what is observed in Niemann–Pick type C disease.

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Abbreviations

NPC:

Niemann–Pick type C

U18666A:

3-β-[2-(Diethylamino)ethoxy]androst-5-en-17-one

ROS:

Reactive oxygen species

CNS:

Central nervous system

ATP:

Adenosine triphosphate

TBARS:

Thiobarbituric acid reactive substances

SOD:

Superoxide dismutase

CAT:

Catalase

H2O2 :

Hydrogen peroxide

NO:

Nitric oxide

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Authors and Affiliations

  1. Biochemistry Department, Lysosomal Storage Diseases Laboratory, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil

    Daniela Copetti-Santos, Vitoria Moraes & Dácio Franco Weiler

  2. Biosciences and Rehabilitation, Centro Universitário Metodista (IPA), Porto Alegre, RS, Brazil

    Alexandre Silva de Mello, Fernanda de Souza Machado, Jéssica Pereira Marinho & Cláudia Funchal

  3. Biochemistry Department, Metabolic Diseases and Neuroprotection Laboratory, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil

    Cassiana Siebert, Janaina Kolling, Angela T. S. Wyse & Janice Carneiro Coelho

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  1. Daniela Copetti-Santos
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  2. Vitoria Moraes
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Correspondence to Daniela Copetti-Santos.

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Copetti-Santos, D., Moraes, V., Weiler, D.F. et al. U18666A Treatment Results in Cholesterol Accumulation, Reduced Na+, K+-ATPase Activity, and Increased Oxidative Stress in Rat Cortical Astrocytes. Lipids 50, 937–944 (2015). https://doi.org/10.1007/s11745-015-4062-4

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